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Die Physiologie des Schmerzes umfasst komplexe immunologische, sensorische und inflammatorische Prozesse im Rückenmark, im Gehirn und in der Peripherie. Wiederholte nozizeptive Stimulation induziert pathophysiologische Veränderungen bei der Schmerzweiterleitung, aus denen eine periphere oder zentrale Sensibilisierung resultiert. Diese kann bei dafür anfälligen Patienten zu der Ausbildung von chronischen Schmerzzuständen führen. Obwohl das Wissen über die genauen molekularen Vorgänge der Schmerz-Chronifizierung noch immer unvollständig ist, sind die Identifizierung von Risikofaktoren vernünftige Schritte, um die individuelle Anfälligkeit für die Entwicklung chronischer Schmerzen zu bestimmen. Das Hauptziel dieser Doktorarbeit bestand daher in der Identifikation humaner genetischer Biomarker für chronische Schmerzzustände.
The fungal interaction with plants is a 400 million years old phenomenon, which presumably assisted in the plants’ establishment on land. In a natural ecosystem, all plant-ranging from large trees to sea-grasses-are colonized by fungal endophytes, which can be detected inter- and intracellularly within the tissues of apparently healthy plants, without causing obvious negative effects on their host. These ubiquitous and diverse microorganisms are likely playing important roles in plant fitness and development. However, the knowledge on the ecological functions of fungal root endophytes is scarce. Among possible functions of endophytes, they are implicated in mutualisms with plants, which may increase plant resistance to biotic stressors like herbivores and pathogens, and/or to abiotic factors like soil salinity and drought. Also, endophytes are fascinating microorganisms in regard to their high potential to produce a great spectrum of secondary metabolites with expected ecological functions. However, evidences suggest that the interactions between host plants and endophytes are not static and endophytes express different symbiotic lifestyles ranging from mutualism to parasitism, which makes difficult to predict the ecological roles of these cryptic microorganisms. To reveal the ecological function of fungal root endophytes, this doctoral thesis aims at assessing fungal root endophytes interactions with different plants and their effects on plant fitness, based on their phylogeny, traits, and competition potential in settings encompassing different abiotic contexts. To understand the cryptic implication of nonmycorrhizal endophytes in ecosystem processes, we isolated a diverse spectrum of fungal endophytes from roots of several plant species growing in different natural contexts and tested their effects on different model plants under axenic laboratory conditions. Additionally,we aimed at investigating the effect of abiotic and biotic variables on the outcome of interactions between fungal root endophytes and plants.
In summary, the morphological and physiological traits of 128 fungal endophyte strains within ten fungal orders were studied and artificial experimental systems were used to reproduce their interactions with three plant species under laboratory conditions. Under defined axenic conditions, most endophytes behaved as weak parasites, but their performance varied across plant species and fungal taxa. The variation in the interactions was partly explained by convergent fungal traits that separate groups of endophytes with potentially different niche preferences. According to my findings, I predict that the functional complementarity of strains is essential in structuring natural root endophytic communities. Additionally, the responses of plant-endophyte interactions to different abiotic factors, namely nutrient availability, light intensity, and substrate’s pH, indicate that the outcome of plant-fungus relationships may be robust to changes in the abiotic environment. The assessment of the responses of plant endophyte interactions to biotic context, as combinations of selected dominant root fungal endophytes with different degrees of trait similarity and shared evolutionary history, indicates that frequently coexisting root-colonizing fungi may avoid competition in inter-specific interactions by occupying specific niches, and that their interactions likely define the structure of root-associated fungal communities and influence the microbiome impacts on plant fitness.
In conclusion, my findings suggest that dominant fungal lineages display different ecological preferences and complementary sets of functional traits, with different niche preferences within root tissues to avoid competition. Also, their diverse effects on plant fitness is likely host-isolate dependent and robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont.
How the brain evolved remains a mystery. The goal of this thesis is to understand the fundamental processes that are behind the evolutionary history of the brain. Amniotes appeared 320 million years ago with the transition from water to land. This early group bifurcated into sauropsids (reptiles and birds) and synapsids (mammals). Amniote brains evolved separately and display obvious structural and functional differences. Although those differences reflect brain diversification, all amniote brains share a common ancestor and their brains show multiple derived similarities: equivalent structures, networks, circuits and cell types have been preserved during millions of years. Finding these differences and similarities will help us understand brain historical evolution and function. Studying brain evolution can be approached from various levels, including brain structure, circuits, cell types, and genes. We propose a focus on cell types for a more comprehensive understanding of brain evolution. Neurons are the basic building blocks and the most diverse cell types in the brain. Their evolution reflects changes in the developmental processes that produce them, which in turn may shape the neural circuits they belong to. However, there is currently a lack of a unified criteria for studying the homology of connectivity and development between neurons. A neuron’s transcriptome is a molecular representation of its identity, connectivity, and developmental/evolutionary history. Hence the comparison of neuronal transcriptomes within and across species is a new and transformative development in the study of brain evolution. As an alternative, comparing neuronal transcriptomes across different species can provide insights into the evolution of the brain. We propose that comparing transcriptomes can be a way to fill this gap and unify these criteria. In previous studies, published in Science (Tosches et al., 2018) and Nature (Norimoto et al., 2020), we leveraged scRNAseq in reptiles to re-evaluate the origins and evolution of the mammalian cerebral cortex and claustrum. Motivated by the success of this approach, in this thesis we have now expanded single-cell profiling to the entire brain of a lizard species, the Australian dragon Pogona vitticeps, with a special focus in thalamus and prethalamus of. This approach allowed us to study the evolution of neuron types in amniotes. Therefore, we aimed to build a multilevel atlas of the lizard brain based on histology and transcriptomic and compare it to an equal mouse dataset (Zeisel et al., 2018).
Our atlas reveals a general structure that is consistent with that for other amniote brains, allowing us to make a direct comparison between lizard and mouse, despite their evolutionary divergence 320 million years ago. Through our analysis of the transcriptomes present in various neuron types, we have uncovered a core of conserved classes and discovered a fascinating dichotomy of new and conserved neuron types throughout the brain. This research challenges the traditional notion that certain brain regions are more conserved than others.
Our research also has uncovered the evolutionary history of the lizard thalamus and prethalamus by comparing them to homologous brain regions of the mouse. This pioneering research sheds new light on our understanding of the evolutionary history of the lizard brain. We propose a new classification of the lizard thalamic nuclei based on
transcriptomics. Our research revealed that the thalamic neuron types in lizards can be grouped into two large, conserved categories from the medial to lateral thalamus. These categories are encoded by a common set of effector genes, linking theories based on connectivity and molecular studies of these areas. In our data we have seen that there is a conservation of the medial-lateral transcriptomic axis in mouse and lizard, this conservation was most likely already present in the common ancestor. Although there is a shared medial-lateral axis, a deeper study of the thalamic cell types has allowed us to see the existence of a partial diversification of the thalamic population, specifically in the sensory-related lateral thalamus; in opposition, the medial thalamic nuclei neuron-types have been preserved.
On the other hand, the comparison with the mammalian prethalamus allowed us to confirm that the lizard ventromedial thalamic neuron types are homologous to mouse reticular thalamic neuron types (Díaz et al., 1994), even if they do not express the classical Reticular thalamic nucleus (RTn) marker PV/pvalb. We also discovered that there has been a simplification in the mammalian prethalamic neuron types in favor of an increase in the number of Interneurons (IN) types within their thalamus. We suggest that the loss of GABAergic neuronal types in the mammalian prethalamus is linked to the need for a more efficient control of the thalamo-pallial communication in mammals, while in lizards, where thalamo-pallial communication is probably simpler, the diversity prethalamus presents a higher diversity.
A novel role for mutant mRNA degradation in triggering transcriptional adaptation to mutations
(2020)
Robustness to mutations promotes organisms’ well-being and fitness. The increasing number of mutants in various model organisms, and humans, showing no obvious phenotype (Bouche and Bouchez, 2001; Chen et al., 2016b; Giaever et al., 2002; Kok et al., 2015) has renewed interest into how organisms adapt to gene loss. In the presence of deleterious mutations, genetic compensation by transcriptional upregulation of related gene(s) (also known as transcriptional adaptation) has been reported in numerous systems (El-Brolosy and Stainier, 2017; Rossi et al., 2015; Tondeleir et al., 2012); however, the molecular mechanisms underlying this response remained unclear. To investigate this phenomenon, I develop and study multiple models of transcriptional adaptation in zebrafish and mouse cell lines. I first show that transcriptional adaptation is not caused by loss of protein function, indicating that the trigger lies upstream, and find that the response involves enhanced transcription of the related gene(s). Furthermore, I observe a correlation between levels of mutant mRNA degradation and upregulation of related genes. To investigate the role of mutant mRNA degradation in triggering the response, I generate mutant alleles that do not transcribe the mutated gene and find that they fail to induce a transcriptional response and display stronger phenotypes. Transcriptome analysis of alleles displaying mutant mRNA degradation revealed upregulation of a significant proportion of genes displaying sequence similarity with the mutated gene’s mRNA, suggesting a model whereby mRNA degradation intermediates induce transcriptional adaptation via sequence similarity. Further mechanistic analyses suggested RNA-decay factors-dependent chromatin remodeling, and repression of antisense RNAs to be implicated in the response. These results identify a novel role for mutant mRNA degradation in buffering against mutations. Besides, they hold huge implications on understanding disease-causing mutations and shall help in designing mutations that lead to minimal transcriptional adaptation-induced compensation, facilitating studying gene function in model organisms.
Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
ADAM15, which belongs to the family of the disintegrin and metalloproteinases, is a multi-domain transmembrane protein. A strongly upregulated expression of ADAM15 is found in inflamed synovial membranes from articular joints affected by osteoarthritis and especially rheumatoid arthritis (RA). During the chronic inflammatory process in RA the synovial membrane gets hyperplastic, resulting eventually in the formation of a pannus tissue, which can invade into the adjacent cartilage and bone thereby destroying their integrity. Previously, the expression of ADAM15 in fibroblasts of the RA synovial membrane was found to confer a significant anti-apoptotic response upon triggering of the Fas receptor, which resulted in the activation of two survival kinases, focal adhesion kinase (FAK) and Src. The Fas receptor, also named CD95, belongs to the death receptor family of the tumor necrosis factor receptors and stimulation of Fas/CD95 by its ligand FasL results in the execution of apoptotic cell death in synovial membranes of RA patients. However, the occurrence of apoptotic cell death in vivo in RA synovial tissues is considerably low despite the presence of FasL at high concentrations in the chronically inflamed joint. Accordingly, a general apoptosis resistance is a characteristic of RA-synovial fibroblasts that contributes considerably to the formation the hyperplastic aggressive pannus tissue. The objective of this study was to investigate the mechanisms underlying the capability of ADAM15 to transform FasL-mediated death- inducing signals into pro-survival activation of Src and FAK in rheumatoid arthritis fibroblasts (RASFs).
In the present study, the down-regulation of ADAM15 by RNA interference resulted in a significant increase of caspase 3/7 activity upon stimulation of the Fas receptor in RASFs. Likewise, chondrocytes expressing a deletion mutant of ADAM15 (ΔC), lacking the cytoplasmic domain, revealed increased caspase activities upon Fas ligation in comparison to cells transfected with full-length ADAM15, clearly demonstrating the importance of the cytoplasmic domain for an increased apoptosis resistance. Furthermore, activation of the Fas receptor triggered the phosphorylation of Src at Y416, which results in the active conformation of Src, as well as the phosphorylation of FAK at Y576/577 and Y861 – the target tyrosines phosphorylated by Src - in full-length ADAM15-transfected chondrocytes. However, cells transfected with ADAM15 mutant (ΔC) or with vector control did not exhibit any activation of Src and FAK upon Fas ligation. This suggested the presence of an as yet unknown protein interaction mediating the Fas triggered activation of the two kinases.
In order to identify this mechanism, the application of signal transduction inhibitors interfering with Calcium signaling either by inhibiting calmodulin with trifluoperazine (TFP) or the Calcium release-activated channel (CRAC/Orai1) with BTP-2 efficiently inhibited the phosphorylation of FAK and Src, revealing a role of calmodulin, the major Ca2+ sensor in cells, in ADAM15-dependent and Fas-elicited activation of the two survival kinases. Also, a direct Ca2+ -dependent binding of calmodulin to ADAM15 could be demonstrated by pull-down assays using calmodulin-conjugated sepharose and by protein binding assays using the recombinant cytoplasmic domain of ADAM15 and calmodulin.
Furthermore, it could be demonstrated in living synovial fibroblasts by double immunofluorescence stainings that triggering the Fas receptor by its ligand FasL or a Fas-activating antibody resulted in the recruitment of calmodulin to ADAM15 as well as to the Fas receptor in patch-like structures at the cell membrane. Simultaneously, Src associated with calmodulin was shown to become engaged in an ADAM15 complex, also containing cytoplasmic-bound FAK, by co-immunoprecipitations.
Additional studies were performed to analyze the efficacy of TFP and BTP-2 on apoptosis induction in synovial fibroblasts from 10 RA patients. Using caspase 3/7 and annexin V stainings for determining apoptosis, it could be shown that both inhibitors did not possess any apoptosis inducing capacity. However, when co-incubated with FasL both compounds synergistically enhanced apoptosis rates in the RASFs. Moreover, an additional silencing of ADAM15 revealed a further significant rise in apoptosis rates upon incubation with FasL/TFP or FasL/BTP-2, providing unequivocal evidence for an involvement of ADAM15 in facilitating apoptosis resistance in RASFs.
Taken together, these results demonstrate that ADAM15 provides a scaffold for the formation of calmodulin-dependent pro-survival signaling complexes upon CRAC/Orai1 coactivation by Fas ligation, which provides a new potential therapeutic target to break the apoptosis resistance in RASFs that critically contributes to joint destruction in RA.
Adaptive Radiation und Zoogeographie anisakider Nematoden verschiedener Klimazonen und Ozeane
(2013)
Anisakide Nematoden sind Parasiten aquatischer Organismen und weltweit in marinen Habitaten verbreitet. Ihre Übertragungswege sind tief im marinen Nahrungsnetz verwurzelt und schließen ein breites Spektrum pelagisch/benthischer Invertebraten (z.B. Cephalopoda, Gastropoda, Crustacea, Polychaeta) und Vertebraten (z.B. Teleostei, Elasmobranchia, Cetacea, Pinnipedia, Aves) als Zwischen- bzw. Endwirte ein. Aufgrund der hohen Befallszahlen u.a. in der Muskulatur und Viszera kommerziell intensiv genutzter Fischarten (z.B. Clupea harengus, Gadus morhua, Salmo salar) sowie ihrer Rolle als Auslöser der menschlichen Anisakiasis nehmen die Vertreter der Gattung Anisakis unter den anisakiden Nematoden eine Sonderstellung ein. Anhand der verbesserten Diagnostik und der Etablierung sowie Weiterentwicklung molekularbiologischer Methoden ist es in den letzten zwei Dekaden gelungen, die bestehende Taxonomie und Systematik der Gattung Anisakis zu erweitern bzw. zu revidieren. Aktuelle molekulare Analysen weisen auf die Existenz von insgesamt neun distinkten Arten hin, welche eine hohe genetische Heterogenität und Wirtsspezifität aufweisen, äußerlich jedoch nahezu identisch sind (sog. kryptische Arten). Trotz kontinuierlicher Forschung auf dem Gebiet ist das Wissen über die Biologie von Anisakis immer noch unzureichend.
Die vorliegende Dissertation ist in kumulativer Form verfasst und umfasst drei (ISI-) Einzelpublikationen. Die Zielsetzung der durchgeführten Studien bestand unter anderem darin, unter Verwendung molekularbiologischer und computergestützter Analyseverfahren, Fragestellungen zur Zoogeographie, (Co-)Phylogenie, Artdiagnostik, Lebenszyklus-Ökologie sowie des bioindikatorischen Potentials dieser Gattung zu bearbeiten und bestehende Wissenslücken zu schließen.
Die Verbreitung von Anisakis, welche bisher ausschließlich anhand von biogeographischen Einzelnachweisen abgeschätzt wurde, konnte durch den angewandten Modellierungsansatz erstmalig interpoliert und in Kartenform vergleichend dargestellt werden. Dabei wurde gezeigt, dass die Verbreitung von Anisakis spp. in den Ozeanen und Klimazonen nicht gleichmäßig ist. Die Analysen deuten auf die Existenz spezies-spezifischer horizontaler und vertikaler Verbreitungsmuster hin, welche neben abiotischen Faktoren durch die Verbreitung und Abundanz der jeweiligen Zwischen- und Endwirte sowie deren Tiefenverteilung und Nahrungspräferenzen geprägt sind.
Durch die umfangreiche Zusammenstellung und anschließende Kategorisierung der (mit molekularen Methoden) geführten Zwischenwirtsnachweise konnten indirekte Rückschlüsse über die vertikale Verbreitung von Anisakis spp. entlang der Tiefenhabitate gezogen werden.
Während Anisakis auf Gattungsebene in der gesamten Wassersäule entlang verschiedener Tiefenhabitate abundant ist, wurde für die stenoxene Art Anisakis paggiae ein meso-/bathypelagisch orientierter Lebenszyklus postuliert. Durch den Einbezug eines breiten Spektrums (paratenischer) Zwischen- und Transportwirte aus unterschiedlichen trophischen Ebenen werden Transmissionslücken im Lebenszyklus der Gattung weitestgehend minimiert und der Transmissionserfolg auf den Endwirt, und damit die Wahrscheinlichkeit einer erfolgreichen Reproduktion, erhöht. Ausgeprägte Wirtspräferenzen sowie phylogenetische Analysen des ribosomalen ITS-Markers stützen eine Theorie zur co-evolutiven Anpassung der Parasiten an ihre Endwirte. Anisakis eignet sich daher unter Einschränkungen als Bioindikator für die vertikale und horizontale Verbreitung und Abundanz der Endwirte und lässt Rückschlüsse auf trophische Interaktionen im Nahrungsnetz zu. Durch die weitere Beprobung von Zwischenwirten aus verschiedenen trophischen Ebenen in zukünftigen Studien, kann eine genauere Bewertung potentiell abweichender Lebenszyklus-Strategien gewährleistet werden. Insbesondere ist die Datenlage zur Prävalenz und Abundanz anisakider Nematoden in Cephalopoda und Crustacea noch unzureichend. Die Probennahme sollte dabei unter besonderer Berücksichtigung bislang wenig oder unbeprobter geographischer Regionen, Tiefenhabitate und Wirtsarten durchgeführt werden.
In the past decades, the use and production of chemicals has been on the rise globally due to increasing industrialization and intensive agriculture; resulting in the occurrence and ecotoxicological risks of chemicals of emerging concern (CECs) in the aquatic compartments. Risks include changes in community structure resulting in the dominance of one species and ecosystem imbalance. When dominant disease-causing organisms are in the environment, the disease transmission is increased. For example, host snails for the schistosomiasis, a human trematode disease, are known to be tolerant to pesticide
exposure compared to the predators. This would therefore result in an increased abundance of snails which consequently increase the disease transmission in the human population.
Kenya, being a low income country faces a lot of challenges with provision of clean water, diseases and sanitation facilities, and increasing population which results in intensive agriculture coupled with pesticide use. Although a lot of research has been carried out on the environmental occurrence and risk of CECs (Chapter 1), most of these studies have been done in developed countries with limited information from Africa. Additionally, research in Africa focused on urban areas with limited number of compounds analyzed and mostly in the water phase, and inadequate information on the effects of CECs on the aquatic organisms. In order to reduce this knowledge gap, this dissertation focused on identification and quantification of CECs present in water, sediment and snails from western Kenya, and the contribution of pesticides to the transmission of schistosomiasis.
Chapter 2 gives a summary of the results and discussion of the dissertation. In Chapter 3, a comprehensive chemical analysis was carried out on 48 water samples to identify compounds, spatial patterns and associated risks for fish, crustacean and algae using toxic unit (TU) approach. A total of 78 compounds were detected with pesticides and biocides being the compounds most frequently detected. Spatial pattern analysis revealed limited compound grouping based on land use. Acute risk for crustaceans and algae were driven by one to three individual compounds. These compounds responsible for toxicity were prioritized as candidate compounds for monitoring and regulation in Kenya.
In Chapter 4, an extension of Chapter 3 was done to cover the CECs present in snails and sediment from the 48 sites. A total of 30 compounds were found in snails and 78 in sediments with 68 additional compounds being found which were not previously detected in water. Higher contaminant concentrations were found in agricultural sites than in areas without anthropogenic activities. The highest acute toxicity (TU 0.99) was determined for crustaceans based on compounds in sediment samples. The risk was driven by diazinon and pirimiphos-methyl. Acute and chronic risks to algae were driven by diuron whereas fish were found to be at low to no acute risk.
In Chapter 5, the effect of pesticide contamination on schistosomiasis transmission was evaluated by applying complimentary laboratory and field studies. In the field studies, the ecological mechanisms through which pesticides and physical chemical parameters affect host snails, predators and competitors were investigated. Pesticide data was obtained from the results in chapter 3. The overall distribution of grazers and predators was not affected by pesticide pollution. However, within the grazers, pesticide pollution increased dominance of host snails. On the contrary, the host-snail competitors were highly sensitive to pesticide exposure. For the laboratory studies, macroinvertebrates including Schistosoma-host snails, competitors and predators were exposed to 6 concentrations levels of imidacloprid and diazinon. Snails showed higher insecticide tolerance compared to competitors and predators. Finally, Chapter 6 summarizes the conclusions of this dissertation, placing it in a broader
context. In this dissertation, a comprehensive chemical characterization and risk assessment of CECs has been carried out in freshwater systems; together with the effects of pesticides on schistosomiasis transmission in rural western Kenya. Results of this dissertation showed that rural areas are contaminated posing a risk to aquatic organisms which contribute to schistosomiasis transmission. This shows the need for regular monitoring and policy formulation to reduce pollutant emissions which contributes negatively to both ecological and human health effects.
The brain vascular system is composed of specialized endothelial cells, which regulate the movement of ions, molecules and cells from the blood lumen to the central nervous system (CNS). Endothelial cells in the brain form the blood-brain barrier (BBB) that is essential to maintain the brain homeostasis and protect the CNS from pathogens and toxins for a proper neurological function. Endothelium together with other cellular components such as pericytes, astrocytes and the basement membrane, forms the neurovascular unit (NVU), the structural unit of the BBB. Breakdown of the BBB occurs in various neurological disorders, leading to edema and neuronal damage. Therapeutic strategies focusing on factors that regulate the permeability of the BBB may help to improve neurological disorders and facilitate drug delivery to the brain.
Angiopoietins (Ang) are potential candidates for therapeutic targeting the BBB due to their role in regulating the vascular permeability in periphery. They are key growth factors that control angiogenesis and vessel maturation. Ang-1 and Ang-2 possess similar binding affinities to the Tie2 receptor tyrosine kinase, which is almost exclusively expressed on endothelial cells. Ang-1 is expressed in smooth muscle cells and pericytes, and binds in a paracrine manner to Tie2. This results in phosphorylation of the receptor and induction of downstream signaling pathways leading to vessel maturation via pericyte recruitment and blood vessel stabilization. Ang-2, on the other hand, is stored in Weibel Palade bodies in endothelial cells and is released upon inflammatory or angiogenic stimuli. Therefore, in mature, stabilized blood vessels, Ang-2 expression is low. Increased level of Ang-2 is only observed during development or in pathology such as ischemia, cancer and inflammation. When Ang-2 is released, it acts in an autocrine manner and interferes with Tie2 phosphorylation in a context-dependent way. Antagonizing the receptor results in de-stabilization of the vessels, often accompanied by reduced numbers of pericytes leading to myeloid cell infiltration. In conjunction with the vascular endothelial growth factor (VEGF), Ang-2 contributes to blood vessel sprouting, whereupon in absence of VEGF it promotes vessel regression. ...
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
Ischemic injuries of the cardiovascular system are still the leading cause of death worldwide. They are often accompanied by loss of cardiomyocytes (CM) and their replacement by non-functional heart tissue. Cardiac fibroblasts (CF) play a major role in the recovery after ischemic injury and in the scar formation. In the last few years researchers were able to reprogram fibroblasts into CM in vitro and in murine models of myocardial infarction using various protocols including a cocktail of microRNAs (miRs). These miRs can target hundreds of messenger RNAs and inhibit their translation into proteins, potentially regulating multiple cellular signaling pathways. Because of this, there has been a rising interest in the use of miRs for therapeutic purposes. However, as different miRs have different effects in different cells, there is the danger of causing serious side effects. These could be alleviated by enacting a cell-specific transport of miRs, for example by using aptamers. Aptamers are usually short strands of DNA or RNA, which can fold into a specific three-dimensional confirmation which allows them to bind specifically to target molecules. Aptamers are commonly selected from a large library for their ability to bind to target molecules using a procedure called SELEX. Aptamers have already been used to transport miRs into cancer cells.
In this thesis, we first established the transport of miRs into cells of the cardiovascular system using aptamers. MiR-126 is an important part of the signaling in endothelial cells (EC), protects from atherosclerosis and supports angiogenesis, which is why we chose it as a candidate to transport into the vasculature. We first tested two aptamers for their ability to internalize into EC and fibroblasts. Both the aptamer for the ubiquitously expressed transferrin receptor (TRA) and a general internalizing RNA motif, but not a control construct, could internalize efficiently into all cell types tested. We then designed three chimeras (Ch) using different strategies to connect TRA to miR-126. While all chimeras could internalize efficiently, only Ch3, which connects TRA to Pre-miR-126 using a sticky bridge structure, had functional effects in EC. Ch3 reduced the protein expression of VCAM-1 in EC and increased the VEGF induced sprouting of EC in a spheroid-sprouting assay. Treatment of breast cancer cells with Ch3 emulated the effects of treatment with classical miR-126-3p and miR-126-5p mimics. In the SK-BR3 cell line Ch3 and miR-126-3p reduce the viability of the cells while they reduce recruitment of EC by the MCF7 cell line. miR-126-5p had no apparent effect in the SK-BR3 line, but increased viability of MCF7 cells, as did Ch3. This implies that Ch3 can be processed to both functional miR-126-3p and miR-126-5p in treated cells.
We were unable to achieve a reprogramming of adult murine cardiac fibroblasts into cells resembling CM using the cocktail of 4 miRs. This indicates that the miR-mediated transdifferentiation is only possible in neonatal fibroblasts. The effects in mice after an AMI might possibly be caused by an enhanced plasticity of fibroblasts in and close to the infarcted area.
We also screened to find aptamers specifically binding to cells of the cardiovascular system. We used two oligonucleotide libraries in a cell-SELEX to select candidates which bind to CF, but not EC. We observed that only the library which contains two randomized regions of 26 bases showed an enrichment of species binding to fibroblasts. We then sequenced rounds 5-7 of the SELEX and analyzed the data bioinfomatically to select 10 candidate aptamers. All candidates showed a strong binding not only to CF, but also EC. This indicates that the selection pressure against species binding to EC was not high enough and would have to be increased to find true CF-aptamers. Four promising candidates were also analyzed for their potential to be internalized and we surprisingly found that all of them were internalized by EC and CF more efficiently than TRA. The similar behavior of the candidates implies that they possibly share a ligand, which is expressed both by EC and CF, but more prominently by the latter.
This work demonstrates the possibility of using aptamers to transport miRs into cells of the cardiovascular system. It also shows that it is possible to select aptamers for non-cancerous mammalian cells, which has not been done before. It is reasonable to assume that a refinement of the cell-SELEX will allow selection of cell-specific aptamers. Due to the failure of reprogramming of adult fibroblasts into induced cardiomyocytes we were unable to test whether a miR-mediated reprogramming might be inducible using aptamer transported-miRs. Ultimately, aptamer mediated transport of miRs is a feasible and promising therapeutic option for the treatment of cardiovascular diseases and other disorders like cancer.
Fossile Rohstoffe dienen in unserer heutigen Gesellschaft als Energiequelle und als Rohstofflieferant für Grund-, Feinchemikalien und Pharmazeutika. Sie tragen jedoch zum Klimawandel und Umweltverschmutzung bei. Lignocellulosische Biomasse ist eine erneuerbare und nachhaltige Alternative, die durch biotechnologische Prozesse erschlossen werden kann. Die Bäckerhefe Saccharomyces cerevisiae ist ein sehr gut untersuchter Modellorganismus, für den es zahlreiche genetische Werkzeuge und Analysemethoden gibt. Zudem wird S. cerevisiae häufig in biotechnologischen Prozessen eingesetzt, da diese Hefe robust gegenüber industriellen Bedingungen wie niedrigen pH-Werten, toxischen Chemikalien, osmotischem und mechanischem Stress ist. Die Pentose D-Xylose ist ein wesentlicher Bestandteil von lignocellulosischer Biomasse, die aber nicht natürlicherweise von der Bäckerhefe verwerten werden kann. Für eine kommerzielle Herstellung von Produkten aus lignocellulosischer Biomasse muss S. cerevisiae D-Xylose effektiv verwerten. Für die Bäckerhefe konnten heterologe Stoffwechselwege etabliert werden, damit diese D-Xylose verwerten kann. Für eine effiziente Xyloseverwertung bleiben dennoch zahlreiche Herausforderungen bestehen. Unter anderem nehmen die Zellen D-Xylose über ihre endogenen Hexosetransporter nur langsam auf. Die heterologe Xylose-Isomerase (XI) besitzt in S. cerevisiae eine geringe Aktivität für die Isomerisierung von D-Xylose. Unspezifische Aldosereduktasen konkurrieren mit der Xylose-Isomerase um das gleiche Substrat und produzieren Xylitol, ein starker Inhibitor der Xylose-Isomerase. Eine Möglichkeit die Umsatzrate von Enzymen zu steigern und Substrate vor Nebenreaktionen zu schützen, ist die Anwendung von Substrate Channeling Strategien. Bei Substrate Channeling befinden sich die beteiligten Enzyme in einem Komplex, wodurch die Substrate lokal angereichert werden und von einem aktiven Zentrum zum nächsten weitergeleitet werden, ohne Diffusion in den restlichen Reaktionsraum. In dieser Arbeit wurde untersucht, ob ein Komplex zwischen einem membranständigen Transporter und einem löslichen Enzym konstruiert werden kann, um durch Substrate Channeling eine verbesserte Substrat-Verwertung zu erreichen. Die Xylose-Isomerase aus C. phytofermentans und die endogene Hexose-Permease Gal2 sollten in dieser Arbeit als Modellproteine in S. cerevisiae-Zellen mit Hilfe von Protein-Protein-Interaktionsmodulen (PPIM) in räumliche Nähe zueinander gebracht werden.
Die Expression verschiedener PPIM konnte in S. cerevisiae mittels Western Blot nachgewiesen werden. Auch Fusionsproteine aus unterschiedlichen PPIM wurden in dieser Hefe exprimiert. Die PPIM binden komplementäre PPIM oder kurze Peptidliganden, welche an die Xylose-Isomerase und an den Gal2-Transporter fusioniert wurden. Die Funktionalität beider Proteine wurde mittels in vivo und in vitro Tests untersucht. Die Xylose-Isomerase mit N-terminalen Liganden des WH1-Protein-Protein-Interaktionsmoduls (WH1L-XI) und der Gal2-Transporter mit N-terminalen SYNZIP2-Protein-Protein-Interaktionsmodul (SZ2-Gal2) erwiesen sich als geeignete Kandidaten für weitere Untersuchungen. Mittels indirekter Immunfluoreszenz konnte die Ko-Lokalisierung von SZ2-Gal2 und WH1L-XI, die einander über ein Scaffold-Protein binden, nachgewiesen werden.
Transformanten, in denen ein Komplex aus Transporter, Scaffold-Protein und Xylose-Isomerase gebildet wurde, zeigten bessere Fermentationseigenschaften gegenüber der Scaffold-freien Kontrolle und dem Wildtyp: Sie verwerteten Xylose schneller, bildeten weniger vom unerwünschten Nebenprodukt Xylitol, produzierten mehr Ethanol und wiesen eine höhere Ethanolausbeute auf. Der beobachtete Substrate Channeling Effekt kompensierte die geringere Enzymaktivität der WH1L-XI im Vergleich zum Wildtyp-Protein. Die Wirksamkeit des Substrate Channeling wurde verringert, wenn die Bildung des Komplexes aus Transporter, Scaffold-Protein und Xylose-Isomerase gestört wurde, indem ein getaggtes GFP mit dem Scaffold-Protein um die Bindungsstelle an Gal2 konkurrierte. Dies zeigt, dass die positive Wirkung auf die Komplex-Bildung zwischen XI und Gal2 zurück zu führen ist. Die Fermentationseigenschaften konnten gesteigert werden, indem der zuvor zwischen SZ2-Zipper und Gal2-Transporter verwendete Linker, der aus zehn Aminosäuren von Glycin, Arginin und Prolin (GRP10) bestand, durch einen aus Glycin und Alanin (GA10) ersetzt wurde. Die verbesserten Fermentationseigenschaften beruhten auf einem Substrate Channeling Effekt und einer gesteigerten Aufnahmerate des SZ2-GA10-Gal2-Transporters. Ein Vergleich der Strukturvorhersagen von SZ2-GRP10-Gal2 und SZ2-GA10-Gal2 zeigte, dass der GRP10-Linker einen unstrukturierten, flexiblen Linker ausbildet, während der GA10-Linker eine starre α-Helix ausbildet. Die Struktur und der Transportprozess von Gal2 sind nicht aufgeklärt. Bei verwandten Transportern geht man davon aus, dass Substrate durch Konformationsänderungen ins Innere der Zelle transportiert werden, indem die beiden Domänen gegeneinander klappen. Die α-Helix könnte die Geschwindigkeit der Konformationsänderungen begünstigen.
Durch Kontrollexperimente konnte ausgeschlossen werden, dass die gesteigerten Fermentationseigenschaften eine Folge der Stabilisierung der XI- und Gal2-Fusionsproteine durch das Anfügen des Liganden oder durch Komplexbildung mit dem Scaffold-Protein waren. Substrate Channeling zwischen Gal2 und XI entsteht durch die Komplexbildung mit dem Scaffold-Protein, wodurch sich Gal2 und XI in räumlicher Nähe zueinander befinden. Dieser Effekt beruht möglicherweise zusätzlich aufgrund einer hohen örtlichen Ansammlung dieser Proteine, da die tetramere XI weitere Scaffold-Proteine binden könnte, welche weitere Gal2-Transporter binden könnte. Darüber hinaus sammeln sich Transporter an bestimmten Orten der Membran an und Transporter mit ähnlicher oder gleicher Transmembransequenz tendieren dazu zu ko-lokalisieren. Hierdurch könnten Gal2-XI-Agglomerate entstehen und Xylose wird mit hoher Wahrscheinlichkeit von einer der vielen Xylose-Isomerasen umgesetzt.
Die Analyse von DNA-Sequenzen steht spätestens seit der Feststellung ihrer tragenden Rolle in der Vererbung organismischer Eigenschaften im Fokus biologischer Fragestellungen. Seit Kurzem wird mit modernsten Methoden die Untersuchung von kompletten Genomen ermöglicht. Dies eröffnet den Zugang zu genomweiten Informationen gegenüber begrenzt aussagekräftigen markerbasierten Analysen. Eine Genomsequenz ist die ultimative Quelle an organismischer Information. Allerdings sind diese Informationen oft aufgrund technischer und biologischer Gründe komplex und werfen meist mehr Fragen auf, als sie beantworten.
Die Rekonstruktion einer bislang unbekannten Genomsequenz aus kurzen Sequenzen stellt eine technische Herausforderung dar, die mit grundlegenden, aber in der Realität nicht zwingend zutreffenden Annahmen verbunden ist. Außerdem können biologische Faktoren, wie Repeatgehalt oder Heterozygotie, die Fehlerrate einer Assemblierung stark beeinflussen. Die Beurteilung der Qualität einer de novo Assemblierung ist herausfordernd, aber zugleich äußerst notwendig. Anschließend ist eine strukturelle und funktionale Annotation von Genen, kodierenden Bereichen und repeats nötig, um umfangreiche biologische Fragestellungen beantworten zu können. Ein qualitativ hochwertiges und annotiertes assembly ermöglicht genomweite Analysen von Individuen und Populationen. Diese Arbeit beinhaltet die Assemblierung und Annotation des Genoms der Süßwasserschnecke Radix auricularia und eine Studie vergleichender Genomik von fünf Individuen aus verschiedenen molekularen Gruppen (MOTUs).
Mollusken beherbergen nach den Insekten die größte Artenvielfalt innerhalb der Tierstämme und besiedeln verschiedenste, teils extreme, Habitate. Trotz der großen Bedeutung für die Biodiversitätsforschung sind verhältnismäßig wenige genomische Daten öffentlich verfügbar. Zudem sind Arten der Gattung Radix auch aufgrund ihrer großen geografischen Verbreitung in diversen biologischen Disziplinen als Modellorganismen etabliert. Eine annotierte Genomsequenz ermöglicht über bereits untersuchte Felder hinaus die Forschung an grundlegenden biologischen Fragestellungen, wie z.B. die Funktionsweise von Hybridisierung und Artbildung. Durch Assemblierung und scaffolding von sechs whole genome shotgun Bibliotheken verschiedener insert sizes und einem transkriptbasiertem scaffolding konnte trotz des hohen Repeatgehalts ein vergleichsweise kontinuierliches assembly erhalten werden. Die erhebliche Differenz zwischen der Gesamtlänge der Assemblierung und der geschätzten Genomgröße konnte zum Großteil auf kollabierte repeats zurückgeführt werden.
Die strukturelle Annotation basierend auf Transkriptomen, Proteinen einer Datenbank und artspezifisch trainierten Genvorhersagemodellen resultierte in 17.338 proteinkodierenden Genen, die etwa 12,5% der geschätzten Genomgröße abdecken. Der Annotation wird u.a. aufgrund beinhaltender Kernrthologen, konservierter Proteindomänenarrangements und der Übereinstimmung mit de novo sequenzierten Peptiden eine hohe Qualität zugesprochen.
Das mapping der Sequenzen von fünf Radix MOTUs gegen die R. auricularia Assemblierung zeigte stark verringerte coverage außerhalb kodierender Bereiche der nicht-Referenz MOTUs aufgrund hoher Nukleotiddiversität. Für 16.039 Gene konnten Topologien berechnet werden und ein Test auf positive Selektion ausgeführt werden. Insgesamt konnte über alle MOTUs hinweg in 678 verschiedenen Genen positive Selektion detektiert werden, wobei jede MOTU ein nahezu einzigartiges Set positiv selektierter Gene beinhaltet. Von allen 16.039 untersuchten Genen konnten 56,4% funktional annotiert werden. Diese niedrige Rate wird vermutlich durch Mangel an genomischer Information in Mollusken verursacht. Anschließende Analysen auf Anreicherungen von Funktionen sind deshalb nur bedingt repräsentativ.
Neben den biologischen Ergebnissen wurden Methoden und Optimierungen genomischer Analysen von Nichtmodellorganismen entwickelt. Dazu zählen eigens angefertigte Skripte, um beispielsweise Transkriptomalignments zu filtern, Trainings eines Genvorhersagemodells automatisiert und parallelisiert auszuführen und Orthogruppen bestimmter Arten aus einer Orthologievorhersage zu extrahieren. Zusätzlich wurden Abläufe entwickelt, um möglichst viele vorhandene Daten in die Assemblierung und Annotation zu integrieren. Etwa wurde ein zusätzliches scaffolding mit eigens assemblierten Transkripten mehrerer MOTUs sequenziell und phylogenetisch begründet ausgeführt.
Insgesamt wird eine umfassende und qualitativ hochwertige Genomsequenz eines Süßwassermollusken präsentiert, welche eine Grundlage für zukünftige Forschungsprojekte z.B. im Bereich der Biodiversität, Populationsgenomik und molekularen Ökologie bietet. Die Ergebnisse dieser Arbeit stellen einen Wissenszuwachs in der Genomik von Mollusken dar, welche bisher trotz ihrer Artenvielfalt deutlich unterrepräsentiert bezüglich assemblierter und annotierter Genome auffallen.
The environmental impact of climate change is meanwhile not only discussed in the scientific community but also in the general public. However, little is known about the interaction between climate change and pollutants like pesticides. A combination of multiple stressors (e.g. temperature, pollutants, predators) may lead to severe alterations for organisms such as changes in time of reproduction, reproductive success and growth performance, mortality and geographic distribution. The questions if aquatic organisms tend to react more sensitive towards incidents under climate change conditions remains. Therefore, within the present thesis the aquatic ecotoxicological profile of the fungicide pyrimethanil, as an exemplarily anthropogenic used contaminant, was examined.
A large test battery of ecotoxicological standard tests and supplement bioassays with non-model species was conducted to investigate if species-specific or life stage-specific differences occur or if temperature alteration may change the impact of the fungicide. Two of the most sensitive species (Chironomus riparius and Daphnia magna) were used to investigate the acute and chronic thermal dependence of pyrimethanil effects. The results clearly depict that the ecotoxicity of pyrimethanil at optimal thermal conditions did not depend on the trophic level, but was species-specific. With regard to EC10 values the acute pyrimethanil toxicity on C. riparius increased with higher temperature (6.78 mg L-1 at 14°C and 3.06 mg L-1 at 26°C). The chronic response of D. magna to the NOEC (no observed effect concentration) of the fungicide (0.5 mg L-1) was examined in an experiment which lasted for several generations under three simulated near-natural temperature regimes (‘cold year, today’ (11 to 22.7°C), ‘warm year, today’ (14 to 25.2°C) and ‘warm year, 2080’ (16.5 to 28.1°C)). A pyrimethanil-induced mortality increase was buffered by the strongly related increase of the general reproductive capacity, while population growth was stronger influenced by temperature than by the fungicide. At a further pyrimethanil concentration (LOEC – lowest observed effect concentration: 1 mg L-1), a second generation could not be established by D. magna under all thermal regimes.
Besides daphnids, the midge C. riparius was used for a second multigeneration study. In a bifactorial test design it was tested if climate change conditions alter or affect the impact of a low fungicide concentration on life history and genetic diversity. The NOAEC/2 (half of the no observed adverse effect concentration derived from a standard toxicity test) was used as a low pyrimethanil concentration to which laboratory populations of the midges were chronically exposed under the mentioned temperature scenarios. During the 140-day-multigeneration study, survival, emergence, reproduction, population growth, and genetic diversity of C. riparius were analyzed. The results reveal that high temperatures and pyrimethanil act synergistically on life history parameters of C. riparius. In simulated present-day scenarios, a NOAEC/2 of pyrimethanil provoked only slight to moderate beneficial or adverse effects. In contrast, an exposure to a NOAEC/2 concentration of pyrimethanil at a thermal situation likely for a summer under the future expactations uncovered adverse effects on mortality and population growth rate. In addition, genetic diversity was considerably reduced by pyrimethanil in the ‘warm year, 2080’ scenario, but only slightly under current climatic conditions. The multigeneration studies under near-natural thermal conditions indicate that not only the impact of climate change, but also low concentrations of pesticides may pose a reasonable risk for aquatic invertebrates in the future. This clearly shows that thermal and multigenerational effects should be considered when appraising the ecotoxicity of pesticides and assessing their future risk for the environment.
In addition to temperature further multiple abiotic and biotic stressors alterate pollutant effects. Moreover, to better discriminate and understand the intrinsic and environmental correlates of changing aquatic ecosystems, it was experimentally unraveled how the effects of a low-dose of pyrimethanil on daphnids becomes modified by different temperatures (15°C, 20°C, 25°C) and in the presence/ absence of predator kairomones of Chaoborus flavicans larvae. The usage of a fractional multifactorial test design provided the possibility to investigate the individual growth, reproduction and population growth rate of Daphnia pulex via different exposure routes to the fungicide pyrimethanil at an environmentally relevant concentration (0.05 mg L-1) - either directly (via the water phase), indirectly (via algae food), dually (via water and food) or for multiple generations (fungicide treated source population).
The number of neonates increased with increasing temperatures. At a temperature of 25°C no significant differences between the individual treatment groups were observed although the growth was overall inhibited due to pyrimethanil. Besides, at 15 and 20°C it is obvious that daphnids which were fed with contaminated algae had the lowest reproduction and growth rate. The obtained results clearly demonstrate that multiple stress factors can modify the response of daphnids to pollutants. The exposure routes of the contaminant are of minor importance, while temperature and the presence of a predator are the dominant factors impacting the reproduction of D. pulex. It can be concluded that low concentrations of pyrimethanil may disturb the zooplankton community at suboptimal temperature conditions, but the effects will become masked if chaoborid larvae are present. Therefore it seems necessary to observe prospectively if the combination of several stress factors like pesticide exposure and suboptimal temperature may influence the life history and sensitivity of several aquatic invertebrates differently.
Besides standard test organisms it is inevitable to conduct test with aquatic invertebrate which are not yet considered regularly in ecotoxicological experiments. For example molluscs represent one of the largest phyla of macroinvertebrates with more than 100.000 species, being ecologically and economically important. Therefore, within the present study embryo, juvenile, half- and full-life cycle toxicity tests with the snail Physella acuta were performed to investigate the impact of pollutants on various life stages. Different concentrations of pyrimethanil (0.06-0.5 or 1.0 mg L-1) assessed at three temperatures (15°C, 20°C, 25°C) revealed that pyrimethanil caused concentration-dependent effects independent of temperature. Interestingly, the ecotoxicity of pyrimethanil was higher at lower temperature for the embryo hatching and F1 reproduction, but its ecotoxicity for the growth of juveniles and the F0 reproduction increased with increasing temperature. More specifically, it could have been observed that especially during the reproduction test high mortality rates occurred at the highest concentration of 1 mg L-1 at all temperatures. Due to high mortality rates no snails were available for the F1 at the highest concentrations (0.5 and 1.0 mg L-1). Compared to the F0, overall more egg masses were produced in the F1, being all fertile and no mortality occurred. For the F1-generation the strongest pyrimethanil effects were detected at 15°C. A comparison of effect concentrations between both generations showed that the F1 is more sensitive than the F0.
These results indicate that an exposure over more than one generation may give a better overview of the impact of xenobiotics. With the establishment of an embryo and reproduction test under different temperatures and various concentrations of pyrimethanil with P. acuta we could successfully show that molluscs can respond more sensitive than model organisms and that both, chemical and thermal stressor strongly influence the behaviour of the pulmonates. It can be concluded that the high susceptibility for the fungicide observed in gastropods clearly demonstrates the complexity of pesticide-temperature interactions and the challenge to draw conclusions for the ecotoxicological risk assessment of pesticides under the impact of global climate change.
Diatoms contribute largely to the total primary production of the ecosphere and are key players in global biogeochemical cycles. Their chloroplasts are surrounded by four membranes owing to their secondary endosymbiotic origin. Their thylakoids are arranged into three parallel bands and differentiation of thylakoid membranes into grana or stroma is not observed. The fucoxanthin chlorophyll a/c binding proteins act as the light harvesting proteins and play a role in photoprotection during excess light as well. The diatom genome encodes three different families of antenna proteins. Family I are the classical light harvesting proteins called "Lhcf". Family II are the red algae related Lhca-R1/2 proteins called "Lhcr" and family III are the photoprotective LI818 related proteins called "Lhcx".
All known Fcps have a molecular weight in the range of 17-23 kDa. They are membrane proteins and have shorter loops and termini compared to LHCs of higher plants and are therefore extremely hydrophobic. This makes the isolation of single specific Fcps using routine protein purification techniques difficult.
The purification of a specific Fcp containing complex has not been achieved so far and until this is done several questions concerning light harvesting antenna systems of diatoms cannot be answered. For e.g. Which proteins interact specifically? Are various Fcps differently pigmented? Which pigments interact with each other and how? Which proteins contribute to photosystem specific antenna systems? Can pure Fcps be reconstituted into crystals like LHCII proteins? In order to answer these questions specific Fcp containing complexes have to be purified. ...
This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
Reading is an essential ability to master everyday life in our society. The ability to read is based on specific connections between brain regions involved in the reading process – so-called cortical networks for reading. These cortical networks for reading allow us to learn the correct identification of visual words. The use of visual words is based on knowledge about the orthography (lexical) and the meaning of words (semantic). This knowledge must be acquired by beginning readers (first grader), i.e. beginning readers learn in a first step to link letters to a whole word and in a second step associate this whole word with meaning. To retrieve this knowledge during visual word recognition (VWR) a cortical network for lexical-semantic process must be activated. However, it is currently unclear whether beginning readers and reading experts activate the same neuronal network during VWR. Therefore, the aim of this thesis was to investigate the question whether beginning readers (first grader, children) and reading experts (adults) use different cortical networks for the lexical-semantic processing in VWR.
To address this question we recorded electroencephalographic (EEG) activity during VWR in children and adults. Children and adults were instructed to read a visualizable word to compare this word with a following picture stimulus. The first part of this thesis is concerned with the analysis of ERPs for visual word recognition in children and adults at sensor level. For both groups we observed the typical ERP components P100 and N170 for visual word recognition. These components differed in amplitude and time course between both groups. The second part of this thesis investigated the neuronal generators (brain areas) of ERPs during VWR and possible differences between children and adults at source level. We observed a high overlap in brain areas involved during VWR in children and adults. However, the brain areas differed in activation and time course between children and adults. Finally, the third and most important part of the thesis investigated the question whether children and adults use different cortical networks for the lexical-semantic processing in VWR over time. To address this question Dynamic Causal Modeling (DCM) and Bayesian model comparison were used. We compared nine biologically plausible cortical network models underlying the ventral lexical-semantic path in VWR. In addition, increasing time intervals were used to consider possible changes of network structure during VWR. The network models included eight brain regions (four bilateral pairs) involved in the lexical-semantic processing in VWR: occipital cortex (OC), temporo-occipital part of inferior temporal gyrus (ITG), temporal pole (TP), and inferior frontal gyrus (IFG). In almost all time intervals we found evidence that children and adults use the same cortical networks for the lexical-semantic processing in VWR. However, we found differences between adults and children in the connection strengths of the favoured model. Interestingly, we found a stronger direct connection from OC to IFG in adults compared to children.
In conclusion, our results suggest that children and adults activate largely the same lexical-semantic networks during VWR over time. This supports the notion that children and adults use the same biological fiber connections for VWR. However in contrast to children, adults showed increased use of the shortcut pathway from OC to IFG. The increased use of the shortcut pathway from OC to IFG in adults can be interpreted as consequence of learning. Learning causes in accordance with the Hebbian learning rule (“neurons that fire together, wire together” (Hebb, 1949)) synaptic change. Consequently the frequent coactivation of the input and output stage of OC and IFG during the lexical-semantic process facilitates the stronger direct connection between both brain areas. The stronger direct connection from OC to IFG most likely allows adult reading experts to speed up the lexical-semantic process during VWR. Accordingly, we conclude that the stronger direct connections from OC to IFG in adults compared to children underlay the different reading capabilities in both groups.
Cardiac trabeculation is one of the essential processes required for the formation of a competent ventricular wall, whereby clusters of ventricular cardiomyocytes (CMs) from a single layer delaminate and expand into the cardiac jelly to form sheet-like projections in the developing heart (Samsa et al., 2013). Several congenital heart diseases are associated with defects in the formation of these trabeculae and lead to embryonic lethality (Jenni et al., 1999; Zhang et al., 2013, Jenni et al., 2001; Towbin 2010). It has been experimentally shown that lack of Nrg1/ErbB2/ErbB4, Angipoetin1/Tie2, EphrinB2/B4, BMP10, or any component of the Notch signaling pathway can cause defective trabeculation. Moreover, changes in blood flow and/or contractility can also affect trabeculation (Samsa et al., 2013). Together, these observations demonstrate that cardiac trabeculation is a highly dynamic and regulated process.
Trabeculation is a morphogenetic process that requires control over cell shape changes and rearrangements, similar to those observed during EMT. Epithelial cells within an epithelium are polarized and establish cell-cell junctions with the neighboring cells (Ikenouchi et al., 2003; Ferrer-vaquer et al., 2010), thus epithelial cell polarity is an important feature to maintain cell shape and tissue structure. During developmental processes such as cell migration and cell division or in disease states epithelial polarity might be disrupted. As a consequence of this alteration, cells lose their tight cell-cell adhesions, undergo cytoskeletal rearrangements, change their shape and gain migratory properties becoming mesenchymal cells (Micalizzi et al., 2010). In epithelial cells, apicobasal polarity is regulated by a conserved set of core complexes, including the PAR, Scribble and Crumbs complexes (Kemphues et al., 1988; Bilder and Perrimon, 2000; Teppas et al., 1984). The polarity proteins composing these complexes interact in a well organized and coordinated-manner creating molecular asymmetry along the apicobasal axis of the cell. In turn, this crosstalk regulates the maturation and stabilization of the junctions between cells and cytoskeleton in order to strengthen cell polarization (Roignot et al., 2013). Amongst the different polarity complex, Crumbs has been shown to be a key regulator of apicobasal polarity during development in both vertebrates and invertebrates (Tepass et al., 1990; Fan et al., 2004).
Here, taking advantage of zebrafish as a model organism, I study in vivo at single cell resolution changes in CM apicobasal polarity during cardiac trabeculation. Moreover, I show which factors regulate CM apicobasal polarity during this process. In addition, I dissect the role of the polarity complex Crumbs in regulating CM junctional rearrangements and the formation of the trabecular network.
The canonical Wnt/β-catenin and the Shh pathway as well as the Notch signaling cascade
are key regulators in stem cell biology and are independently associated with the development
of cancer. Despite the knowledge of a balanced signaling for cellular maintenance, the
fundamental biochemical mechanisms of crosstalk are still poorly understood. This study
demonstrates that the outcome of interaction between Wnt and Shh is cell type specific. A
combined inhibitory mechanism of the Shh and Notch2/Jagged2 pathways on dominant
active β-catenin signaling in the adult tongue epithelium keeps Wnt/β-catenin signaling
restricted to physiological tolerable levels. In the opposite crosstalk the activation of
Wnt/β-catenin signaling in medulloblastoma (MB) of the Shh subtype, in turn inhibits the Hh
pathway.
The inhibitory mechanism of Shh and Notch2/Jagged2 on Wnt/β-catenin signaling is
independent of the degradation complex of β-catenin and takes place inside the nucleus.
Furthermore, the negative feedback on Wnt/β-catenin signaling by the Shh pathway relies
on transcriptional activity of Gli1/2A. Inhibition of Gli1/2A with the specific inhibitor GANT61
abrogated the negative impact of Shh on β-catenin signaling in vitro. Although the negative
feedback loop of Shh is still functional in human SCC25 cells, the inhibitory effect of
Notch2/Jagged2 is lost and contributes to the cancerogenic phenotype of these cells. In the
inverse situation, the activation of β−catenin signaling has a negative feedback on
constantly active Shh signaling and significantly inhibits the Hh pathway. This was shown in
Ptch+/- and Math1-Cre:SmoM2Fl/+ MB tumor spheres in vitro, in which inhibition of sphere
formation and growth was observed and Hh target gene transcription was down-regulated.
This demonstrates for the first time that the activation of canonical Wnt/β-catenin signaling
in primary MB cells with a Hh pathway over-activation has a negative effect on the growth of
these cells in vitro.
In summary the results show that crosstalk of Wnt/β-catenin and Shh signaling has context
specific outcome on pathway activity. Elucidation of the molecular interactions will improve
our understanding of Wnt and Hh associated tumors and contribute to the development of
new therapeutic strategies.
Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). TAA-producing bacteria are the causative agent of many human diseases and TAA targeted anti-adhesive compounds might counteract such bacterial infections. The modularly structured Bartonella adhesin A (BadA) is one of the best characterised TAAs and serves as an attractive adhesin to study the domain-function relationship of TAAs during infection. BadA is a major virulence factor of B. henselae and is essential for the initial attachment to host cells via adhesion to extracellular matrix proteins. B. henselae is the causative agent of cat scratch disease and adheres to fibronectin using its long BadA fibres. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations.
Human, feline, and laboratory adapted B. henselae isolates display genomic and phenotypic differences. By analysing the genomes of eight B. henselae strains using long-read sequencing, a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes was identified. Moreover, numerous conserved flanking genes were characterised, however, their influence on the regulation of badA expression and modification remains to be explored. It seems that B. henselae G 5436 is the evolutionary ancestor of the other B. henselae strains analysed in this work. The diversity of the badA island among the B. henselae strains indicates that the downstream badA-like domain region might be used as a ‘toolbox’ for rearrangements in the badA gene. Overall, it is suggested that badA-domain duplications, insertions, and/or deletions are the result of active phase variation via site-specific recombination and contribute to rapid host adaptation in the scope of pathogenicity, immune evasion, and/or enhanced long-term colonisation.
The model strain B. henselae Marseille expresses a badA gene that includes 30 repetitive neck/stalk domains, each consisting of several predicted structural motifs. To further elucidate the motif sequences that mediate fibronectin binding, various modified badA constructs were generated. Their ability to bind fibronectin was assessed via whole-cell ELISA and fluorescence microscopy. In conclusion, it is suggested that BadA adheres to fibronectin in a cumulative fashion with quick saturation via unpaired β-strands appearing in structural motifs present in BadA neck/stalk domains 19, 27, and other homologous domains. Furthermore, antibodies targeting a 15-mer amino acid sequence in the DALL motif of BadA neck/stalk domain 27 were able to reduce fibronectin binding of the B. henselae mutant strain S27. Moreover, this DALL motif sequence is conserved in the genome of all analysed B. henselae strains. The identification of common binding motifs between BadA and fibronectin supports the development of new anti-adhesive compounds that might inhibit the initial adherence of B. henselae and other TAA-producing pathogens during infection.
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
Die Spinozerebelläre Ataxie Typ 2 (SCA2) ist eine autosomal dominant vererbte neurodegenerative Krankheit, welche durch die Expansion des Trinukleotids Cytosin-Adenin-Guanin von ~22/23 auf >32 im Ataxin-2 Gen (ATXN2) verursacht wird. Dieses Trinukleotid codiert für die Aminosäure Glutamin weshalb SCA2 auch zu den Polyglutaminerkrankungen zählt. Zu dieser Gruppe zählen außerdem fünf weitere SCA-Subtypen sowie drei weitere neurodegenerative Erkrankungen, darunter die Huntington-Krankheit.
SCA2 wurde 1971 zum ersten Mal von Wadia und Swami beschrieben und unterscheidet sich von den anderen SCAs aufgrund der typischen Störung der sakkadischen Augenbewegungen. Weitere klinische Symptome von SCA2 sind Ataxie, Tremor, Dysmetrie, Dysarthrie, Hyporeflexie und Dysdiadochokinese. Die Symptome gehen auf einen neuronalen Verlust insbesondere im Cerebellum, aber auch in anderen Hirnregionen wie zum Beispiel dem Hirnstamm zurück.
Atxn2 wird in weiten Teilen des Zentralnervensystems aber auch in vielen nicht-neuronalen Geweben exprimiert. Es handelt sich um ein überwiegend cytoplasmatisch lokalisiertes Protein, welches im Gegensatz zu vielen anderen SCA-Proteinen cytoplasmatische und nur selten nukleäre Aggregate bildet. Die exakte Funktion von Atxn2 ist bisher unklar, es wurde allerdings mehrfach gezeigt, dass es in die mRNA Translation involviert ist aufgrund seiner Interaktion mit dem PolyA-bindenden Protein PABPC1.
Eine Expansion des Trinukleotids in Ataxin-2 kann nicht nur zu SCA2 führen, sondern stellt bei Wiederholungen zwischen 27 und 32 CAGs auch ein erhöhtes Risiko für eine Erkrankung an Amyotropher Lateralsklerose (ALS) und anderen neurodegenerativen Krankheiten dar. Eine Interaktion zwischen ATXN2 und dem ALS-verursachenden TDP43 (Tardbp) wurde bereits zahlreich beforscht, da Aggregate von ATXN2 in Motoneuronen des Rückenmarks von ALS-Patienten und aggregiertes TDP43 in SCA2-Neuronen beobachtet wurden.
Generell sind die Mechanismen, die zur Pathologie von SCA2 und ALS führen, noch weitgehend unklar. Ziel dieser Arbeit war es daher auf der einen Seite einen Einblick in den Pathomechanismus von SCA2 zu erhalten, indem mögliche oder bereits bekannte Interaktoren in etablierten Atxn2-Mausmodellen untersucht wurden. Auf der anderen Seite wurden zwei neue Mausmodelle charakterisiert, um ihre Eignung für die Erforschung von ALS und SCA2 zu prüfen.
Für den ersten Teil der Arbeit dienten Daten aus mehreren Transkriptomstudien von Atxn2-Knock-Out (KO) und Atxn2-CAG42-Knock-In (KIN) Mäusen als Grundlage. Konnten die Daten mit einer unabhängigen Methode bestätigt werden, folgten weitere Untersuchungen auf mRNA und Proteinebene sowie unter zusätzlicher Verwendung von Zellkultur und Patientenmaterial. Dadurch konnten neue Interaktionspartner von ATXN2 identifiziert und bereits bekannte in diesen Mausmodellen bestätigt werden.
So wurde zum Beispiel eine Interaktion von ATXN2 mit der E3-Ubiquitin-Protein-Ligasekomponente FBXW8 gezeigt und deren Beteiligung am Abbau von expandiertem ATXN2. Außerdem wurde eine Interaktion von FBXW8 mit dem bereits bekannten ATXN2-degradierenden Protein PARK2 gezeigt. Eine Hochregulierung des Fbxw8 Transkripts wurde sowohl im Atxn2-CAG42-KIN-Mausmodell als auch in SCA2-Patientenfibroblasten gefunden, während Park2 in keinem der Modelle signifikant veränderte Transkriptspiegel aufwies. Diese Daten belegen die Relevanz von Fbxw8 für den Abbau von moderat-expandiertem Atxn2 und begründen weitere Studien zur genauen Funktion dieses Proteins im Pathomechanismus von Atxn2.
Des Weiteren wurden diverse Kalziumhomöostasefaktoren untersucht, welche eine konsistente Herunterregulierung der Transkripte in beiden Mausmodellen aufwiesen. Auf Proteinebene zeigten sich jedoch Unterschiede zwischen den Modellen. Diese Daten belegen, dass zwar ähnliche Transkriptveränderungen im KIN- und KO-Modell auftreten, diesen aber vermutlich verschiedene Mechanismen zugrunde liegen. Welche Mechanismen dies genau sind bleibt zu klären, es ist jedoch wahrscheinlich, dass im KIN-Modell die Aggregatbildung sowie in beiden Modellen die Beteiligung von ATXN2 an der Translationregulation eine Rolle spielen. Die Ergebnisse dieser Studie unterstreichen die Relevanz des Ca2+ Signalwegs für die Entwicklung von SCA2.
Der zweite Teil der Arbeit beinhaltet die Charakterisierung einer ATXN2/TDP43 Doppelmutante auf Verhaltensebene sowie die gründliche Evaluierung des Phänotyps einer vollkommen neuen SCA2 Mausmutante. Während in der Doppelmutante trotz doppelter Genmutation nur ein sehr schwacher Phänotyp auf Verhaltensebene festgestellt werden konnte und bis zu einem Alter von 12 Monaten keine Potenzierung der Mutationen zu beobachten war, zeigte die Atxn2-CAG100-KIN Maus signifikante und früh auftretende Pathologie. Neben einer verminderten Überlebensrate, einem Gewichtsverlust und diversen motorischen Störungen, konnten auch Aggregate des mutierten Proteins in diversen Hirnregionen identifiziert werden. Der Atxn2-CAG100-KIN Phänotyp spiegelt die humanen Symptome daher recht gut wider, weshalb diese Mausmutante ein wertvolles Modell für die weitere SCA2-Forschung darstellt.
Zusammengefasst zeigt diese Arbeit die Bedeutung des ATXN2-Interaktors FBXW8 im SCA2-Mausmodell als auch im Patientenmaterial. Sie betont die Relevanz des Atxn2-KO-Modells in Bezug auf Störungen der Kalziumhomöostase und dokumentiert die Alters- und Gewebespezifität dieser Veränderungen. Außerdem beinhaltet sie die vorläufige Beschreibung eines kombinierten Atxn2/TDP43-Mausmodells und schließlich die ausführliche Charakterisierung eines vollkommen neuen und äußerst wertvollen SCA2-Mausmodells.
This work deals with the characterization of three different type II polyketide synthase systems (PKS II) from the Gram-negative bacteria Xenorhabdus and Photorhabdus.
Particular attention was paid to a biochemically underexplored class of aryl polyene (APE) pigments. Bioinformatic analysis of enzymes involved in the biosynthesis and the in vitro reconstruction proved that the synthesis of APEs involves an unusual fatty acid-like elongation mechanism. Furthermore, the discovery of unexpected protein-protein interactions provided new insights into the multienzyme complex formation of this unusual PKS II system. Through collaboration with the groups from Prof. Michael Groll and junior Prof. Nina Morgner, two protein complexes were structurally solved and several native protein multimerization events were identified and allowed us to suggest a possible protein-interaction network. The results are summarized in publication ‘An Uncommon Type II PKS Catalyzes Biosynthesis of Aryl Polyene Pigments’ (first author; J. Am. Chem. Soc.).
In addition to in vitro-analysis, in vivo-studies were used to investigate the APE compound produced by X. doucetiae in more detail. The activation of the silent biosynthetic gene cluster (BGC) led to the detection of the APE compound in the homologous host. Further combination of homologous expression and targeted deletions of the APE BGC revealed an APE-lipid-like structure. MS-based analyses and purification of intermediates allowed us to deduce structural building blocks of the APE-lipid, which is composed of an APE structural core, a glucosamine residue and an unusual long-chain fatty acid with unusual conjugated double bonds and a phosphoethanolamine head group. In combination with the above stated in vitro-data, we assumed a plausible biosynthetic mechanism of the APE-lipid. The results are summarized in the section ‘Additional Results: Tracing the Full-length APE’.
The biosynthesis of isopropylstilbene (IPS) has already been well-studied by the Bode laboratory and the group of Prof. Ikuro Abe. Studies with Photorhabdus laumondii TT01 by the Bode group revealed the distributed locations and functions of the genes involved in biosynthesis, which originate from two pathways. Particularly, the Bode group first demonstrated that an unusual ketosynthase/cyclase (StlD) catalyzes the condensation of 5-phenyl-2,4-pentadienoyl-ACP and isovaleryl-beta-ketoacyl-ACP via a Michael addition. Such a pathway for stilbene formation is distinct from those widespread in plants. The Abe group solved the structure and biochemical mechanism of StlD and further investigated the aromatization reaction of the aromatase StlC. However, the generation of the required cinnamoyl-precursor 5-phenyl-2,4-pentadienoyl-ACP as a Michael acceptor for this cyclization reaction remained elusive. In this work, we were able to reconstitute the synthesis of the Michael acceptor in vitro, by the action of enzymes from the fatty acid biosynthesis. With the knowledge about the crucial cross-talk from primary and specialized metabolism, we further determined the minimal endowment for stilbene production in a heterologous host. Here, the discovered AasS enzyme StlB is responsible for the generation of cinnamoyl-ACP and among others, plFabH plays a key role as gatekeeper enzyme for further processing. With this information in hand, we were able to obtain IPS production in E. coli. These results are presented in the manuscript ‘Biosynthesis of the Multifunctional Isopropylstilbene in Photorhabdus laumondii Involves Cross-talk Between Specialized and Primary Metabolism’ (co-first author, manuscript).
The biosynthesis of the orange-to-red-pigmented anthraquinones (AQs) is the best-studied type II PKS system according to preliminary results. While several investigations by Brachmann et al. discovered the BGC and the overall product spectrum of the main AQ-256 and its methylated derivatives, data of Quiqin Zhou (Bode group) performed biochemical in vitro analysis paired with in vivo heterologous expression of the ant-genes antA-I. This led to the identification of shunt products that indicated an AQ-scaffold derived from an octaketide intermediate that gets shortened to a heptaketide by the hydrolase AntI, resulting in the main anthraquinone AQ-256. This PKS-shortening mechanism was further confirmed by the protein crystal structure of AntI by the Groll group (publication, minor contributions, co-author, Chem Sci. ‘Molecular Mechanism of Polyketide Shortening in Anthraquinone Biosynthesis of Photorhabdus luminescens’). Further substrate analysis of the P. luminescens AQ-producer and mutants revealed an inhibitory effect of cinnamic acid against the hydrolase AntI. Cinnamic acid might therefore be involved in regulation of AQ biosynthesis (‘Anthraquinone Production is Influenced by Cinnamic Acid’, first author, manuscript).
Biochemical analysis from Quiqin Zhou with the minimal PKS of the AQ-synthase further revealed the exclusive activation of the AQ-ACP by the PPTase AntB. The PPTase is insoluble alone but gets stabilized by the CoA-ligase, most likely inactive, working as a chaperone. Thus, the minimal PKS endowment to produce the octaketide scaffold compromises, besides the ACP, the KS:CLF heterodimer and the MCAT, the co-occurrence of the PPTase AntB and the CoA-ligase AntG. For the first time, X-ray crystallography depicted a minimal PKS in action, by obtaining the structural data of native complexes from an ACP:KS:CLF, the KS:CLF alone and an ACP:MCAT in their non-active and active forms. It was possible to confirm a KS-bound hexaketide, which was built upon heterologous expression of the KS:CLF. Mutagenesis with amino-acids proposed to be involved in protein-protein interactions in the ACP:KS:CLF complex revealed some interesting protein-interaction sites. Additionally, an induced-fit mechanism of the MCAT with the ACP during the malonylation reaction confirmed a monodirectional transfer reaction (‘Structural Snapshots of the Minimal PKS System Responsible for Octaketide Biosynthesis’ co-author, manuscript under review).
Heat stress transcription factors (Hsfs) have an essential role in heat stress response (HSR) and thermotolerance by controlling the expression of hundreds of genes including heat shock proteins (Hsps) with molecular chaperone functions. Hsf family in plants shows a striking multiplicity, with more than 20 members in many species. In Solanum lycopersicum HsfA1a was reported to act as the master regulator of the onset of HSR and therefore is essential for basal thermotolerance. Evidence for this was provided by the analysis of HsfA1a co-suppression (A1CS) transgenic plants, which exhibited hypersensitivity upon exposure to heat stress (HS) due to the inability of the plants to induce the expression of many HS-genes including HsfA2, HsfB1 and several Hsps. Completion of tomato genome sequencing allowed the completion of the Hsf inventory, which is consisted of 27 members, including another three HsfA1 genes, namely HsfA1b, HsfA1c and HsfA1e.
Consequently, the suppression effect of the short interference RNA in A1CS lin e was re-evaluated for all HsfA1 genes. We found that expression of all HsfA1 proteins was suppressed in A1CS protoplasts. This result suggested that the model of single master regulator needs to be re-examined.
Expression analysis revealed that HsfA1a is constitutively expressed in different tissues and in response to HS, while HsfA1c and HsfA1e are minimally expressed in general, and show an induction during fruit ripening and a weak upregulation in late HSR. Instead HsfA1b shows preferential expression in specific tissues and is strongly and rapidly induced in response to HS. At the protein level HsfA1b and HsfA1e are rapidly degraded while HsfA1a and HsfA1c show a higher stability. In addition, HsfA1a and HsfA1c show a nucleocytosolic distribution, while HsfA1b and HsfA1e a strong nuclear retention.
A major property of a master regulator in HSR is thought to be its ability to cause a strong transactivation of a wide range of genes required for the initial activation of protective mechanisms. GUS reporter assays as well as analysis of transcript levels of several endogenous transcripts in protoplasts transiently expressing HsfA1 proteins revealed that HsfA1a can stimulate the transcription of many genes, while the other Hsfs have weaker activity and only on limited set of target genes. The low activity of HsfA1c and HsfA1e can be attributed to the lower DNA capacity of the two factors as judged by a GUS reporter repressor assay.
HsfA1a has been shown to have synergistic activity with the stress induced HsfA2 and HsfB1. The formation of such complexes is considered as important for stimulation of transcription and long term stress adaptation. All HsfA1 members show synergistic activity with HsfA2, while only HsfA1a act as co-activator of HsfB1 and HsfA7. Interestingly, HsfA1b shows an exceptional synergistic activity with HsfA3, suggesting that different Hsf complexes might regulate different HS-related gene networks. Altogether these results suggest that HsfA1a has unique characteristics within HsfA1 subfamily. This result is interesting considering the very high sequencing similarity among HsfA1s, and particularly among HsfA1a and HsfA1c.
To understand the molecular basis of this discrepancy, a series of domain swapping mutants between HsfA1a and HsfA1c were generated. Oligomerization domain and C-terminal swaps did not affect the basal activity or co-activity of the proteins. Remarkably, an HsfA1a mutant harbouring the N-terminus of HsfA1c shows reduced activity and co-activity, while the reciprocal HsfA1c with the N-terminus of HsfA1a cause a gain of activity and enhanced DNA binding capacity.
Sequence analysis of the DBD of HsfA1 proteins revealed a divergence in the highly conserved C-terminus of the turn of β3-β4 sheet. As the vast majority of HsfA1 proteins, HsfA1a at this position comprises an Arg residue (R107), while HsfA1c a Leu and HsfA1e a Cys. An HsfA1a-R107L mutant has reduced DNA binding capacity and consequently activity. Therefore, the results presented here point to the essential function of this amino acid residue for DNA binding function. Interestingly, the mutation did not affect the activity of the protein on Hsp70-1, suggesting that the functionality of the DBD and consequently the transcription factor on different promoters with variable heat stress element number and architecture is dependent on structural peculiarities of the DBD.
In conclusion, the unique properties including expression pattern, transcriptional activities, stability, DBD-peculiarities are likely responsible for the dominant function of HsfA1a as a master regulator of HSR in tomato. Instead, other HsfA1-members are only participating in HSR or developmental regulations by regulating a specific set of genes. Furthermore, HsfA1b and HsfA1e are likely function as stress primers in specific tissues while HsfA1c as a co-regulator in mild HSR. Thereby, tomato subclass A1 presents another example of function diversity not only within the Hsf family but also within the Hsf-subfamily of closely related members. The diversification based on DBD peculiarities is likely to occur in potato as well. Therefore this might have eliminated the functional redundancy observed in other species such as Arabidopsis thaliana but has probably allowed the more refined regulation of Hsf networks possibly under different stress regimes, tissues and cell types.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Die oxygene Photosynthese bildet den Grundpfeiler des heutigen Ökosystems unseres Planeten. Neben den gut untersuchten Landpflanzen bilden Mikroalgen eine äußerst bedeutende Organismengruppe der phototrophen Lebewesen. Zu den Mikroalgen zählen die Diatomeen, welche sich beispielsweise durch eine Silikatschale und spezielle Lichtsammelkomplexe auszeichnen und für einen Großteil der marinen Primärproduktion verantwortlich sind. Die stoffwechselphysiologischen Grundlagen des ökologischen Erfolgs der Kieselalgen sind bislang noch unzureichend erforscht. Ein Vertreter der zentrischen Diatomeen, Cyclotella, wurde bereits zur Jahrtausendwende zur biochemischen Charakterisierung der Diatomeen Photosynthese verwendet (Eppard und Rhiel, 1998; Eppard und Rhiel, 2000), das Genom des Organismus aber erst vor kurzem sequenziert (Traller et al., 2016). Die Sequenzierung des Genoms konnte einige Gene für Lichtsammelproteine identifizieren, die Homologie zu den LhcSR-Proteinen aus C. reinhardtii aufweisen, welche nachweislich eine photoprotektive Funktion besitzen (Peers et al., 2009). Diese sogenannten Lhcx-Proteine der Diatomeen sind in den zwei Gruppen der Kieselalgen, den zentrischen und pennaten Diatomeen zu finden, unterscheiden sich aber in ihren jeweiligen Lhcx-Kandidaten. So können in der pennaten Diatomee P. tricornutum vier lhcx-Gene ausgemacht werden, während die zentrische Kieselalge T. pseudonana sechs lhcx-Gene besitzt und C. cryptica vier verschiedene lhcx-Kandidaten genomisch aufweist (Armbrust et al., 2004; Bowler et al., 2008; Traller et al., 2016). Die beschriebenen Diatomeen weisen alle eine Homologie im Lhcx1 auf, während sich die übrigen Lhcx-Kandidaten zwischen pennaten und zentrischen Diatomeen unterscheiden. Ein zwischen T. pseudonana und C. cryptica konserviertes Lhcx ist das Lhcx6_1, welches 2011 das erste Mal massenspektrometrisch an Photosystemen von T. pseudonana nachgewiesen wurde (Grouneva et al., 2011) und in weiteren Massenspektrometrie-gestützten Untersuchungen in beiden zentrischen Diatomeen an Photosynthese-Komplexen gefunden werden konnte (Gundermann et al., 2019; Calvaruso et al., 2020). Die Funktion des Lhcx6_1 ist bislang unklar.
Diese Arbeit konnte das Lhcx6_1 aus C. meneghiniana charakterisieren und Antikörper-gestützt genauer lokalisieren, eine nicht dynamische Phosphorylierung der Thylakoidmembran-Proteine der zentrischen Diatomee nachweisen und die molekularbiologische Zugänglichkeit des Organismus optimieren. qRT-PCR gestützte Expressions-Analysen konnten eine unerwartete Expression des lhcx6_1-Gens aufdecken. Dieses weist, im Vergleich zum Lhcx1, keine Starklicht induzierte Expression auf. Die Expression des Gens konnte nach wenigen Stunden Schwachlicht als maximal bestimmt werden, während sie im Starklicht abnimmt. Das Muster der Genexpression glich im Schwachlicht eher der des lhcf1-Gens. Die Sequenzierung des lhcx6_1 aus C. meneghiniana identifizierte eine verlängerte N-terminale Sequenz des Proteins, welche Homologie zu den minoren Antennen aus A. thaliana besitzt und Teil des reifen Proteins ist. Mittels eines C-terminalen Epitops wurde ein Antikörper gegen das Lhcx6_1 entworfen, welcher das Protein in C. meneghiniana spezifisch nachweisen kann. Die Isolation von Thylakoidmembranen der zentrischen Diatomee und weitergehende Aufreinigung mittels Saccharosedichtegradienten und lpBN-PAGE konnten die Lokalisation des Lhcx6_1 eingrenzen. Das Protein zeigt dabei keine Unterschiede in seiner Lokalisation nach Inkubation in Schwach-, Stark- und Fernrot-Licht und ist vorrangig mit Photosystem I assoziiert. In geringerer Menge konnte es zudem an Photosystem II nachgewiesen werden, während der immunologische Nachweis in Lichtsammelkomplexen (FCPs) minimale Mengen erbrachte. Ferner konnte eine Phosphorylierung des Lhcx6_1 an Threonin-Resten nachgewiesen werden, während die meisten anderen Thylakoidmembran-Proteine mittels Phospho-Serin Antikörper detektiert werden konnten. Weder die Phosphorylierung des Lhcx6_1, noch der anderen Thylakoidmembran-Proteine, zeigt eine dynamische Regulation, im Stile einer state-transition ähnlichen Kinase auf. Die Qualität des Umgebungslichts führte zu keinerlei Unterschieden in Phosphorylierungsmustern. Weiterführende Untersuchungen der Lhcx6_1-Phosphorylierung mittels Phos-tag PAGE identifizieren eine unphosphorylierte und eine einfach phosphorylierte Form des Proteins. Dabei kann an PSI ausschließlich die phosphorylierte Version des Lhcx6_1 gefunden werden. Im Zuge der Arbeit konnte zudem erstmalig die Elektroporation und Konjugation für C. meneghiniana als Transformations-Methoden etabliert werden, während das Protokoll für die biolistische Transformation optimiert wurde. Die Elektroporation erbrachte die höchste Transformationseffizienz. Molekularbiologische Unterfangen eines Lhcx6_1-Knockdowns mittels Antisense-RNA erzielten zunächst, aufgrund der starken Gegenregulation der Diatomee, keinen Erfolg...
Im Rahmen dieser Arbeit wurden sRNAs des halophilen Archaeons Haloferax volcanii hinsichtlich ihrer biologischen und ihrer regulatorischen Funktion charakterisiert.
Um einen Überblick über die biologischen Funktionen archaealer sRNAs zu erhalten, wurde eine umfassende phänotypische Charakterisierung von 27 sRNA-Deletionsmutanten im Vergleich zum Wildtyp ausgewertet. Im Zuge dieser phänotypischen Charakterisierungen wurden zehn verschiedene Wachstumsbedingungen, morphologische Unterschiede und Veränderungen in der Zellmotilität untersucht. Hierbei zeigten nahezu alle Deletionsmutanten unter mindestens einer der getesteten Bedingungen phänotypische Unterschiede. Durch den Verlust von sRNAs wurden sowohl sogenannte Gain-of-function als auch Loss-of-function Phänotypen beobachtet. Haloarchaeale sRNAs spielen eine wichtige Rolle beim Wachstum mit verschiedenen Salzkonzentrationen, mit verschiedenen Kohlenstoffquellen und beim Schwärmverhalten, sind jedoch weniger in die Adaptation an diverse Stressbedingungen involviert.
Zur näheren Charakterisierung der regulatorischen Funktion archaealer sRNAs wurden sRNA362, sRNAhtsf468 und sRNA479 mittels molekulargenetischer Methoden wie Northern Blot-Analyse und DNA-Mikroarray sowie bioinformatischer in silico-Analyse untersucht. Das Expressionslevel von sRNA362 konnte bestimmt und potentielle Zielgene für sRNAhtsf468 und sRNA479 identifiziert werden.
Eine vorangegangene Studie zeigte den Einfluss von sRNA30 unter Hitzestress und führte zur Identifikation differentiell produzierter Proteine in Abwesenheit der sRNA. In dieser Arbeit wurde mittels Northern Blot-Analysen die Expression der sRNA30 charakterisiert. Das Wachstum in An- und Abwesenheit von sRNA30 wurde bei 42°C und 51°C phänotypisch charakterisiert und der regulatorische Einfluss der sRNA auf die mRNA differentiell regulierter Proteine durch Northern Blot-Analyse überprüft. Eine Transkriptomanalyse mittels DNA-Mikroarray nach Hitzeschock-Induktion führte zur Identifikation differentiell regulierter Gene involviert in Transportprozesse, Metabolismus, Transkriptionsregulation und die Expression anderer sRNAs. Die differentielle Regulation des Proteoms nach Hitzeschockinduktion in An- und Abwesenheit von sRNA30 konnte bestätigt werden.
Desweiteren wurde in dieser Arbeit sRNA132 und deren phosphatabhängige Regulation der Ziel-mRNA HVO_A0477-80 näher charakterisiert. Eine Induktionskinetik nach Phosphatentzug bestätigte die Bedeutung von sRNA132 für die verstärkte Expression des Operons HVO_A0477-80 unter Phosphatmangel-Bedingungen und verwies auf die Existenz weiterer Regulationsmechanismen. Während vor und nach Phosphatentzug kein Unterschied bezüglich der Zellmorphologie von Wildtyp und Deletionsmutante zu erkennen war, führte das Wachstum mit einem starken Phosphatüberschuss von 5 mM zu einer Zellverlängerung der Deletionsmutante. Die Kompetition der nativen 3‘-UTR des Operons HVO_A0477-80 mit einer Vektor-kodierten artifiziellen 3‘-UTR legt eine Regulation über die Bindung von sRNA132 an die 3‘-UTR nahe. Der Transkriptomvergleich nach Phosphatentzug in An- und Abwesenheit von sRNA132 führte zur Identifikation des Phosphoregulons der sRNA. Zu diesem Phosphoregulon gehören unter anderem zwei Glycerinphosphat-Dehydrogenasen, Transkriptionsregulatoren, eine Polyphosphatkinase und eine Glycerolphosphodiesterase. Zudem waren die Transkriptlevel der beiden ABC-Transporter HVO_A0477-80 und HVO_2375-8 für anorganisches Phosphat und des Transporters HVO_B0292-5 für Glycerinaldehyd-3-Phosphat in Abwesenheit der sRNA verringert. Die beiden ABC-Transportsysteme für anorganisches Phosphat wurden im Rahmen dieser Arbeit deletiert und weiter charakterisiert. Es konnte gezeigt werden, dass das ABC-Transportsystem HVO_2375-8 bei geringen Phosphatkonzentrationen leicht induziert wird und das Transkriptlevel in Anwesenheit von sRNA132 erhöht ist. Wachstumsversuche der jeweiligen Deletionsmutante in direkter Konkurrenz mit dem Wildtyp zeigten, dass keiner der beiden ABC-Transporter den anderen vollständig ersetzen kann und der Wildtyp mit beiden intakten ABC-Transportern unter phosphatlimitierenden Bedingungen einen Wachstumsvorteil besitzt. In silico-Analysen der Promotorbereiche von sRNA und ABC-Transporter legen zudem die Existenz von P-Boxen nahe.
Der erste Teil der vorliegenden Arbeit beinhaltet die funktionelle Analyse von fünf Oberflächenproteinen von B. recurrentis die die Fähigkeit besitzen, die Aktivierung von humanen Komplement zu inhibieren und Borrelien vor Bakteriolyse zu schützen. Im zweiten Teil der Arbeit wurden zwei immunologische Testverfahren mit hoher Sensitivität sowie Spezifität entwickelt und mit zahlreichen Patientenseren evaluiert. Die entwickelten Tests könnten in Zukunft als zuverlässige Instrumente für eine gesicherte Diagnose von LRF eingesetzt werden.
Eine Sequenzanalyse führte zur Identifizierung eines neuen Proteinclusters, welches die fünf untersuchten Komplement-inhibierenden Proteine als „Cluster of Complement-targeting and Host-interacting Proteins“ oder „Chi-Gencluster“, zusammenfasst. Diese Oberflächenproteine wurden als ChiA, ChiB, ChiC, ChiD und ChiE bezeichnet. Weiterführende Sequenzanalysen ergaben, dass das Chi-Gencluster extrem hoch konserviert ist und sowohl in den ersten B. recurrentis-Isolaten aus den 1990er Jahren als auch in B. recurrentis-Stämmen nachgewiesen werden konnte, die 2015 aus Patienten isoliert wurden.
Durch funktionelle Analysen konnte gezeigt werden, dass alle fünf Chi-Proteine in der Lage sind den alternativen und terminalen Komplementweg zu inhibieren. Ebenfalls konnte für die Proteine ChiB, ChiD sowie ChiE nachgewiesen werden, dass die Interaktion mit der Komplementkomponente C5 dosisabhängig verläuft.
Die strukturelle Aufklärung des Proteins ChiB ermöglichte es Aminosäuren zu identifizieren, von denen angenommen wurde, dass sie für die Interaktion mit Komplement eine Rolle spielen könnten. Durch in vitro Mutagenese konnten insgesamt fünf verschiedene Varianten von ChiB generiert werden, die jedoch keine Veränderungen in ihrem Komplement-inhibierenden Potential gegenüber dem unveränderten ChiB-Protein aufwiesen. Weder in der Inhibition des alternativen oder des terminalen Komplementweges, noch in der Interaktion mit den untersuchten Komplementkomponenten C3b, C5 und C9.
Weiter konnte gezeigt werden, dass die lytische Aktivität von Humanserum durch Vorinkubation mit ChiB, ChiC, ChiD und ChiE drastisch reduziert werden konnte, sodass Serum-sensible Borrelienzellen in Gegenwart von Komplement überlebten. „Gain-of-function“ B. garinii-Transformanten, welche mit dem entsprechendem Chi-kodierenden Gen transformiert wurden, bestätigten die mit den gereinigten Proteinen erhobenen Ergebnisse.So konnte nachgewiesen werden, dass ChiB-, ChiC- oder ChiD-produzierende „Gain-of-function“ B. garinii Transformanten, nicht jedoch ChiE- produzierende Zellen, in der Lage waren einen Serum-resistenten Phänotypen auszubilden. Für Transformanten, die zwei-, drei- oder vier Chi-Proteine in verschiedenen Kombinationen gleichzeitig produzierten, konnte allerdings die Fähigkeit in Gegenwart von Humanserum zu überleben nicht bestätigt werden.
Molekulare Analysen mit verschiedenen RF-Borrelienstämmen führten zum Nachweis, dass die fünf Chi-kodierenden Gene bei allen Isolaten vorhanden sind und unter in vitro Bedingungen exprimiert werden. Im Gegensatz zu B. recurrentis PAbJ, ließ sich das HcpA kodierende Gen in B. duttonii LAI nicht nachweisen, jedoch alle dem Chi-Cluster zugehörigen Gene. Bei B. duttoni V fehlte das gesamte Chi-Cluster sowie die für CihC- und HcpA-kodierenden Gene. Durch eine Western Blot-Analyse konnte mit spezifischen Antikörpern bestätigt werden, dass die Proteine CihC, HcpA und ChiB in B. recurrentis A17 unter in vitro Bedingungen produziert wurden.
Im zweiten Teil der vorliegenden Arbeit wurden durch die Analyse der IgM- und IgG-Immunreaktivitäten der LRF-Patientenseren zwei Proteine identifiziert, CihC und GlpQ, die als potenzielle Antigene für die Serodiagnostik des LRF evaluiert wurden. Eine initiale Evaluierung des IgM Lineblot-Immmunoassays zeigte jedoch nur eine geringe Sensitivität für die beiden Antigene, während der IgG Lineblot-Immunoassay eine sehr hohe Sensitivität aufwies. Der ELISA hingegen zeigte bei einer Kombination beider Antigene sehr gute Sensitivitäten und Spezifitäten. Um die starke Hintergrundfärbung bei den Lineblot-Immunoassays, welche eine korrekte Bewertung der Reaktivitäten gegenüber CihC erheblich erschwerten, zu minimieren, wurde ein „Epitop-Mapping“ durchgeführt, um immunogene Regionen innerhalb des CihC-Proteins zu lokalisieren. Eine zweite Evaluierung mit dem immunreaktiven N-terminalen CihC-Fragment CihC-N führte zu einer deutlichen Verbesserung der IgG Lineblot-Immunoassays mit einer Sensitivität von 100 % und einer starken Reduktion der Hintergrundfärbung. Zusätzlich konnte die Sensitivität der IgM-ELISA deutlich verbessert werden. Die Verwendung von CihC-N führte beim IgG-ELISA zur Herabsetzung des Cut-off-Wertes und zu einer besseren Unterscheidung zwischen den positiven LRF-Seren und den verwendeten Kontrollseren. Im Rahmen dieser Arbeit konnten somit zwei serologische in vitro Diagnostika entwickelt werden, die als zuverlässige Point-of-Care-Diagnostik in klinischen Studien eingesetzt werden könnten. Zur Steigerung der Sensitivität des IgM-Lineblot-Immunoassays sollten allerdings weiterführende Untersuchungen mit weiteren immunreaktiven Antigenen, wie z.B. den Vmp-Proteinen von B. recurrentis, angestrebt werden.
Seit Jahrzehnten finden Kunststoffe aufgrund ihrer vorteilhaften Materialeigenschaften wie z. B. Formbarkeit und im Vergleich zu Glas oder Metall geringe Kosten und leichtes Gewicht, vermehrt Anwendung in allen Bereichen des täglichen Lebens. Einhergehend gelangen Kunststoffe zunehmend in die Umwelt, und reichern sich dort an. Besondere Aufmerksamkeit erfahren Partikel im Größenbereich von 1-1000 µm, sogenanntes Mikroplastik (MP), welches entweder direkt eingetragen wird oder in der Umwelt durch Fragmentierung größerer Plastikteile entsteht. Lange Zeit fokussierte sich die MP Forschung vorrangig auf aquatische Ökosysteme, obwohl Schätzungen davon ausgehen, dass die Kunststoffeinträge in terrestrischen Ökosystemen um ein Vielfaches höher sind. Besonders relevante Eintragspfade sind neben der unsachgemäßen Entsorgung von Abfällen, die landwirtschaftliche Klärschlamm- und Kompostdüngung und der zunehmende Einsatz von Mulchfolien, sowie der im Straßenverkehr generierte Reifenabrieb.
Für eine Abschätzung und Bewertung der MP-Belastung in Böden sind analytische Messungen von MP in Umweltproben essenziell, derzeit jedoch kaum existent, da MP im Boden partikulär und heterogen verteilt vorliegt und deshalb nur schwierig zu detektieren ist. Die für viele Analyseverfahren notwendige Isolation der Kunststoffpartikel, sowie die für repräsentative Messungen erforderliche Aufbereitung großer Probenvolumina stellen besondere analytische Herausforderungen mit großem Kosten- und Zeitaufwand dar. Chromatografische Verfahren finden wenig Anwendung, bieten aber vorteilhafte Voraussetzungen als Screeningverfahren für die Untersuchung von Böden, da sie nicht zwangsweise eine Partikelisolation verlangen, und zudem als Ergebnis einen Massegehalt liefern.
Diese Dissertation zeigt drei Anwendungen Chromatografie basierter Analyseverfahren zur Charakterisierung von MP im Boden. Erstmalig wurde die Thermo-Extraktion-Desorption-Gaschromatografie-Massenspektrometrie (TED-GC/MS) für die Analytik von Reifenabrieb in realen Umweltproben angewandt bei minimaler Probenaufbereitung. Dafür wurde ein Straßenrandboden umfangreich beprobt und analysiert, und es konnte neben der Eignung der analytischen Methode auch eine repräsentative Probenahmestrategie und räumliche Verteilungsmuster von Reifenabrieb im Boden demonstriert werden.
Der zweite Forschungsschwerpunkt lag auf der Methodenentwicklung und validierung eines neuartigen chemischen Extraktionsverfahrens für die Bestimmung von Polyestern in Bodenproben. Das Verfahren basiert auf der hydrolytischen Spaltung von Polyestern in ihre Monomere, deren flüssigchromatografische Abtrennung von Matrixbestandteilen und der Detektion mittels UV-Absorption. Das Verfahren verlangt neben der Extraktion keine weiteren Probenaufbereitungsschritte, ist für unterschiedliche Umweltmatrizes geeignet und ist damit z. B. prädestiniert für den Nachweis von Polyesterfasern auf gedüngten landwirtschaftlichen Flächen.
MP ist nicht nur aufgrund seiner Persistenz problematisch, sondern auch, weil es hydrophobe organische Schadstoffe aus dem Umweltmedium anreichern und transportieren kann. Maßgeblich für das Sorptionsverhalten sind die Materialeigenschaften des zugrunde liegenden Kunststoffes, welche Änderungen durch Alterungsprozessen unterliegen. Der Zusammenhang zwischen Materialalterung und Sorptionsverhalten wurde in früheren Studien kontrovers diskutiert und ist der dritte Teil dieser Arbeit. In einem Sorptionsexperiment konnte mittels Headspace-Gaschromatografie mit Flammenionisations-Detektion die Aufnahme von Aromaten an den Kunststoffen Polypropylen und Polystyrol quantifiziert werden. Die Kunststoffe wurden materialwissenschaftlich charakterisiert, teilweise künstlich gealtert und die daraus resultierende Änderungen der Materialeigenschaften sowie einhergehenden Änderungen des Sorptionsverhaltens erfasst. Dadurch war es möglich den Einfluss einzelner Materialeigenschaften auf das Sorptionsverhalten zu bewerten, Rückschlüsse auf zugrunde liegende Sorptionsmechanismen zu treffen und zu zeigen, dass in vorliegendem Experiment die Polymeralterung bei MP nicht zu einer erhöhten Schadstoffsorption führte.
Sleep is one of the fundamental requirements of all animals from nematodes to humans. It appears in different formats with shared features such as reduced muscle activities and reduced responsiveness to the environment. Despite the long history of sleep research, why a brain must be taken offline for a large portion of each day remains unknown. Moreover, sleep research focused on mammals and birds reveals two stages, rapid-eye-movement (REM) and slow-wave (SW) sleep, alternating during sleep. Whether these two stages of sleep exist in other vertebrates, particularly reptiles, is debated, as is the evolution of sleep in general.
Recordings from the brain of a lizard, the Australian bearded dragon Pogona vitticeps, indicate the presence of two electrophysiological states and provides a better picture of their sleep. Local field potential (LFP) signals, head velocity, eye movements, and heart rate during sleep match the pattern of REM and SW sleep in mammals. The SW and REM sleep patterns that we observed in lizards oscillated continuously for 6 to 10 hours with a period of 80-100 seconds when the ambient temperature was ~27°C. Lizard SW dynamics closely resemble those observed in rodent hippocampal CA1, yet originated from a brain area, the dorsal ventricular ridge (DVR), that does not correspond anatomically or transcriptomically to the mammalian hippocampus. This finding pushes back the probable evolution of these dynamics to the emergence of amniotes, at least 300 million years ago.
Unlike mammals and birds, REM and SW sleep in lizards occupy an almost equal amount of time during sleep. The clock-like alternation between these two sleep states was found initially by measuring the power modulation of two frequency bands, delta and beta. I recorded the full-band LFP and found an infra-slow oscillation (ISO) in the frequency range between 5 and 20 milli-Hz during sleep. The magnitude of ISO increased during sleep and decreased during both wakefulness and arousal during sleep. The up- and down-states of ISO were synchronized with the sleep state alternating rhythm but with a significant time lag dependent on the locations of the recording electrodes. Multi-site LFP recordings indicated that this ISO is a putative propagation wave sweeping extremely slowly, 30-67 µm/sec, from the posterior-dorsal pole to the anterior-ventral pole of the DVR.
Previous studies in other animals showed that brainstem areas such as the locus coeruleus, laterodorsal tegmentum, and periaqueductal gray are involved in sleep states regulation. It is sadly impossible to carry out in vivo recordings in the lizard brainstem without severely affecting them and their quality of life. I thus carried out ex vivo recordings in both DVR and brainstem. Pharmacological stimulation of the brainstem could reversibly silence one distinct EEG pattern characteristic of SW sleep, the sharp-wave and ripple complex, in DVR. An ISO could be recorded simultaneously in both DVR and brainstem. From data collected in both intact and split ex vivo brains, I concluded that there are independent ISO generators in at least two areas, the brainstem and the telencephalon. Their signals may normally be synchronized by long-range connections. The DVR ISO leads the brainstem ISO by ~29 sec. Optogenetic stimulation of brainstem neurons was able to disrupt the ISO in DVR reversibly.
In conclusion, the lizard brain offers a relatively simple model system to study sleep. Despite a diversity of results in different lizard species, my results revealed a number of new findings. Relevant for sleep research in general: 1) REM and SW sleep exist in a reptile. Since they also exist in birds and mammals, they probably existed in their common amniote ancestor, if not earlier. 2) REM and SW occupy equal amounts of time during sleep (50% duty cycle), a unique feature among all described sleep electrophysiological patterns, suggesting the possible existence of a simple central pattern generator of sleep, possibly ancestral. 3) I discovered the existence, in the local field potential, of an infra slow oscillation with extremely slow propagation, locked to the SW-REM alternating rhythm. The causes and mechanisms of this ISO remain to be understood. To my knowledge, the correlation between sleep states and a slow rhythm has only been reported in human scalp EEG recordings so far.
Climate and subsequent environmental changes are regarded as one driver of species evolution. Against this background the present study investigates the evolutionary history of the mammalian family Bovidae (Cetartiodactyla, Mammalia), today the most species-rich family of large herbivores on the African continent. Temporal and spatial patterns in that group’s evolution are the focus of the present study and were investigated using methods and data deriving from multiple disciplines (palaeontology, genetics, climatology, conservation biology). The results serve as a validation of macroevolutionary hypotheses of species evolution.
A major proportion of African mammalian fossils can be assigned to that family. Due to their morphological adaptations, bovid species are highly indicative of their habitats. Hence, bovids are of great importance for paleontology. However, a strong taphonomic bias is present in the fossil record of bovids, favoring large and arid- adapted species. Molecular phylogenies of extant species and species distribution modelling combined with climate reconstructions can help to overcome these limitations.
A molecular phylogeny, based on the cytochrome b gene of 136 bovid species served as basis for analysis of temporal patterns. Divergence events were dated using the relaxed molecular clock approach. The tree was time calibrated at 30 nodes using information inferred from the fossil record. Lineage-Through-Time plots and the respective statistical analyses reveal detailed temporal patterns in the evolutionary history of tribes and groups combining arid- and humid-adapted tribes. The resulting pattern shows three distinct phases. Phase 1 (P1) is dominated by speciation events within the humid group, while the second phase (P2) is marked by a dominance of speciation within the arid group. The switch in diversification rates (BDS) from P1 to P2 is dated to 2.8 million years ago. The third phase (P3) shows low diversification rates for all groups, starting around 1.4 million year ago and culminates in a significantly reduced diversification rate for the complete family at 0.8 million years ago. Both transitions are contemporaneous with global climate changes and turnover events in fossil faunal communities.
To investigate the impact of climate changes onto the habitat availability within the last 3 million years and its putative influence on diversification rates, the species distribution modeling method was applied. For 85 African species and subspecies the climate niches were established and grouped into 5 climate-groups based on their climate preferences. For each group the available habitat for the period before and after the BDS was calculated on continental scale using reconstructed climate scenarios. To evaluate the modeled habitat distributions, regional analyses were performed in test areas surrounding well studied fossil sites (Laetoli, Olduvai, Chiwondo Beds, Lothagam, Koobi Fora, West Turkana, Swartkrans, Sterkfontain und Toros-Menalla). Habitat profiles (HP) permitted the comparison of the model based habitat reconstruction with the interpretations of classic paleontological reconstruction. The validity of the habitat modeling has been shown in particular for East African test areas. The reconstructions for the northern and southern fossil sites does not support the modeled habitats in these areas. Yet, the method of habitat- profiling may serve as suitable tool for environmental reconstruction of areas lacking sufficient paleontological material. A comparison of habitat availability before and after the BDS on continental scale identified a significant loss of habitat for humid adapted groups (7-22%) and habitat gain for arid adapted groups (19-173%). The climatically intermediate group experiences a tremendous gain of habitat (3366%). The greatest environmental change was modeled for East Africa, initiated by a progressive regional aridification.
In addition to the distribution modeling for past climate conditions, the geographical distribution was modeled for the future, i.e. for climate scenarios representing the years 2050 and 2080 under a putative climate change scenario (global surface warming). It was shown that in particular the arid groups have to expect a remarkable loss of habitat (41-76%), while a gain of available habitat can be expected for the humid adapted groups (114-577%). The climatically intermediate group suffers the strongest habitat loss (85%). Regions with locally stable climate conditions were detected and may serve as potential refugia and are already today known as Africa’s hot spots of biodiversity.
The results show a positive correlation of high diversification rates and increasing habitat availability. None of the tested speciation hypotheses taken alone explains the observations (e.g., Turnover-pulse Hypothesis, Relay Model). A major element in these hypotheses is the passive fragmentation of populations induced by unfavorable climate changes. In contrast, the Periodic Model (Grubb 1999) considers natural, periodically recurring climate changes and moreover, the active dispersal of individuals and resulting founder events. I added the effect of a superimposed directed climate trend – like the progressive aridification since the late Pliocene in Africa – which leads to a bias in the proportion and probability towards leading edge effects. This Directed Periodic Model explains the patterns found in the evolution of Bovidae.
The combination of a molecular phylogeny and species distribution modeling, together with information inferred from the fossil record, reveals remarkable temporal and spatial patterns in the evolution of bovids, and helps overcome the limitations of the fossil record. The present study highlights the importance of active dispersal and founder populations in speciation processes. A point widely unattended in speciation hypotheses. The fully dated molecular phylogeny is the most densely sampled tree for the family Bovidae to date and may serve as a framework for a connection of present and future population studies, permitting the connection of medium-scale with long- term effects induced by climate and environmental changes.
Gravitropism is a fundamental process in plants that allows shoots to grow upward and roots to grow downward. Protein phosphorylation has been postulated to participate in the intricate signaling cascade of gravitropism. In order to elucidate the underlying mechanisms governing the gravitropic signaling and unearth novel protein constituents, an exhaustive investigation employing microgravity-induced phosphoproteomics was undertaken. The significantly phosphorylated proteins unraveled in this study can be effectively divided into two groups through clustering analysis. Furthermore, the elucidation of Gene Ontology (GO) enrichment analysis disclosed the conspicuous overrepresentation of these clustered phosphoproteins in cytoskeletal organization and in hormone-mediated responses intimately intertwined with the intricate phenomenon of gravitropism. Motif enrichment analysis unveiled the overrepresentation of [-pS-P-] and [-R-x-x-pS-] motifs. Notably, the [-pS-P-] motif has been suggested as the substrate for the Casein kinase II (CK II) and Cyclin-dependent kinase (CDK). Kinase-inhibitor assays confirmed the pivotal role played by CK II and CDK in root gravitropism. Mutant gravitropism assays validated the functional significance of identified phosphoproteins, with some mutants exhibiting altered bending kinetics using a custom-developed platform. The study also compared phosphoproteomics data from different platforms, revealing variations in the detected phosphopeptides and highlighting the impact of treatment differences. Furthermore, the involvement of TOR signaling in microgravity-induced phosphorylation changes was uncovered, expanding the understanding of plant gravitropism responses.
To fulfill the large-scale verification of interesting candidates from the phosphoproteomics study, a novel root and hypocotyl gravitropism phenotyping platform was developed. This platform integrated cost-effective hardware, including Raspberry Pi, a high-quality camera, an Arduino board, a rotation stage (obtained from Prof. Dr. Maik Böhmer), and programmable green light (modified by Sven Plath). In addition, through collaboration with a software developer, machine-learning-based software was developed for data analysis. This platform tested the gravitropic response of candidate mutants identified in the phosphoproteomics study. Furthermore, the capabilities of this platform were expanded to investigate tropisms in other species and organs. To find novel proteins that might act as partners of a key protein that is involved in gravitropism signaling, ALTERED RESPONSE TO GRAVITY 1 (ARG1), immunoprecipitation coupled with Mass Spectrometry (IP-MS) was performed and identified ARG1-LIKE1 (ARL1) as a potential interacting protein with ARG1. This interaction was further confirmed through in vivo pull-down assays and bimolecular fluorescence complementation assays. In addition, the interaction between ARG1 and HSP70-1 was also validated.
Overall, this thesis sheds light on the molecular components and signaling events involved in plant gravitropism. It contributes to existing knowledge and opens up new ways to investigate this fascinating area of plant biology.
The continuous conversion of natural wildlife habitats into agricultural areas, as well as the fragmentation of the last wildlife refuges, is increasing the interface between people and wildlife. When wildlife negatively impacts on people and vice versa, we speak about human-wildlife conflicts (HWCs). This definition includes losses on both sides and takes into consideration the rooting of most of these conflicts between different groups of interest, such as advocates for nature conservation and economic groups. The centres of highest biodiversity are located in developing countries, which are also characterized by poverty. In African and Asian countries, people living in the vicinity of national parks and other conservation areas mostly receive only little support through the government or conservation organisations. Especially for those people who are dependent on agriculture, damage to fields and harvests can have catastrophic consequences. If the species causing damage is protected by national or even international law, the farmer is not allowed to use lethal methods, but has to approach the authority in charge. If this agency, however, cannot offer appropriate support, resentment, anger or even hate develops, and the support for wildlife conservation activities declines. For this reason, HWCs were declared as one of the most important conservation topics today, being particularly relevant for large and threatened species such as the African and Asian elephant, hippopotamus and the greater one-horned rhino, as well as for large predators. Up to today, no general assessment scheme has been recommended for damage caused by protected wildlife species.
In my study, HWCs in Asia and Africa are compared, focussing on all herbivorous species identified which damaged crops. For the French NGO Awely, des animaux et des hommes, I developed a detailed assessment scheme suitable for all terrestrial ecosystems, and any type of HWCs and any species (Chapter 2). This HWC assessment scheme was used in four different study areas located in two African countries (South Luangwa/Zambia (SL), Tarangire/Tanzania (TA)) and two Asian countries (Bardia/Nepal (BA) and Manas/India (MA)). This scheme ran for six consecutive years (2009 to 2014) for Zambia, Nepal and India and two years (2010 to 2011) for Tanzania. To carry out the assessments, I trained local HWC officers (Awely Red Caps) to assess HWCs by field observations (measurement of damage, identification of species through signs of presence, landscape attributes etc.) and interviews with aggrieved parties (socio economic data). Results of this assessment are presented in Chapters 2-4.
To determine whether elephants prefer or avoid specific crop species, two field experiments were carried out, one in SL and one in BA (Chapter 5 and 6). For this, two test plots were set up and damage by elephants (and other herbivores) were quantified.
Within this doctoral thesis, 3306 damage events of 7408 aggrieved parties were analysed. In three out of the four study areas (SL, BA, MA), elephants caused the highest number of damage events compared to all other wildlife species, however, in TA, most fields were damaged by zebra. Furthermore, the greater one-horned rhino, hippopotamus, wild boar, bushpig, deer and antelope, as well as primates, caused damage to fields and harvests. Damage to houses and other property were nearly exclusively caused by elephants.
With this doctoral thesis I was able to show that season, crop availability, type and the phenological stage of the crop played an important role for crop damaging behavior of herbivores (Chapter 2). Elephants especially damaged rice, maize and wheat and preferred all crop types in a mature stage of growth. In contrast, rhinos preferred wheat to rice and similar to antelope and deer, they preferred crops at earlier stages of growth, before ripening. Crop damage by wildlife species varied strongly in size; most damages fell below 40% of the total harvest per farmer, but in several cases (3 to 8% depending on the study area), harvests were completely destroyed. Interestingly, during times of low nutritional availability in the natural habitat (dry season), crop damages in all four study areas were significantly less than during other seasons.
In all four study areas, crop protection strategies, such as active guarding in the fields, chasing wildlife with noise or fire torches or erecting barriers, were used. In some cases protection strategies were combined. Analysis of data revealed that traditional protection strategies did not reduce the costs of damage (Chapter 3). In some cases, costs of damage, on protected fields were even higher than for unprotected fields. Only in MA did strategic and cohesive guarding significantly reduce crop damage by wildlife species.
Besides damage in the fields, elephants also caused damage to properties in the villages. In search for stored staple crops, they damaged houses, grain stores and kitchens. Such damage was analysed in three study areas (SL, BA, MA) (Chapter 4). Although property damage occurred less frequently compared to crop damage in the fields, the mean cost of this damage was found to be double in BA/MA and four times higher in SL, compared to the costs of crop damage in the fields. It is further remarkable that property damage significantly increased towards the dry season, when the harvest was brought into the villages.
The findings of this study underpin the assumption that wildlife herbivores, especially elephants, are lured to fields and crops because the highly nutritional food (crop) being readily available. Traditional crop protection is cost and labour intensive and does not reduce the costs of damage. For this reason, crop types, which are thought to be not consumed by elephants were systematically tested on their attractiveness in field experiments in SL and BA (Chapter 5 and 6). In SL, lemon grass, ginger and garlic were proven to be less attractive to African elephants than maize and in BA, basil, turmeric, chamomile, coriander, mint, citronella and lemon grass were found to be less attractive to Asian elephants than rice.
The results of this doctoral thesis are relevant for the management of wildlife conservation as they can lead to new approaches to the mitigation of HWCs in African and Asian countries. Finally, specific needs for more scientific research in this field have been identified.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
Alternative splicing (AS) is a co- or post-transcriptional process by which one gene gives rise to multiple isoforms. This ‘split and combine’ step multiplies eukaryotic proteome diversity several fold and is implicated in several diseases given its pervasive impact. Control of alternative splicing is brought about by cis-regulatory elements, such as RNA sequence and structure, which recruit trans-acting RNA-binding proteins (RBPs). Although several of these interactions are already described in detail, we lack a comprehensive understanding of the regulatory code that underlies a splicing decision.
Here, we have established a high-throughput screen to comprehensively identify and characterise cis-regulatory elements that control a specific splicing decision. A cancer-relevant splicing event in proto-oncogene RON was picked as a minigene prototype for initialising the screening approach. Then, we transfected a library of thousands of randomly mutagenised minigene variants as a pool into human cells, and subsequently quantified the spliced isoforms by RNA sequencing. Importantly, we used a barcode sequence to tag the minigene variants and thereby linked mutations to their corresponding spliced products. By using a linear regression-based modelling approach, we were able to determine the effects of single mutations on RON AS. In total, more than 700 mutations were found to significantly affect the splicing regulation of the RON alternative exon. In addition, mutation effects quantified from the screening approach correlate with RON alternative splicing in cancer patients. We discovered numerous previously unknown cis-regulatory elements in both introns and exons, and found that the RBP heterogeneous nuclear ribonucleoprotein H (HNRNPH) extensively regulates RON AS at multiple levels in both cell lines and cancer. Furthermore, the large number of RBPs involved in the process, point to a complex splicing regulatory network involved in the control of RON splicing. iCLIP and synergy analysis between mutations and HNRNPH knockdown data pinpointed the most relevant HNRNPH binding sites across RON. Finally, cooperative HNRNPH binding was shown to mediate a splicing switch of RON alternative exon. In summary, our results provide an unprecedented view on the complexity of splicing regulation of an alternative exon. The novel screening approach introduces a tool to study the relationship of RNA sequence variants along with trans-acting regulators to their impact on the splicing outcome, offering insights on alternative splicing regulation and the relevance of mutations in human disease.
RNA modification is a dynamic and complex process that involves the addition of various chemical groups to RNA molecules, contributing to their diversity and functional complexity. Among all the RNA modifications, N6-methyladenosine (m6A) is the most common post-transcriptional modification found in mRNA molecules, particularly in eukaryotic mRNA. It involves methylation of the adenosine base at the nitrogen-6 position. This modification plays a crucial role in many aspects of RNA metabolism, including splicing, stability, translation, and the cellular response to stress. With the development of m6A sequencing technologies, our knowledge of m6A has evolved rapidly over the past two decades. However, one of the most widely used m6A profiling techniques termed “m6A individual-nucleotide resolution UV cross-linking and immunoprecipitation (miCLIP)” suffers from a high unspecific background signal due to the limited antibody binding specificity.
To accurately discriminate m6A sites from the background signal in miCLIP data, in Chapter 4, I first developed different strategies to identify the true miCLIP2 signal changes that are corrected for the underlying transcript abundance changes. I performed this analysis on data that generated with an improved experiment protocol, named miCLIP2. With the best performing strategy, the Bin-based method, I detected more than 10,000 genuine m6A sites. I then used the information embedded in the genuine m6A sites to train a machine learning model - named "m6Aboost" - to enable accurate m6A site detection from the miCLIP2 data without a control dataset from an m6A depletion cell line. To allow an easy access for future users, I packaged the m6Aboost model into an R package that is available on Bioconductor.
Although previous studies have reported that m6A is involved in three different RNA decay pathways, it remains unclear how a pathway is selected for a specific transcript or m6A site. In Chapter 5, I reveal that m6A sites in the coding sequence (CDS) induce a stronger and faster RNA decay than the m6A sites in the 3’ untranslated region (3’UTR). Through an in-depth investigation, I found that m6A sites in CDS trigger a novel mRNA decay pathway, which I termed CDS-m6A decay (CMD). Importantly, CMD is distinct from the three previously reported m6A-mediated decay pathways. In terms of its mechanism, CMD relies on translation, where m6A sites in the CDS lead to ribosome pausing and subsequent destabilization of the transcript. The transcripts targeted by CMD are identified by the m6A reader protein YTHDF2, preferentially localized to processing bodies (P-bodies), and undergo degradation facilitated by the decapping factor DCP2. CMD provides a flexible way to control the expression of CDS m6A-containing transcripts which include many developmental regulators and retrogenes.
In summary, this PhD thesis introduces a novel workflow for identifying m6A sites in miCLIP data through the implementation of the m6Aboost machine learning model. Using the m6A sites identified by m6Aboost and additional data, a newly uncovered m6A-mediated mRNA decay pathway, CMD, is elucidated, providing valuable insights into m6A-mediated decay processes.
Most cellular processes are regulated by RNA-binding proteins (RBPs). These RBPs usually use defined binding sites to recognize and directly interact with their target RNA molecule. Individual-nucleotide resolution UV crosslinking and immunoprecipitation (iCLIP) experiments are an important tool to de- scribe such interactions in cell cultures in-vivo. This experimental protocol yields millions of individual sequencing reads from which the binding spec- trum of the RBP under study can be deduced. In this PhD thesis I studied how RNA processing is driven from RBP binding by analyzing iCLIP-derived sequencing datasets.
First, I described a complete data analysis pipeline to detect RBP binding sites from iCLIP sequencing reads. This workflow covers all essential process- ing steps, from the first quality control to the final annotation of binding sites. I described the accurate integration of biological iCLIP replicates to boost the initial peak calling step while ensuring high specificity through replicate re- producibility analysis. Further I proposed a routine to level binding site width to streamline downstream analysis processes. This was exemplified in the re- analysis of the binding spectrum of the U2 small nuclear RNA auxiliary factor 2 (U2AF2, U2AF65). I recaptured the known dominance of U2AF65 to bind to intronic sequences of protein-coding genes, where it likely recognizes the polypyrimidine tract as part of the core spliceosome machinery.
In the second part of my thesis, I analyzed the binding spectrum of the serine and arginine rich splicing factor 6 (SRSF6) in the context of diabetes. In pancreatic beta-cells, the expression of SRSF6 is regulated by the transcription factor GLIS3, which encodes for a diabetes susceptibility gene. It is known that SRSF6 promotes beta-cell death through the splicing dysregulation of genes essential to beta-cell function and survival. However, the exact mechanism of how these RNAs are targeted by SRSF6 remains poorly understood. Here, I applied the defined iCLIP processing pipeline to describe the binding landscape of the splicing factor SRSF6 in the human pancreatic beta-cell line EndoC-H1. The initial binding sites definition revealed a predominant binding to coding sequences (CDS) of protein-coding genes. This was followed up by extensive motif analysis which revealed a so far, in human, unknown purine-rich binding motif. SRSF6 seemed to specifically recognize repetitions of the triplet GAA. I also showed that the number of contiguous triplets correlated with increasing binding site strength. I further integrated RNA-sequencing data from the same cell type, with SRSF6 in KD and in basal conditions, to analyze SRSF6- related splicing changes. I showed that the exact positioning of SRSF6 on alternatively spliced exons regulates the produced transcript isoforms. This mechanism seemed to control exons in several known susceptibility genes for diabetes.
In summary, in my PhD thesis, I presented a comprehensive workflow for the processing of iCLIP-derived sequencing data. I applied this pipeline on a dataset from pancreatic beta-cells to unveil the impact of SRSF6-mediated splicing changes. Thus, my analysis provides novel insights into the regulation of diabetes susceptibility genes.
Die Funktion der äußeren Haarsinneszellen geht weit über die normale Rezeptoreigenschaft der Kategorie Mechanorezeptor hinaus. Äußere Haarzellen mit ihrer reichhaltigen efferenten Innervierung sind nicht nur für die sensorische Aufnahme mechanischer Bewegung zuständig, sondern ermöglichen aufgrund ihrer motorischen Funktionen die mechanische Verstärkung der Wanderwelle in der Cochlea. Äußere Haarzellen sind eine maßgebliche Komponente des ´cochleären Verstärkers` und ihr Ausfall führt zur Schwerhörigkeit. Beiprodukte des cochleä-ren Verstärkers sind otoakustische Emissionen, deren Messung Aufschluss über aktive mechanische Prozesse im Innenohr gibt.
Die äußeren Haarsinneszellen bilden Synapsen mit dem olivo-cochleären efferenten System, welches im Zentrum der vorliegenden Untersuchung steht. Es vermittelt den Einfluss des Zentralnervensystems auf das Corti-Organ des Innenohrs. Über die akustische Reizung des olivo-cochleären Reflexbogens ist man in der Lage, das efferente System zu aktivieren und gleichzeitig die Antworteigenschaften der Cochlea zu verändern. Efferente Modulationen des cochleären Verstärkers können sich z. B. in einer Veränderung des Emissionspegels bemerk-bar machen. Die Fledermausspezies Carollia perspicillata ist aufgrund ihres Echoortungs-systems mit einem sehr sensitiven und hochauflösenden Hörvermögen ausgestattet und eignet sich hervorragend als Modelltier in der Hörforschung, insbesondere auch deshalb, da oto-akustische Emissionen sehr gut messbar sind.
Das efferente System von C. perspicillata wurde in dieser Untersuchung durch akustische Stimulation der kontralateralen Cochlea angeregt. Die Stimuli, die nicht nur in ihrem Pegel sondern auch in ihrer Bandbreite und in der Mittelfrequenz in Relation zu den ipsilateralen Stimulusfrequenzen variierten, beeinflussten dabei die Generierung der otoakustischen Emis-sionen (DPOAE, engl: distortion product otoacoustic emissions) im ipsilateralen Ohr: akustische Stimulation der kontralateralen Cochlea bewirkte zuverlässig eine Änderung der DPOAE- Amplitude im kontralateralen Ohr. Vor allem eine Suppression des cochleären Verstärkers in Form von DPOAE-Pegelverminderungen wurde beobachtet. Die supprimieren-den Effekte erreichten trotz leiser bis moderater kontralateraler Rauschpegel (bis maximal 54 dB SPL) Werte von bis zu 14, 17.1 und 13.9 dB SPL (bei f2 = 20, 40 und 60 kHz und effek-tivstem kontralateralen Rauschstimulus) und waren damit deutlich größer als in vorangegang-enen Studien an anderen Spezies. Die DPOAE-Pegelverminderungen waren positiv mit dem x Pegel der kontralateralen akustischen Stimulation, ebenso wie seiner Bandbreite und der Mittelfrequenzen in Relation zu den ipsilateralen Stimulusfrequenzen korreliert. Es gab keinen absoluten Frequenzbereich, in dem die efferenten Effekte am größten gewesen wären. Vielmehr traten maximale Effekte immer durch etwas oberhalb der ipsilateralen Stimulusfre-quenzen gelegene kontralaterale Rauschstimuli auf. Die Effekte waren auch abhängig von der Bandbreite des kontralateralen Rauschstimulus und maximal bei einer relativen Bandbreite von 1.5 Oktaven. Die Verschiebung des efferenten Effekts hin zu hohen Frequenzen und die Bandbreitenabhängigkeit sind vereinbar mit den anatomischen Eigenschaften der Projektio-nen der medialen olivo-cochleären Efferenzen in der Säugetiercochlea. Kontralaterale akusti-sche Reizung bewirkte auch eine Verschiebung der Wachstumsfunktionen der 2f1-f2 -DPOAE in einen unsensitiven Bereich und außerdem eine Verformung der Wachstumsfunktion. Bei-des könnte durch Beeinträchtigung des cochleären Verstärkers verursacht sein. Eine Beteili-gung des Mittelohrmuskels an den Effekten kann nahezu ausgeschlossen werden und die beobachteten Effekte sind höchstwahrscheinlich dem olivo-cochleären System zuzuschreiben.
Funktionell ist denkbar, dass bei C. perspicillata das mediale olivo-cochleäre System im Kontext einer Frequenzverschärfung bei der cochleären Verstärkung der Basilarmembranbe-wegung aktiv wird. Aus diesem Grund wurden ipsilateral sogenannte DPOAE-Suppressions- Abstimmkurven gemessen, welche die mechanische Abstimmschärfe im Innenohr beschrei-ben. Während und nach kontralateraler Reizung kam es zu Veränderungen der Abstimmkur-ven. Signifikante Effekte konnten allerdings nicht festgestellt werden, da die Veränderungen der Suppressions-Abstimmkurven variabel und schlecht kategorisierbar war.
Die vorliegenden Ergebnisse unterstützen weit verbreitete Hypothesen zur Funktion der medialen olivo-cochleären Effernzen in Bezug auf mechanische Suppression, Verbesserung des cochleären Signal-Rauschverhältnisses und einer generellen frequenzspezifischen Wirkung.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
Der DNA-Translokator von T. thermophilus HB27, ebenso wie Typ-IV-Pili (T4P), sind Multiproteinkomplexe, die die Membranen und das Periplasma durchspannen. Sie sind ähnlich aufgebaut und enthalten identische Proteine. Der DNA-Translokator vermittelt Transport von DNA in das Zellinnere während der natürlichen Transformation. T4P sind filamentöse Zellorganellen, die an der inneren Membran assembliert werden und bis zu mehrere Mikrometer aus der Zelle hinausragen. Sie dienen der Anhaftung und Fortbewegung der Zellen auf Oberflächen.
Das Ziel dieser Arbeit war es, die Funktionen einzelner Komponenten der Komplexe und ihrer Proteindomänen bei der natürlichen Transformation, der T4P-Assemblierung und den durch T4P vermittelten Funktionen Adhäsion und „twitching motility“ aufzuklären.
Es sind neun Proteine bekannt, die eine duale Rolle als Komponenten des DNA-Translokators und des T4P spielen. Eines dieser Proteine ist die Assemblierungs-ATPase PilF, die Hexamere bildet. Diese cytoplasmatischen ATPase-Komplexe stellen die Energie für die Assemblierung der T4P bereit, ebenso wie für die Aufnahme freier DNA. Es ist jedoch bisher nicht geklärt, wie die durch PilF bereitgestellte Energie auf die anderen Komponenten des DNA-Translokators/T4P übertragen wird.
In dieser Arbeit konnte gezeigt werden, dass PilF an das cytoplasmatische Protein PilM des T4P und DNA-Translokators bindet. Zudem konnten Proteinkomplexe bestehend aus den Proteinen PilM, PilN und PilO heterolog produziert und aus Zellmembranen koisoliert werden. PilF interagierte mit diesen PilMNO-Komplexen via PilM. Diese Interaktionen führt zur Stimulierung der ATPase-Aktivität von PilF. Dies deutet an, dass PilM ein Kupplungsprotein ist, welches die Assemblierungs-ATPase PilF physisch und funktionell mit dem T4P/DNA-Translokator über den PilMNO-Komplex verbindet.
Neben PilF standen Präpiline von T. thermophilus im Fokus dieser Arbeit. Präpiline sind Vorläuferproteine, die zu Pilinen prozessiert werden und als solche dann die Untereinheiten der Pilus-Strukturen bilden.
Zusammenfassend konnten die Rollen einzelner Präpilin-ähnlicher Proteine bei T4P-assoziierten Funktionen geklärt werden und es konnten erste Analysen zur Charakterisierung des weitestgehend unbekannten Proteins ComZ durchgeführt werden. Desweiteren liefert diese Arbeit Hinweise darauf, dass die membranassoziierten Proteine PilM, PilN und PilO Kupplungsproteine sind, die PilF mit den periplasmatischen Komponenten des T4P/DNA-Translokators verbinden und dadurch die ATPase-Aktivität von PilF stimulieren. Die Rollen einzelner Proteindomänen von PilF und PilM bei der Protein-Protein-Interaktion und der Bindung von Liganden wurden aufgeklärt, sowie ihre Funktionen bei den T4P-vermittelten Funktionen und der natürlichen Transformation.
Die Psoriasis vulgaris (PsV) ist eine immunvermittelte entzündliche Erkrankung der Haut mit einer Prävalenzrate von 2-3 %, sodass etwa zwei Millionen Menschen in Deutschland an dieser erkrankt sind. Charakteristisch für die PsV sind veränderte Hautareale (Plaques), die im Rahmen der der entzündungsbedingten Durchblutungssteigerung gerötet erscheinen und eine silbrig-weiße Schuppung als Resultat einer vermehrten Abschilferung abgestorbener Keratinozyten aus der hyperproliferativen Epidermis aufweisen.
In dieser Arbeit wurde die Bedeutung des proinflammatorischen Zytokins granulocyte-macrophage colony-stimulating factor (GM-CSF) in der Pathogenese einer modellhaften Experimentalerkrankung der PsV untersucht. GM-CSF wird unter anderem von Interleukin (IL-) 17 produzierenden T-Helferzellen (Th17-Zellen) sezerniert, deren pathogenetische Bedeutung für die PsV gut etabliert ist. Die pathogene Wirkung von GM-CSF als Effektorzytokin konnte bereits in Tiermodellen anderer Th17-vermittelter Autoimmunerkrankungen wie der multiplen Sklerose und der rheumatoiden Arthritis (RA) gezeigt und die therapeutische Wirkung von GM-CSF-neutralisierenden Antikörpern in klinischen Studien an RA-Patienten demonstriert werden.
Das in dieser Arbeit angewendete murine Krankheitsmodell der Imiquimod (IMQ-) induzierten psoriasiformen Dermatitis wird durch die topische Anwendung des Medikaments Aldara®, dessen Wirkstoff IMQ ist, ausgelöst und führt zu einer Entzündung der Haut, die in vielen Aspekten dem humanen Krankheitsbild einer PsV ähnelt. Die pathogenetische Bedeutung von GM-CSF für die IMQ-induzierte psoriasiforme Dermatitis wurde über zwei unterschiedliche experimentelle Ansätze untersucht. So wurde GM-CSF in C57Bl/6J Mäusen mittels eines spezifischen, rekombinanten murinen Antikörpers in der Induktionsphase des Krankheitsmodells neutralisiert und zeitgleich der modifizierte Psoriasis Area Severity Index (PASI-)Score als Parameter des Schweregrades der klinischen Manifestationen ermittelt. Des Weiteren wurde am Versuchsende die Infiltration von Immunzellen in das entzündete Gewebeareal untersucht. Diese Ergebnisse wurden mit den Daten einer Behandlungsgruppe, nach Applikation eines IgG-Isotyp identischen Kontrollantikörpers verglichen. Dabei zeigte die Neutralisierung des Zytokins einen therapeutischen Effekt, der in einem signifikant niedrigeren PASI-Score, einer verringerten Tnfa mRNA Expression und einer reduzierten Infiltration mit neutrophilen Granulozyten resultierte.
Parallel zu diesen Versuchen wurde die Modellerkrankung auch in einer GM-CSF-defizienten C57Bl/6J Mauslinien (GM-CSF-/-) studiert. Die funktionelle Inaktivität des GM-CSF-kodierenden Csf2 Gens wurde 1994 durch gezielte genetische Manipulation etabliert. Unter den experimentellen Bedingungen war der Schweregrad der IMQ-induzierten psoriasiformen Dermatitis in GM-CSF-/- Mäusen nicht signifikant different von dem der wildtypischen (Wt) Mäuse und zeigte somit im Gegensatz zu den Ergebnissen aus den Versuchsreihen der Antikörper vermittelten Zytokinneutralisierung keinen offensichtlichen Hinweis auf eine GM-CSF-Abhängigkeit. In den GM-CSF-defizienten Tieren war jedoch nach IMQ-Induktion eine signifikant höhere Il6 und Il22 mRNA Expression am Entzündungsort im Vergleich zu den Wt Mäusen auffällig. Aufgrund dieser Ergebnisse wurde der Phänotyp der GM-CSF-defizienten Mäuse genauer untersucht und eine vermehrte Anzahl plasmazytoider dendritischen Zellen (pDCs) in Milz und Lymphknoten nachgewiesen. Diese Zellen werden im Rahmen ihrer Differenzierung aus Vorläuferzellen durch GM-CSF suppressiv reguliert und sind sowohl in die Entwicklung der PsV im Menschen als auch die Pathogenese der IMQ-induzierten psoriasiformen Dermatitis involviert. Aufgrund des in den sekundären lymphatischen Organen GM-CSF-defizienter Mäuse expandierten pDC-Kompartiments wurde die Beteiligung dieser Zellen in der Initiationsphase des Modells analysiert. Im Vergleich mit GM-CSF-suffizienten C57Bl/6J Mäusen weisen die Tiere der GM-CSF-defizienten Mauslinie zu diesen Zeitpunkten eine verstärkte Infiltration von pDCs in die Haut auf. Für pDCs ist bekannt, dass sie über die Produktion von IL-6 und TNF die Effektorzelldifferenzierung aktivierter, naiver T-Lymphozyten in Richtung Th22-Zellen polarisieren können. Dieser Mechanismus liefert ein hypothetisches Konzept, das die Ergebnisse zur gesteigerten IL-6-Produktion und Differenzierung IL-22-produzierender T-Zellen in IMQ-behandelten GM-CSF-/- Mäusen im Kontext der nachweisbaren Expansion von pDCs, erklären könnte. Dieser in den GM-CSF-/- Mäusen nachweisbare alternative Pathogenesemechanismus, ist offenbar geeignet die proinflammatorische Wirkung des genetisch fehlenden Zytokins zu kompensieren, aber hinsichtlich seiner Etablierung über ein verändertes pDC-Kompartiment von Dauer und Ausmaß der GM-CSF-Defizienz abhängig. So erklärt sich, warum die zeitlich limitierte Antikörper vermittelte GM-CSF-Neutralisierung in GM-CSF-suffizienten-Mäusen zu keiner pDC-Expansion und Steigerung von IL-6 und IL-22 Expression nach IMQ-Induktion führt.
Die GM-CSF-Neutralisierung durch einen rekombinanten murinen Antikörper reduziert deutlich die Krankheitsschwere der IMQ-induzierten psoriasiformen Dermatitis und belegt damit das therapeutische Potenzial dieses Therapieansatzes für die Humanerkrankung der PsV. Die unter angeborener GM-CSF-Defizienz in den Studien darüber hinaus aufgedeckten Veränderungen des pDC-Kompartiments sind von potenzieller Relevanz für zukünftige therapeutische Anwendungen dieses Prinzips, da unter einer dauerhaften GM-CSF-Neutralisierung mit therapeutischen Antikörpern ein Monitoring dieser Zellpopulation empfehlenswert erscheint z.B. über veränderte Interferonsignaturen durch pDCs, um mögliche Wirkverluste, aber auch unerwünschte Effekte zu erkennen.
In dieser Arbeit wurde der Hefepilz Xanthophyllomyces dendrorhous als vielseitige biotechnologische Plattform für die Produktion von Carotinoiden verwendet. Durch genetische Modifikationen der Carotinoidbiosynthese wurde ein Astaxanthin-Hochproduzent zur Akkumulation des farblosen Phytoens, das die menschliche Haut vor der schädlichen Wirkung der UV-Strahlung schützt und des gelben Zeaxanthins, das zur Förderung und Erhalt der Sehfähigkeit beiträgt, befähigt. Zur Generierung eines Phytoen-Hochproduzenten wurde das Gen crtI (Phytoen-Desaturase) inaktiviert und der Phytoengehalt durch Überexpression der Gene HMGR, crtE und crtYB gesteigert. Die Generierung eines Zeaxanthin-Hochproduzenten beinhaltete die Inaktivierung des Gens asy (Astaxanthin-Synthase) und die heterologe Expression einer bakteriellen ß-Carotin-Hydroxylase CrtZoXd.
Die Inaktivierung der Gene erfolgte mit spezifischen Knock-Out-Konstrukten, die mittels homologer Rekombination in crtI oder asy integrierten. Nachdem die Transgene auf Vektoren mit verschiedenen Antibiotikaresistenzen kloniert wurden, wurde die Überexpression durch genomische Integration in die ribosomale DNA erreicht. Anschließend wurde die Carotinoidzusammensetzung der Zellextrakte durch Hochleistungsflüssigkeitschromatographie an einer C18-Trennsäule oder durch Dünnschichtchromatographie bestimmt. Der Knock-Out-Nachweis erfolgte mittels Polymerase-Kettenreaktion und Amplifikation der Genloci, während die Anzahl integrierter Carotinoidgene durch quantitative Real-Time-PCR bestimmt wurde. Die Kultivierungen von X. dendrorhous wurden sowohl in Schikanekolben als auch in einem 2L-Bioreaktor durchgeführt.
Im Zuge der genetischen Modifikationen konnte der Ploidiegrad des Wildtyps bestimmt werden, der bis dahin unbekannt war. Durch das Auftreten von instabilen heterozygoten Stämmen und deren Überführung zu stabilen Homozygoten wurde die Existenz eines diploiden Genoms nachgewiesen. Um die für die biotechnologische Anwendung notwendige Stabilität der Carotinoidbiosyntheseleistung zu erreichen, wurden zwei Strategien entwickelt. Hierbei erfolgte die Stabilisierung der Stämme als Folge mitotischer Rekombination nach Subkultivierung und anschließender Farbselektion oder durch Induktion des sexuellen Zyklus und Sporulation.
Der crtI-Knock-Out führte zur Akkumulation von 3,6 mg/g dw Phytoen. Anschließend wurde die Limitierung der Phytoensynthese durch crtYB-Überexpression aufgehoben und die Versorgung der Carotinoidbiosynthese mit Vorläufermolekülen durch HMGR- und crtE-Überexpression erhöht. Im Bioreaktor wurde durch die Anwendung eines dreistufigen Fed-Batch-Prozesses, der eine effiziente Glucoseverwertung sicherstellte, mit 10,4 mg/g dw die höchste bis dato publizierte zelluläre Phytoenkonzentration im stabilisierten Hochproduzenten erreicht.
Der asy-Knock-Out führte zur Akkumulation von 4,5 mg/g dw ß-Carotin, das anschließend durch heterologe Expression der codon-optimierten ß-3,3-ß-Hydroxylase crtZoXd im Hochproduzenten zu 3,5 mg/g dw Zeaxanthin umgesetzt wurde. Zur Optimierung des Vorgehens wurden Knock-In-Konstrukte entwickelt, mit denen beide Schritte (Knock-Out und Integration von Carotinoidgenen) in nur einem molekular-biologischen Schritt durchgeführt und 94 % des in einem Wildtypstamm vorhanden ß-Carotins zu Zeaxanthin umgesetzt wurden. Die Optimierung der Wachstumsbedingungen bei der Bioreaktor-Kultivierung des stabilisierten Zeaxanthinproduzenten führte mit 10,8 mg/L zu einem 5-fach höheren Zeaxanthingehalt im Vergleich zur Schikane-Kultivierung.
Durch den Einsatz der Pentosen Arabinose und Xylose als alternative Kohlenstoffquellen wurde der Carotinoidgehalt der Phytoen- und Zeaxanthin-Hochproduzenten um 70 bzw. 92 % im Vergleich zur Glucose-Kultivierung gesteigert, wobei die Gründe für diesen Effekt in einer stärkeren Kohlenstoffverwertung und der Hemmwirkung von Glucose vermutet wurden. Aus verschiedenen pflanzlichen Abfallstoffen kann Xylose durch Hydrolyse freigesetzt werden, deren Nutzung zum Aufbau einer nachhaltigen und kostengünstigen biotechnologischen Carotinoidproduktion beitragen kann.
Darüber hinaus wurden multioxigenierte Zeaxanthinderivate, von denen eine positive Wirkung auf die menschliche Gesundheit vermutet wird, durch kombinatorische Biosynthese erhalten. Durch die schrittweise Integration der Gene crtZoXd, crtG (ß-2,2-Hydroxylase) und bkt (ß-4,4-Ketolase) in eine ß-Carotinmutante wurde die Biosynthese von Zeaxanthin, Nostoxanthin und schließlich von 4-Keto-Nostoxanthin und 4,4-Diketo-Nostoxanthin erreicht. Anschließend erfolgte die chemische Reduktion zu den neuartigen Carotinoiden 4-Hydroxy-Nostoxanthin und 4,4-Dihydroxy-Nostoxanthin und der zweifelsfreie Nachweis aller vier Carotinoide anhand der mittels Massenspektrometrie bestimmten Molekülmassen und Fragmentierungsmuster.
Die Analyse früher Entwicklungsstadien von Säugetierembryonen und daraus gewonnener Stammzelllinien kann entscheidende Erkenntnisse im Bereich der Reproduktionsbiologie und der regenerativen Medizin hervorbringen. Dabei spielt die Maus, als geeignetes Modellsystem für die Übertragbarkeit auf den Menschen eine wichtige Rolle, in erster Linie weil die Blastozysten der Maus verglichen mit menschliche Blastozysten eine morphologische Ähnlichkeit aufweisen. Humane embryonale Stammzelllinien haben großes Potential für die Anwendung in der regenerativen Medizin und vergleichend dazu wurde Gen-Targeting in embryonalen Stammzellen verwendet, um tausende neuer Mausstämme zu generieren. Die Gewinnung embryonaler Stammzellen erfolgt im Blastozystenstadium, diese können dann nach Injektion in eine andere Blastozyste zur Entwicklung aller Gewebearten, einschließlich der Keimbahngewebe, beitragen (Martin, 1981; Evans and Kaufman 1981).
Ursache einer Fehlgeburt können vor allem Defekte in der Entwicklung des Trophoblasten und des primitive Entoderms (PrE) sein, dabei sind ca. 5 % der Paare betroffen die versuchen ein Kind zu bekommen (Stephenson and Kutteh, 2007). Eine Untersuchung dieser Zelllinien im Mausmodell könnte weitere Erkenntnisse für die Gründe einer Fehlentwicklung liefern. Trophoblasten Stammzelllinien können aus den Blastozysten der Maus und dem extraembryonalen Ektoderm von bereits implantieren Embryonen gewonnen werden (Tanaka et al., 1998). Diese Zelllinien geben Aufschluss über die Entwicklung des Trophoblasten, fördern die Entwicklung der Plazenta und sind gleichzeitig ein gutes Modellsystem um die Implantation des Embryos im Uterus näher zu untersuchen. Zellen des primitive Entoderms (PrE) beeinflussen das im Dottersack vorhandene extraembryonale Entoderm, welches dort als “frühe Plazenta” fungiert und für die Versorgung des Embryos mit Nährstoffen zuständig ist (Cross et al., 1994). Des Weiteren besitzt das Entoderm einen induktiven Einfluss auf die Bildung von anterioren Strukturen und die Bildung von Endothelzellen sowie Blutinseln (Byrd et al., 2002).
Extraembryonale Endodermstammzellen (XEN Zellen) können aus Blastozysten gewonnen und in embryonale Stammzellen (ES-Zellen) umgewandelt werden (Fujikura et al., 2002; Kunath et al., 2005). Es war jedoch nicht bekannt, ob XEN-Zellen auch aus Postimplantations-Embryonen gewonnen werden können. XEN-Zellen tragen in vivo zur Entwicklung des Darmendoderms bei (Kwon et al., 2008; Viotti et al., 2014) und könnten als alternative, selbsterneuernde Quelle für extraembryonale Endoderm-abgeleitete Zellen dienen, die zur Herstellung von Geweben für die regenerative Medizin verwendet werden könnten (Niakan et al., 2013).
In der Embryogenese der Maus zeigt sich an Tag E3.0 eine kompakte Morula die sich allmählich in das Trophektoderm (TE) differenziert, welches wiederum den Embryonalknoten (“innere Zellmasse”) umschließt (Johnson and Ziomek, 1981). Ein wichtiger Schritt im Rahmen der Entwicklung findet an Tag E3.5 statt, in diesem Zeitraum gehen aus dem Embryonalknoten der pluripotente Epiblast und das primitive Entoderm hervor. Im späten Blastozystenstadium an Tag E4.5 liegt das PrE als Zellschicht entlang der Oberfläche der Blastocoel-Höhle. Aus dem Epiblast entwickeln sich im weiteren Verlauf der Embryo, das Amnion und das extraembryonale Mesoderm des Dottersacks. Die Zellen des Trophektoderm führen zur Entwicklung der Plazenta. Das PrE differenziert sich im Zuge der Weiterentwicklung in das viszerale Entoderm (VE) und das parietale Entoderm (PE) des Dottersacks (Chazaud et al., 2006; Gardner and Rossant, 1979; Plusa et al., 2008). VE umgibt den Epiblast und extraembryonisches Ektoderm (ExE). PE-Zellen wandern entlang der inneren Oberfläche von TE und sezernieren zusammen mit Trophoblasten-Riesenzellen Basalmembranproteine, um die Reichert-Membran zu bilden (Hogan et al., 1980). Die Reichert-Membran besteht aus Basalmembranproteinen, einschließlich Kollagenen und Lamininen, die zwischen den parietalen Endoderm- und Trophoblastzellen liegen. Diese Membran wirkt als ein Filter, der dem Embryo den Zugang zu Nährstoffen ermöglicht, während er eine Barriere zu den Zellen der Mutter bildet (Gardner, 1983).
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Octanoic acid (C8 FA) is a medium-chain fatty acid which, in nature, mainly occurs in palm kernel oil and coconuts. It is used in various products including cleaning agents, cosmetics, pesticides and herbicides as well as in foods for preservation or flavoring. Furthermore, it is investigated for medical treatments, for instance, of high cholesterol levels. The cultivation of palm oil plants has surged in the last years to satisfy an increasing market demand. However, concerns about extensive monocultures, which often come along with deforestation of rainforest, have driven the search for more environmentally friendly production methods. A biotechnological production with microbial organisms presents an attractive, more sustainable alternative.
Traditionally, the yeast Saccharomyces cerevisiae has been utilized by mankind in bread, wine, and beer making. Based on comprehensive knowledge about its metabolism and genetics, it can nowadays be metabolically engineered to produce a plethora of compounds of industrial interest. To produce octanoic acid, the cytosolic fatty acid synthase (FAS) of S. cerevisiae was utilized and engineered. Naturally, the yeast produces mostly long-chain fatty acids with chain lengths of C16 and C18, and only trace amounts of medium-chain fatty acids, i.e. C8-C14 fatty acids. To generate an S. cerevisiae strain that produces primarily octanoic acid, a mutated version of the FAS was generated (Gajewski et al., 2017) and the resulting S. cerevisiae FASR1834K strain was utilized in this work as a starting strain.
The goal of this thesis was to develop and implement strategies to improve the production level of this strain. The current mode of quantification of octanoic acid includes labor-intensive, low-throughput sample preparation and measurement – a main obstacle in generating and screening for improved strain variants. To this end, a main objective of this thesis was the development of a biosensor. The biosensor was based on the pPDR12 promotor, which is regulated by the transcription factor War1. Coupling pPDR12 to GFP as the reporter gene on a multicopy plasmid allowed in vivo detection via fluorescence intensity. The developed biosensor enabled rapid and facile quantification of the short- and medium-chain fatty acids C6, C7 and C8 fatty acids (Baumann et al., 2018). This is the first biosensor that can quantify externally supplied octanoic acid as well as octanoic acid present in the culture supernatant of producer strains with a high linear and dynamic range. Its reliability was validated by correlation of the biosensor signal to the octanoic acid concentrations extracted from culture supernatants as determined by gas chromatography. The biosensor’s ability to detect octanoic acid in a linear range of 0.01-0.75 mM (≈1-110 mg/L), which is within the production range of the starting strain, and a response of up to 10-fold increase in fluorescence after activation was demonstrated.
A high-throughput FACS (fluorescence-activated cell sorting) screening of an octanoic acid producer strain library was performed with the biosensor to detect improved strain variants (Baumann et al., 2020a). For this purpose, the biosensor was genomically integrated into an octanoic acid producer strain, resulting in drastically reduced single cell noise. The additional knockout of FAA2 successfully prevented medium-chain fatty acid degradation. A high-throughput screening protocol was designed to include iterative enrichment rounds which decreased false positives. The functionality of the biosensor on single cell level was validated by adding octanoic acid in the range of 0-80 mg/L and subsequent flow cytometric analysis. The biosensor-assisted FACS screening of a plasmid overexpression library of the yeast genome led to the detection of two genetic targets, FSH2 and KCS1, that in combined overexpression enhanced octanoic acid titers by 55 % compared to the parental strain. This was the first report of an effect of FSH2 and KCS1 on fatty acid titers. The presented method can also be utilized to screen other genetic libraries and is a means to facilitate future engineering efforts.
In growth tests, the previously reported toxicity of octanoic acid on S. cerevisiae was confirmed. Different strategies were harnessed to create more robust strains. An adaptive laboratory evolution (ALE) experiment was conducted and several rational targets including transporter- (PDR12, TPO1) and transcription factor-encoding genes (PDR1, PDR3, WAR1) as well as the mutated acetyl-CoA carboxylase encoding gene ACC1S1157A were overexpressed or knocked out in producer or non-producer strains, respectively. Despite contrary previous reports for other strain backgrounds, an enhanced robustness was not observable. Suspecting that the utilized laboratory strains have a natively low tolerance level, four industrial S. cerevisiae strains were evaluated in growth assays with octanoic acid and inherently more robust strains were detected, which are suitable future production hosts.
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Biotechnological processes offer better production conditions for a wide variety of goods of industrial interest. The production of aromatic compounds, for example, involves molecules of great value for cosmetic, plastic, agrochemical and pharmaceutic industries. However, the yield of such processes frequently prevents a proper implementtation that would allow the replacement of traditional production processes.
Numerous rational engineering approaches have been attempted to enhance metabolic pathways associated with desired products. Unfortunately, genetic modifications and heterologous pathway expression often lead to a higher metabolic burden on the producing organisms, ultimately leading to reduced production levels and fitness.
This project utilised adaptive laboratory evolution to better understand the development of synthetic cooperative consortia, using S. cerevisiae as a model organism. Specifically, a synthetic cooperative consortium was developed around the exchange of lysine and tyrosine, which was subjected to adaptive laboratory evolution aiming to induce mutations that would improve the system’s fitness either by enhanced production or upgraded stress resistance. Consequently, the mutant strains isolated after the evolution rounds were sequenced to identify relevant variations that could be related to the growth and production phenotypes observed.
The insights derived from this project are expected to contribute to further developing synthetic cooperative consortia with utilitarian purposes.
Derzeit breiten sich gebietsfremde Stechmücken (Diptera: Culicidae) aufgrund von Globalisierung und Klimawandel auf der ganzen Welt aus und bilden neue, stabile Populationen. Wegen ihrer hämatophagen Ernährungsweise sind sie Überträger von Pathogenen, die teilweise schwere bis tödliche Krankheiten beim Menschen, seinen Haustieren oder auch Wildtieren auslösen können. Mit den Stechmücken treten daher auch Infektionskrankheiten vermehrt in Gebieten auf, in denen sie vorher nicht vorkamen oder als bereits ausgerottet galten. Da die meisten im Menschen wirksamen Pathogene nicht durch Impfungen kontrolliert werden können, bleibt als eine der wenigen Möglichkeit der Krankheitsprävention die Dezimierung der Stechmückenpopulation. Daher sind Stechmücken momentan im Fokus von biologischer und epidemiologischer Forschung. Diese hat zum Ziel epidemische Krankheitsausbrüche vektorübertragener Krankheiten in der menschlichen Population zu verhindern. Eine Verringerung der lokalen Stechmückenpopulation bis hin zum Aussterben kann durch die Verwendung von Insektiziden, die Vernichtung von Bruthabitaten oder anderen Kontrollmaßnahmen erreicht werden. Jedoch sind diese Maßnahmen unterschiedlich effektiv, haben zum Teil unerwünsch-te ökologische und gesundheitsschädigende Folgen und sind unterschiedlich aufwendig und kostenintensiv in der Anwendung. Für die Entwicklung eines integrierten, effektiven, zielgerichteten und kostengünstigen Vektormanagements fehlen bislang jedoch die populationsbiologischen Grundlagen.
Ziel dieser Arbeit ist daher die Schaffung der Datengrundlage eines Integrierten Stechmückenmanagements für die Asiatische Buschmücke (Aedes japonicus japonicus THEOBALD 1901), die am weitesten verbreitete exotische Stechmücke in Deutschland. Schwerpunkte dafür wurden auf das zeitliche und räumliche Vorkommen, die Temperaturabhängigkeit des Lebenszyklus, sowie die Wirksamkeit von Kontrollmethoden gelegt.
Die Kenntnis der räumlichen Verbreitung und saisonalen Häufigkeit der Stechmücken ist notwendig, um befallene Standorte und Zeitpunkte des größten Populationszuwachses definieren zu können. Die Verbreitung und die Häufigkeit der endothermen Stechmücken sind stark von der Umgebungstemperatur abhängig, die beispielsweise deren Entwicklungsdauer und Sterblichkeit beeinflusst. Dabei entwickeln sich die verschiedenen Stadien (Ei, Larven, Puppe, Imago), die eine Stechmücke während ihres Lebens durchläuft, in Abhängigkeit von der Umgebungstemperatur unterschiedlich und haben jeweils andere Temperaturpräferenzen. Lebenszyklustabellen geben die Entwicklungsdauer und Mortalität pro Stadium in Abhängigkeit von der Temperatur an. Mit ihrer Hilfe können somit die räumlichen und zeitlichen Vorkommen und Häufigkeiten einer Stechmückenart berechnet werden. Dies ist insbesondere für Stechmücken in Gebieten mit jahreszeitlichen Temperaturveränderungen wichtig. Um Daten für eine solche Lebenszyklustabelle aufnehmen zu können, ist es notwendig Laborexperimente bei festgelegten Temperaturen durchzuführen. Die Voraussetzung dafür ist, dass die Stechmückenart im Labor optimale Bedingungen erhält, um ihren Lebenszyklus abschließen zu können. In dieser Arbeit wurde daher ein Laborprotokoll entwickelt, mithilfe dessen der Lebenszyklus der Asiatischen Buschmücke im Labor untersucht werden kann. Dazu wurden systematisch die Fütterung, die innerartliche Konkurrenz und das Wasservolumen des Brutge-fäßes für die aquatischen Stadien erprobt. Auf Basis dieses Protokolls wurden anschließend die Temperatureinflüsse auf die Entwicklung aller Stadien aufgenommen. Diese Daten dienten der Parametrisierung eines populationsdynamischen Modells. Dieses wurde verwendet, um Standorte mehrjähriger Populationen zu definieren, saisonale Häufigkeiten für Deutschland zu berechnen, durch Temperaturveränderungen hervorgerufene zukünftige Verbreitungsgebiete vorherzusagen, sowie Effekte von Kontrollmaßnahmen auf die Häufigkeit der Asiatischen Buschmücke zu modellieren.
Um eine dauerhafte Kontrolle der Stechmückenvektoren zu gewährleisten, ist weiterhin die permanente Neuentwicklung von wirksamen Kontrollmethoden notwendig. Dazu gehört die präventive Vermeidung von Bruthabitaten der aquatischen Stadien von Stechmücken. Die exotischen Stechmücken, die in Deutschland etabliert sind, gehören mehrheitlich der Gattung Aedes an und sind sogenannte Gefäßbrüter. Ihre bevorzugten Bruthabitate sind kleine Was-seransammlungen wie sie in Baumhöhlen, Gesteinsauswaschungen, Gießkannen, Regentonnen und Blumenuntersetzern vorkommen. In dieser Arbeit wurde untersucht, welche Farben und Volumina von Plastikbechern die Asiatische Buschmücke zur Eiablage bevorzugt, um präferierte Bruthabitate gezielt zu identifizieren und verringern zu können. Auch die Bereitstellung von Insektiziden wird durch in Stechmücken auftretende Insektizidresistenzen erschwert. Insektizide sollen dabei umweltfreundlich, spezifisch für den Zielorganismus und nicht gesundheitsschädlich für den Menschen sein. Weiterhin sind eine gute Anwendbarkeit, geringe Kosten und eine hohe Effizienz wünschenswert. Eine Quelle für potentielle Insektizide sind pflanzliche Stoffe, zum Beispiel ätherische Öle. Diese sind leicht erhältlich, natürlichen Ursprungs und wirksame Vergrämungsmittel gegen stechbereite Stechmückenweibchen. In dieser Arbeit wurde nach einer Literaturrecherche Nelkenöl ausgewählt und als Insektizid gegen Larven der Asiatischen Buschmücke getestet. Dafür wurden die akute toxische Wirkung von Nelkenöl bei drei Temperaturen untersucht und zusätzlich die Wirkung von Nelkenöl auf die Eiablage im Freiland. Nelkenöl zeigte dabei sowohl eine larvizide als auch eine eiablagehemmende Wirkung. Weiterhin wurde Kupfer in Form von kupferhaltigen Euromünzen als Larvizid untersucht. Kupfer ist ein wirksamer Stoff gegen die aquatischen Stadien von Stechmücken. Allerdings wurde der Stoff noch nicht in Form der einfach zu handhabenden, leicht erhältlichen Kupfermünzen getestet. Dazu wurden Vorexperimente durchgeführt, um herauszufinden, wieviel Kupferionen sich aus den Münzen lösen lassen. Anschließend wurde der akut toxische Effekt auf Larven der Asiatischen Buschmücke untersucht.
Ein Integriertes Stechmückenmanagement hat zum Ziel, die lokale Stechmückenpopulation zu kontrollieren, um so Stichen und daraus resultierender Krankheitsübertragung vorzubeugen. Dies erfolgt über die Aufklärung von Betroffenen, der Überwachung der Stechmückenpopulation, dem Testen auf Pathogenbefall und der direkten Kontrolle von Stechmücken. Diese Arbeit leistet einen Beitrag zu den Kenntnissen über die Laborhaltung einer exotischen Stechmückenart, zur Identifizierung von Bruthabitaten, zur zeitlichen und räumlichen Festlegung von Kontrollmaßnahmen und zur Anwendung von Larviziden und eines Vergrämungsmittels. Mit dieser Arbeit wurde die Grundlage eines faktenbasierten Integrativen Stechmückenmanagements für die Asiatische Buschmücke entwickelt, das eventuell auch auf weitere Aedes-Arten übertragbar ist, und als Handlungsempfehlung für politische Entscheidungstragende dienen kann.
Mitglieder der ubiquitär verbreiteten Cryptochrom-Photolyase-Familie sind Blaulicht-absorbierende Flavoproteine mit hoher Sequenzhomologie aber diversen Funktionen. Photolyasen katalysieren die Reparatur UV-Licht-induzierter DNA-Schäden. Cryptochrome (CRYs) wirken als lichtunabhängige Transkriptionsrepressoren innerhalb des Kern-Oszillators der circadianen Uhr oder als primäre Photorezeptoren zur Synchronisation dieser mit dem äußeren Tag-Nacht-Rhythmus und steuern durch Regulation der Genexpression Wachstum und Entwicklung. Gemeinsames Strukturmerkmal aller CPF-Vertreter ist die Photolyase- homologe Region (PHR), die das Chromophor Flavinadenindinukleotid (FAD) bindet, das lichtabhängig zwischen den Redoxformen oxidiert (FADox), semireduziert (FAD●- bzw. FADH●) und vollreduziert (FADH-) wechseln kann und damit die CRY-Konformation und -Aktivität beeinflusst. Unterscheidungsmerkmale sind die spezifische C-terminale Erweiterung (CTE) sowie die Komposition der FAD-Bindetasche, die unterschiedliche FAD-Redoxformen stabilisiert. Die Mechanismen der CRY-Photosignaltransduktion sind nicht völlig erforscht.
CryP ist eines von vier CRYs in der Diatomee Phaeodactylum tricornutum und gehört zur bislang nicht charakterisierten Gruppe pflanzenähnlicher CRYs. In vorhergehenden Untersuchungen wurde für CryP eine nukleare Lokalisation und damit verbunden eine blaulicht- sowie dunkelabhängige Regulation der Transkription unterschiedlichster Gene gezeigt. Zudem reguliert CryP das Proteinlevel photosynthetischer Lichtsammelkomplexe. CryP interagiert mit bisher nicht charakterisierten Proteinen aus dem Bereich DNA und Regulation sowie Ribosomen und Translation. Heterolog exprimiertes und isoliertes CryP stabilisiert das Neutralradikal FADH● und das Antennenchromophor Methenyltetrahydrofolat (MTHF).
In vorliegender Dissertation wurde die Bedeutung des FAD-Redoxzustands und der C-terminalen Proteindomäne für Strukturänderungen hinsichtlich der Oligomerisierung und Konformation sowie für das CryP-Interaktionsverhalten untersucht. Hierzu wurden rekombinante CryP-Varianten heterolog isoliert, die Mutationen in für die FAD-Reduzierbarkeit entscheidenden Aminosäuren oder eine Deletion der CTE tragen.
Die Analyse der CryP-Oligomerisierungsstufe und Konformation erfolgte mittels Ko-Präzipitation, nativen und zweidimensionalen PAGEs sowie partieller Proteolyse. Dabei wurde heterolog isoliertes CryP in seinen drei Redoxformen oxidiert (mit FADox), semireduziert (mit FADH●) und vollreduziert (mit FADH-) sowie das um die CTE-verkürzte CryP-PHR verglichen. Für CryP wurde eine redoxunabhängige, PHR-vermittelte Di- und Tetramerisierung über elektrostatische Wechselwirkung der Monomere beobachtet. Die CTE bindet spezifisch und redoxunabhängig an die PHR in einem Bereich um die FAD-Bindetasche. Dies schließt eine großräumige Konformationsänderung zwischen PHR und CTE infolge einer FAD-Photoreduktion wie für pflanzliche und viele tierische CRYs als Aktivierungsmechanismus für CryP aus.
Interaktionsstudien mittels zweidimensionaler PAGE gaben Aufschluss über unterschiedliche Bindeverhalten der beiden betrachteten Interaktionspartner an CryP. Sowohl BolA, ein potentieller redoxregulierter Transkriptionsfaktor, als auch ID42612 mit unbekannter Funktion interagieren mit CryP unabhängig von der FAD-Redoxform. Dabei bindet BolA an die CTE des CryP-Dimers und -Monomers, während ID42612 einen Komplex mit dem CryP-Dimer bildet.
Mittels in vitro Absorptions- und Fluoreszenzspektroskopie wurde die FAD-Redoxchemie von CryP und CryP-PHR verglichen. Die beiden Varianten unterscheiden sich in der FAD-Photoreduzierbarkeit und -Oxidationskinetik. Das Volllängenprotein CryP kann ohne externes Reduktionsmittel zum semireduzierten FADH● phototreduziert werden, das im Gegensatz zu bekannten CRYs über Tage im Dunkeln stabil gegen aerobe Oxidation ist. Eine Belichtung mit Reduktionsmittel führt zur Bildung des vollreduzierten FADH-, das innerhalb von Minuten zu FADH● rückoxidiert. Das um die CTE verkürzte CryP-PHR kann nur mit externem Reduktionsmittel zu FADH● photoreduziert werden, der vollreduzierte Zustand wird nie erreicht. Die Stabilisierung von FADH● gegen aerobe Oxidation im CryP-Holoprotein ist vergleichbar zur FAD-Redoxchemie von Photolyasen. Verglichen mit sonstigen charakterisierten CRYs ist die Wichtigkeit der CTE für eine effiziente FAD-Photoreduktion und FADH●-Stabilisierung eine CryP-spezifische Charakteristik.
Neben der CTE trägt die zu FAD-N5 proximal gelegene Position zur FADH●-Stabilisierung bei, wie Absorptionsmessungen an CryP_N417C zeigten. CryP weist mit Asparagin die gleiche Konservierung an dieser Position wie Photolyasen auf und unterscheidet sich damit ebenfalls von klassischen CRYs.
Analysen zur cryp-Transkription mittels qRT-PCR zeigten eine rhythmische Expression mit maximalen Transkriptmengen in der Nacht und eine rasche photoinduzierte Herunterregulation der Transkription...
In der vorliegenden Arbeit konnte gezeigt werden, dass bestimmte neuronale microRNAs im Rückenmark und in den Spinalganglien konstitutiv exprimiert und nach peripherer Entzündung mit Formalin oder Zymosan differenziell reguliert werden. Bei der SNI-induzierten Neuropathie konnte indessen keine signifikante Regulation der untersuchten microRNAs nachgewiesen werden. Aufgrund der Lokalisation in den Neuronen der Schmerz-verarbeitenden Laminae I und II des Dorsalhorns des Rückenmarks und angesichts der Regulation in entzündlich stimulierten Neuronen und Mikroglia wurde der Fokus der Arbeit auf die Untersuchung von microRNA-124a gelegt. Anhand von Expressionsanalysen konnte gezeigt werden, dass eine periphere entzündliche Stimulation mit Formalin oder Zymosan microRNA-124a im Rückenmark inhibiert, die Expression pro-inflammatorischer und pro-nozizeptiver Gene hiernach ermöglicht und ein vermehrtes Schmerzverhalten bewirkt. Die funktionelle Relevanz von microRNA-124a wurde in vivo mittels intravenöser Applikation von microRNA-124a-Modulatoren bei einem Modell für entzündliche Schmerzen, dem Formalin-Modell untersucht. Dabei führte die Hemmung von microRNA-124a zu einem verstärkten Schmerzverhalten, welches mit einer Hochregulation verschiedener Entzündungsmarker einherging. Die Überexpression von microRNA-124a dagegen antagonisierte die Hochregulation entzündlicher Mediatoren und führte zu einer Schmerzhemmung. Darüber hinaus konnte in der vorliegenden Arbeit der antinozizeptive Effekt von microRNA-124a mit der Regulation der Epigenetik-regulierenden Targets MeCP2, HDAC5 und MYST2 assoziiert werden und u.a. über die Hemmung des neuromodulierenden, pro-inflammatorischen Peptids BDNF verifiziert werden. Die spezielle Darreichung von microRNA-124a könnte demzufolge einen vielversprechenden Ansatz zur Therapie chronisch-entzündlicher Schmerzen liefern. Zukünftig werden weitere Studien notwendig sein um die eindeutige Funktion, die individuelle Wirkung sowie die therapeutische Relevanz von microRNA-124a zu analysieren. Darüber hinaus müssten Dosis-Wirkungs-Beziehungen und Nebenwirkungsprofile für microRNA-124a erstellt werden, um potenzielle Risiken, Chancen und Vorteile der microRNA-Modulation hinsichtlich einer humanen Schmerztherapie bewerten zu können.
In der vorliegenden Doktorarbeit zur Untersuchung der Rolle der Superoxid-Dismutasen in P. anserina lieferten die durchgeführten Analysen folgende Ergebnisse:
1)Sowohl in P. anserina als auch in S. cerevisiae wurde eine gemeinsame Regulation von SODs nachgewiesen: Stämme, die die mitochondriale MnSod (PaSod3 bzw. ScSod2) überexprimieren zeigen eine erhöhte Cu/ZnSOD-Aktivität (PaSOD1 bzw. ScSOD1).
2)Es konnte keine SOD-Aktivität für die putativen SODs Pa_1_10620, Pa_1_10630 und Pa_1_6300 detektiert werden. Für Pa_1_10620, dessen Überexpression unter Standardbedingungen zu einer Lebensverlängerung führt, wird eine Funktion als mitochondriales ribosomales Protein angenommen.
3)Der ∆PaSod3-Stamm weist keinen Unterschied im Phänotyp, der Wuchsrate und der Lebensspanne unter Standardbedingungen zum Wildtyp auf. Paraquat-Stress führt allerdings zu einer Kurzlebigkeit des ∆PaSod3-Stammes, wohingegen diese Mutante eine höhere Resistenz gegenüber Wasserstoffperoxid aufweist als der Wildtyp.
4)Transkriptomanalysen des Wildtyps und der ∆PaSod3-Mutante lassen vermuten, dass eine Hochregulation von Detoxifizierungs- und Energie-abhängigen Prozessen die durch den Verlust der mitochondrialen PaSOD3 vermuteten negativen Auswirkungen kompensieren.
5)PaSod3_OEx-Stämme weisen unter Standardbedingungen aufgrund der erhöhten intrazellulären Wasserstoffperoxid-Menge, bedingt durch die vermehrte Umsetzung von Superoxid, diverse negative Auswirkungen auf: Eine reduzierte Wuchsrate, verkürzte Lebensspanne, geringere Fertilität, stärkere Pigmentierung, vermehrt fragmentierte Mitochondrien, mehr unprozessierte mitochondriale Proteine und weniger Komplex IV der Atmungskette als der Wildtyp. Zusätzlich wird vermehrt über die alternative Oxidase geatmet, um die ROS-Generierung zu reduzieren.
6)Oxidativer Stress in Form von Paraquat führt in PaSod3_OEx-Stämmen zu einer weiteren Verkürzung der medianen Lebensspanne, während die maximale Lebensspanne von PaSod3_OEx3-Stämmen im Vergleich zum Wildtyp sogar verlängert ist. Wasserstoff-peroxid resultiert in stark verringerten medianen und maximalen Lebensspannen beider PaSod3-überexprimierenden Stämme.
7)Die Anzucht auf Medium mit zusätzlichem Mangan (80 µM MnSO4) kann die beobachteten Defekte der PaSod3_OEx-Stämme fast vollständig auf Wildtyp-Niveau revertieren: Die Wuchsrate, die Lebensspanne, der Phänotyp, die Mitochondrien-morphologie, die Prozessierung mitochondrialer Proteine und die Atmung entsprechen dem Wildtyp. Lediglich die Fertilität erreicht nicht das Wildtyp-Niveau. Diese positiven Effekte von Mangan werden erzielt, da die erhöhte Wasserstoffperoxid-Menge in PaSod3_OEx-Stämmen entsprechend ihrer Entstehung detoxifiziert wird, denn Mangan führt zu einer gesteigerten Transkription bzw. Aktivität von Katalasen und Peroxidasen sowie zu einer erhöhten Peroxiredoxin-Menge.
8)Die Anzucht des Wildtyps unter Wasserstoffperoxid-Stress resultiert in einer Lebens-spannenverkürzung. Diese kann durch Supplementation mit Mangan revertiert werden. Unter diesen Bedingungen weisen u. a. Peroxidasen eine erhöhte Aktivität auf.
Insgesamt ließen die gewonnenen Daten den Schluss zu, dass das Genom von P. anserina für drei aktive SODs kodiert. Ein Verlust der einzigen mitochondrial lokalisierten SOD kann durch die Induktion von Energie-abhängigen Prozessen sowie von Detoxifizierungsprozessen kompensiert werden. Ferner weisen die durchgeführten Studien darauf hin, dass PaSod3_OEx-Stämme als Modell für erhöhte intrazelluläre Wasserstoffperoxid-Mengen in P. anserina verwendet werden können. Darüber hinaus wurde ein Zusammenhang zwischen Mangan und dem Detoxifizierungs-netzwerk in P. anserina nachgewiesen. Dabei können zwei Mechanismen zur Reduktion der Wasserstoffperoxid-Mengen unterschieden werden: Bei Vorhandensein ausreichender Mengen Mangan kommt es zu einer stärkeren Detoxifizierung von Wasserstoffperoxid. Ist Mangan allerdings limitiert und die Detoxifizierung kann nicht gesteigert werden, wird eine Umstellung der Atmung eingeleitet, um die neu entstehende ROS-Menge zu minimieren.
Das maligne Gliom, auch Glioblastom multiforme (GBM) genannt, ist der häufigste und gleichzeitig auch bösartigste hirneigene Tumor und macht rund 2% aller Krebsneuerkrankungen aus. Die Weltgesundheitsorganisation (world health organisation, WHO) stuft das GBM als Grad IV Tumor ein, was es als hochmalignen Tumor auszeichnet der infiltrativ in das umliegende Hirnparenchym einwandert und mit den gegenwärtigen Behandlungsmethoden, bestehend aus Resektion des Tumors, Chemotherapie und Strahlentherapie nicht kuriert werden kann. Das aggressive Wachstum und die ausgeprägte Resistenz dieses astrozytären Tumors gegenüber den verfügbaren Therapien der Bestrahlung und Chemotherapie sind Hauptgründe für die schlechte Prognose für Patienten mit Glioblastomen, deren medianes Überleben immer noch unter der Zwei-Jahres-Grenze liegt. Daher ist es von Nöten neue therapeutische Strategien auf Grundlage der Chemotherapie zu entwickeln, die selektiv wichtige, deregulierte Signalwege der Krebszelle angreifen. Einer dieser Signalwege in Gliomen ist der Stat3-Signalweg (signal transducer and activator of transcription). Stat3, ein latenter zytoplasmatischer Transkriptionsfaktor liegt in Gliomen oftmals konstitutiv aktiv vor. Diese Deregulation des Signalweges führt zur dauerhaften Transkription proonkogener Zielgene die in transformierten Zellen zu Proliferation, Apoptoseresistenz, Neoangiogenese und Immunsupprimierung führen können. In der vorliegenden Arbeit wurde untersucht, inwiefern eine pharmakologische oder gentechnische Inhibierung von Stat3 molekulare und zelluläre Charakteristika von Gliomen beeinflusst. Dazu wurde für die in-vivo Versuche ein syngenes, murines Gliom-Transplantationsmodell verwendet dessen Pathologie der eines humanen Glioms gleicht und den Vorteil besitzt keine immunsupprimierten Tiere verwenden zu müssen. Die murinen Gliomzelllinien, gewonnen aus spontanen Gliomen von GFAP-v-Src überexprimierenden Mäusen, wurden vorher in-vitro und auch exvivo bezüglich ihres Verhaltens auf die pharmakologische oder gentechnische Inhibierung von Stat3 charakterisiert. Für die pharmakologische Inhibierung wurde Kurkumin gewählt, der biologische aktive Wirkstoff der Pflanze Curcuma longa. In der vorliegenden Arbeit konnte gezeigt werden, dass eine Behandlung mit Kurkumin konzentrationsabhängig die Phosphorylierung von Stat3 in drei murinen Gliomzelllinien hemmt. Des Weiteren zeigte sich, dass auch die Proliferation der untersuchten transformierten Zellen sowie ihre Fähigkeit zur Invasion und Migration konzentrationsabhängig durch den Einsatz von Kurkumin inhibiert werden konnte, ohne dabei allerdings die Proliferation von primären Astrozyten im gleichen Maße zu hemmen. Kurkumin induziert zusätzlich in den überaus aopotoseresistenten Gliomzellen einen G2/M Zellzyklusarrest. Diese beobachteten Effekte stehen im Zusammenhang mit der konzentrationsabhängigen transkriptionellen Beeinflussung Kurkumins der tumorpromotenden Stat3- Zielgene. Durch Einsatz einer Stat3-Mutante, Stat3C, die ohne Phosphorylierung konstitutiv aktiv in der Zelle vorliegt, konnte in der vorliegenden Arbeit gezeigt werden, dass Kurkumin seinen Einfluss auf die Invasion und Migration der murinen Gliomzelllinien auch über den Stat3-Signalweg vermittelt, zeigte sich, dass durch Einbringung dieser Mutante trotz Kurkuminbehandlung die Migrations- und Invasionsfähigkeit partiell retabliert werden konnte. Durch dietätische Gabe von Kurkumin konnte in tumortragenden Mäusen gezeigt werden, dass die invitro ermittelten Effekte an einem längeren Überleben jener Mäuse beteiligt waren, deren Futter das Kurkumin enthielt. Die Administration des Kurkumins wurde entsprechend einer für die Klinik bevorzugten Darreichugsform gewählt. Im zweiten Teil dieser Arbeit wurde Stat3 in den murinen Gliomzelllinien durch Transduktion mit shRNA gerichtet gegen die Stat3-mRNA stabil depletiert um im Folgenden untersuchen zu können, welche zellulären und molekularen Konsequenzen konstitutiv aktives Stat3 für die Gliomzellen hat. Es zeigte sich, dass der Wegfall von Stat3 das Migrations- und Invasionspotential signifikant verringerte und die Expression tumorfördernder Zielgene ebenfalls in den Stat3-defizienten Zellen auf Protein- und mRNA-Ebene signifikant reduziert war. Der Einfluss von Stat3 auf die Hif1α-Expression, ein Transkriptionsfaktor der die Anpassung der Gliomzellen an ein hypoxisches Milieu und damit verbunden auch Migration und Invasion induziert kann, macht deutlich, dass konstitutiv aktives Stat3 unter normoxischen sowie auch hypoxischen Bedingungen upstream entscheidender Transkriptionsfaktoren liegt und sich somit als Zielmolekül für eine therapeutische Intervention anbietet. Eine ex-vivo Applikation auf organotypischen Schnittkulturen zeigte, dass durch den Wegfall von Stat3 in den murinen Gliomzellen die Einzelzellinvasion unterbunden werden konnte was entscheidend für das klinisch hochrelevante Problem der Rezidive sein könnte. Transplantierte man nun Kontroll- und Stat3-defiziente Zellen orthotrop in die immunkompetenten Mäuse zeigte die Kaplan-Meier-Kurve, dass der Krankheitsbeginn so wie das mediane Überleben in den Mäusen mit Stat3-defizientem Tumor zeitlich deutlich nach hinten verschoben war. Neben den invitro und ex-vivo ermittelten Effekte des Stat3-Wegfalls ist anzunehmen, dass das verlängerte Überleben dieser Mäuse auch mit der fehlenden Immunsupprimierung der Stat3-defizienten Tumore zusammenhängt. Es zeigte sich, dass eine Intervention gegen Stat3, ob nun pharmakologisch oder gentechnisch, die malignen Charakteristika des Glioblastoms positiv beeinflussen kann. Stat3, bestätigt als onkogener Transkriptionsfaktor, stellt damit eine lohnenden Zielstruktur in Gliomen dar.
Even one century after Santiago Ramón y Cajal’s groundbreaking contribu- tions to neuroscience, one of the most fundamental questions in the field is still largely open, namely understanding how the shape of a dendrite is adapted to its specific biological function. A systematic investigation of this problem is challenging both technically and conceptually because neurons have diverse genetic, molecular, morphological, connectional and functional properties.
In the light of the preceding, dendritic arborisation (da) neurons of the Drosophila melanogaster larva PNS have proven to be an excellent model system for the study of such growth and patterning processes. Structure and function in these cell classes are intimately intertwined, as class type-specific dendritic arbour differentiation processes are required to satisfy a given phys- iological need. Also, there is a remarkable genetic toolkit that enables one to selectively and reproducibly label, image and manipulate each one of these sensory neuron classes. In this thesis, I address the aforementioned open problem by linking single-cell patterning, information processing and wiring optimisation in sensory da neurons to behaviour in Drosophila larva.
In particular, I study Class I ventral peripherical dendritic arborisation (c1vpda) neurons. These are a class of proprioceptive neurons that relay information on the position of the larva’s body back to the CNS during crawling behaviour to assure proper locomotion. Their stereotypical comb- like shaped dendritic branches spread along the body-wall, and they get noticeably deformed during crawling behaviour. The bending of the den- dritic branches is hypothesised to be a possible mechanism to transduce the mechanosensory inputs arising from cuticle folding. Interestingly, c1vpda neurons do not necessarily satisfy optimal wiring constraints since they are required to pattern into a specific shape to fulfil their function. Therefore, I considered the da system to study how the specific functional requirements may be combined with optimal wiring constraints during development.
Although the molecular machinery of dendrite patterning in c1vpda neurons is well studied, the precise elaboration of the comb-like shaped dendrites of these cells remains elusive. Moreover, even though a lot of work has been put into the description and quantification of growth processes of the nervous system, there are still few solid and standardised models of arbour staging and patterning. Importantly, the defining parameters that determine the dendrite elaboration program that in turn is responsible for creating the final arbour morphology are still unknown. As a result, unraveling possible universal stages of dendrite elaboration shared between different model systems and cell types is challenging.
Thus, in order to understand the development of the fine regulation of branch outgrowth that leads to the observed terminal arbour morphology in the mature cell, I collected in vivo, long-term, non-invasive high temporal res- olution time-lapse recordings of dendritic trees during the differentiation process in the embryo and its maturation phase in the larva. For further analysis, I developed new algorithms that quantified the structural changes in dendrite morphology in the time-lapse videos. My approach provides a framework to analyse such developmental data, or any dataset comprising continuous morphological dynamical processes in an unbiased way. Using these newly developed methods, I examined the development of a sample of c1vpda cells and identified five stages of differentiation in these data: initial stem polarization, extension, pruning, stabilization, and isometric stretching during larval stages.
The beginning of the growth process is marked by the polarisation of the main stem. Subsequently, during the extension phase, branches emerge interstitially from the existing main stem. Later, higher-order branches sprout from pre-existing lateral branches, increasing arbour complexity. This is followed by a pruning stage where developmental intermediate dendritic branches are removed. This step leads to a spatial rearrangement of the dendritic tree. The end of the pruning step is followed by a stabilisation period where arbour morphology remains virtually unaltered in the embryo. After hatching, c1vpda dendrites experience an isometric scaling, with their branching complexity and pattern being invariant across all larval stages.
After dissecting the c1vpda dendrites spatiotemporal differentiation process, I established a link between dendritic shape and behaviour. I measured intra- cellular Ca++ activity in the dendrite branches of l1 larvae during forward locomotion, while simultaneously recording branch deformation using a dual genetic line. I reported that post-embryonic c1vpda dendrites Ca++ responses increased in freely crawling larvae. Furthermore, I showed strong correlations between Ca++ signal and deformation of the comb-like dendritic ranches during body-wall contractions.
Then, using a geometrical model, I provided evidence that the pruning stage could reorganise the dendrite morphology to maximise mechanosensory re- sponses during body wall contraction. I showed that the angle orientation of each side branch correlates with the bending curvature and thus with the me- chanical displacement of the cell membrane during locomotion. During the pruning phase, I observed a preferential reduction of less efficient branches with low bending curvature, influencing the mechanisms of dendritic sig- nal integration of c1vpda sensory neurons. I proceeded to quantify branch dynamics at single tip resolution during pruning, providing evidence that a simple random pruning mechanism is sufficient to remodel the tree structure compatible with the observed way.
I used these time-lapse data to constrain a new computational noisy growth model with random pruning based on optimal wiring principles. This model is able to generate highly realistic synthetic c1vpda morphologies. The model furthermore requires few parameters to generate highly accurate temporal development trajectories and morphologies at single-cell level. Utilising this data and model enabled me to investigate upon the hypothesis that a noisy dendrite growth and random pruning mechanism synergise to achieve den- dritic trees efficient in terms of both wiring and function. My findings show how single neurons can create functionally specialised dendrites while min- imising wiring costs, elucidating how general principles of self-organisation may be involved in the generation of these structures.
The main goal of this work is to contribute to the existing knowledge of soil micro-fungi in Panama and Germany. Studies about soil degradation and its influents in the soil fungi diversity have not been investigated as extensively in these countries. This is an extensive and challenging topic to examine since there is an immense phenotypic and genetic diversity in the soil fungal community and relating this community together with factors of soil degradation is an extensive task. For this reason, the present thesis studies the species identified in the study areas, in other words, the soil fungal diversity in relation to environmental factors in the Taunus Mountain range in Frankfurt, Germany, and in the Majagua valley in Chiriquí, Panama. Two complementary objectives were achieved, the first was the development of a theoretical irrigation model for degraded soils. The second was the development of a mobile application to facilitate laboratory work in the cultivation of soil micro-fungi.
The design of the methodology was based on identifying the species and relating the diversity found to soil factors. Soil samples were taken in both countries: the Taunus Mountain range was sampled eight times from January to November 2012 and the Majagua valley was sampled on three occasions between February and July 2012. In both studies, the areas included three different vegetation types (forest, grassland, and bare soil). Samples were separated for two purposes: the assessment of fungal diversity by molecular and morphological methods and soil characterization.
Soil samples used in the methodology of pyrosequencing were related to global climatic factors. Morphological identification was achieved with identification keys. Micro-fungi were cultivated in different media until obtaining pure cultures. Molecular identification was performed by getting the DNA sequences using the ITS1 and ITS4 primers and comparing the sequences with other reference sequences from GenBank. This was done considering the BLAST algorithm, which considered sequences that matched 98 % or more of maximum identity as reliable identifications.
Soil characterization was carried out to measure the soil's Physico-chemical properties; those abiotic factors were compaction, temperature, pH, moisture, and soil composition.
Species richness was calculated in each study area with the estimators Chao, Jackknife, and Bootstrap. Furthermore, the species accumulation curves were performed to observe the species discovery rate and estimate sample completeness. Estimate linear regression models correlated the influence between the soil factors (temperature, moisture, pH, soil compaction, and soil composition) and the species richness. In the same way, an analysis of ecological distance was undertaken based on the similarity in the species composition, compared across samples, and correlated with soil factors, using non-metric multidimensional scaling (NMDs).
Study of abundance showed differences between the bare soil abundances and the forest abundances in Germany and Panama; the grasslands in both countries work as transitional areas in the fungi abundance. The key stone species in Germany were Penicillium daleae, and Pochonia bulbillosa, whereas in Panama were Purpureocillium lilacinum and Trichoderma harzianum. Based on Pareto analysis, a theoretical irrigation model was developed to counteract the degradation effects on the abundance of micro-fungi in the soil.
Applications for mobile devices dealing with the cultivation of soil micro fungi were sought. Due to the small number of existing applications, a new App called Soil-Fungi-Cultures (SFC) was developed to facilitate data collection of cultivated soil micro fungi. App Inventor was the program used to design, program, test, and publish the application developed. The developed application was compared with other applications used in identifying bacteria cultures. The results showed that the new application needed more time to capture the records because it saves more information, the navigation flow was acceptable, the number of clicks was high, but it is due to the usefulness in data capture, and finally, the users rated it as a good application with an eight out of ten rating.
Pyrosequencing resulted in 204 Operational Taxonomic Units (OTUs) considering the two study areas (the Taunus Mountain range and the Majagua valley). The Pyrosequencing database was used to contribute to the most important study of fungal diversity globally based on OTUs, which surpasses any study of molecular and taxonomic diversity previously conducted. The principal result in this study was that the climatic factor is the best predictor of fungal richness and community composition on a global scale. However, the part of the research that focused on the local scale, that is to say, on the correlation patterns between the distribution of fungal species and abiotic factors, showed that the soil properties and degradation levels were not associated with fungal richness, diversity or soil composition in the study areas in Germany or Panama. The above confirms that there are exceptions to the way relationships between soil factors with fungal diversity are established at the local level.
In the case of soil samples used for morphological identification, 71 fungal species were obtained, 47 from Germany, and 32 from Panama.
Many metabolic pathways of eukaryotes are carried out in form of interconnected pathways, which take place in organelles. The organelle membrane separates the reaction compartments from each other, making it a key feature of organelle existence in the cell. To maintain cellular homeostasis, organelle positioning in and transport through the cell as well as organelle interaction are important for the organisms. In plants, organellar movement of peroxisomes, Golgi stacks and mitochondria was shown to be mediated by the actin-myosin machinery. The molecular mechanisms are not elucidated, but working models comprise classical movement mechanisms of motor proteins pulling their cargo on cytoskeletal filaments. In contrast, many mechanisms of chloroplasts movement, which are regulated by blue and red light, are deciphered but follow a different molecular mechanism. Plastidal relatives of the chloroplast have long been disregarded by scientific research but carry out important metabolic reactions to maintain cellular homeostasis. The cellular transport and movement mechanisms of root plastids have not been described in detail until now. Additionally, all plastid subspecies can form tubular structures, called stromules. Those are thought to be involved in the organelle communication and metabolite exchange. Since they are very mobile structures, they influence the organellar dynamic of plastids. This work aimed for an in-detail description of the cellular movements of root plastids in the plant Arabidopsis thaliana to elucidate underlying mechanisms of their movement. Additionally, the dynamics of root plastid stromules were investigated, led by the questions, if and how stromules are involved in the mediation of plastidal movement and their overall dynamics. Plastidal movement in Arabidopsis thaliana was captured using light sheet-based fluorescence microscopy. 4D image data was automatically analyzed using the program Arivis Vision 4D with subsequent manual correction. Additionally to the 4D approach, a manual 3D analysis of plastid and stromule dynamics was performed. The results of the semiautomated analysis displayed heterologous distribution of the plastidal movement. Using a combination of the vector length of each motion event and the angle in relation to previous motion vectors, the proportions of different movement patterns were determined. Main fractions of the data showed undirected motion of plastids, whereas small proportions displayed directed movement with speed up to 8.5 µm/sec. Directed motion was shown to be carried out on defined routes in the cell. Salt stress did not affect plastidal motion, whereas drought stress lead to its reduction. Sucrose depletion led to a drastic decrease of plastidal movement. Additionally, stromule dynamics were investigated using the acquired image data. Stromules were observed in high frequency mainly at stationary plastids giving them the opportunity of dynamic interaction in their cellular surrounding. Stromules reached lengths of up to 60 µm. Additionally, they displayed a variety of movement patterns that contributed greatly to the overall plastid dynamics. Stromule related motion events were captured reaching up to 3.2 µm/sec. Similar to determined plastid dynamics, stromule motions were reduced during drought stress and sucrose depletion, but also were negatively influenced by salt stress. Those results strongly favor an actin-myosin mediated movement machinery mediating the plastidal and stromule movement. This stands in contrast to previous results describing the movement mechanisms of light induced chloroplast movement.
In an additional approach, the molecular mechanisms underlying stromule formation were analyzed. Previous results describe that stromule formation can be induced at isolated chloroplasts of the plant Nicotiana benthamiana by mixing it with concentrated cell extract. During this work, a variation of the described assay was established using the plant Pisum sativum. It was shown that an unknown protein factor presumably undergoing protein-lipid interaction is responsible for in vitro stromule formation. Using a combination of sucrose gradient centrifugation and anion exchange chromatography, the desired factor could be enriched, while the majority of unwanted proteins could be reduced drastically. A following LC-MS analysis revealed a selection of proteins with membrane interaction- and unknown functions that might be involved in in vitro stromule formation.
Cerebellar ataxias are a group of neurodegenerative disorders primarily affecting the cerebellum. Although causative mutations in several genes have been identified there is currently no cure for ataxias.
The first part of this dissertation is focused on Spinocerebellar ataxia type 2 (SCA2). SCA2 is a dominant ataxia caused by repeat expansion mutations in the ATXN2 gene, which encodes the protein Ataxin2 (ATXN2). A polyglutamine (polyQ) tract consisting of CAG repeats interrupted by CAA was identified at exon 1 of ATXN2. Healthy individuals have between 22 and 23 glutamines, while expansions longer than 33 CAG repeats cause SCA2. The most noticeable symptom that SCA2 patients show is ataxic gait; however, they also show cerebellar dysarthria, dysdiadochokinesia, and ocular dysmetria caused by the progressive cerebellar degeneration.
To model the SCA2 disease, we generated a new mouse model where 100 CAG repeats were introduced in the mouse Atxn2 gene via homologous recombination. The characterization of this mouse model, Atxn2-CAG100-KIN, demonstrated that it reproduces the symptomatology observed in SCA2 patients. These animals showed significant loss of weight over time, brain atrophy, and motor deficits.
In addition, ATXN2 intermediate expansions have been linked to the pathology of Amyotrophic lateral sclerosis (ALS) as a risk factor. ALS is a fatal neurodegenerative disease where the motor neurons in the brain and spinal cord degenerate. A hallmark of ALS is the presence of TDP43-positive inclusions in neurons and glia. Further studies of post mortem spinal cord samples from SCA2 patients showed severe and widespread neurodegeneration of the central somatosensory system. Therefore, it was of interest to further investigate the pathology affection of this tissue in the Atxn2-CAG100-KIN line and the relationship between ATXN2 and TDP43. The characterization of the spinal cord pathology via protein quantification, transcript quantification, and immunohistochemistry showed a preferential affection of RNA binding proteins (RBP) in the spinal cord rather than the cerebellum. The ALS-linked factors TDP43 and TIA1 showed time-dependent co-aggregation with ATXN2 in spinal cord sections together with an increase of CASP3 levels. Therefore, this mouse model can help develop new therapies and evaluate their effect in differently affected areas.
A transcriptome data set from Atxn2-CAG100-KIN spinal cord samples at the final disease stage of this mouse model showed a strong up-regulation of RNA toxicity-, immune- and lysosome-implicated factors. These data pointed to a pathological reactivation of the synaptic pruning and phagocytosis in microglia. ATXN2-positive aggregates were found in microglia from spinal cord sections of 14-month-old Atxn2-CAG100-KIN via immunohistochemistry. The characterization of microglial response and the potentially deleterious effects of the expanded ATXN2 in this cell type could lead to therapies to improve patients’ living standards or delay the symptoms’ onset.
The second part of this thesis was focused on an autosomal recessive form of cerebellar ataxia, Ataxia Telangiectasia (A-T), with childhood onset. A-T patients show severe cerebellar atrophy manifesting as ataxia when the child starts to walk. The genetic cause of A-T is loss-of-function-mutations in the Ataxia Telangiectasia Mutated gene (ATM). ATM is a kinase involved in DNA damage response, oxidative stress, insulin resistance, autophagy via mTOR signaling, and synaptic function.
Working with proteome data from cerebrospinal fluid of 12 A-T patients and 12 healthy controls, we aimed to define novel biomarkers that would allow following the neurodegeneration in extracellular fluid. Additional validation efforts with ~2-month-old Atm-knock-out (Atm-/-) cerebellar samples helped us to define a scenario were the deficit of vesicle-associated ATM alters the secretion of ApoB, reelin, and glutamate. As extracellular factors, apolipoproteins and their cargo such as vitamin E may be useful for neuroprotective interventions.
Die klimatische Nische beschreibt die klimatischen Bedingungen, unter denen eine Art eine stabile Population aufrechterhalten kann. Die Quantifizierung von Klimanischen ist ein wichtiges Werkzeug, um tiefergehende Einsichten in individuelle Art-Umwelt Beziehungen zu erlangen, um den Effekt des Klimawandels effektiv zu bewerten, und um Arten- und Naturschutz zu unterstützen. Ein makroökologischer Ansatz ist von Vorteil um Ökosysteme über ein breites taxonomisches, geographisches und zeitliches Spektrum zu untersuchen, und damit die klimatischen Nischen vieler Arten auf eine konsistente Art und Weise zu quantifizieren und vergleichen.
Im Kontext des aktuellen Klimawandels ist es wichtig zu verstehen, ob Arten in der Lage sind ihre Klima-nische anzupassen. Viele bisherige Vorhersagen über klimawandelbedingte Veränderungen von Artverbreitungen beruhen auf der Annahme, dass die klimatische Nische einer Art konstant ist. Allerdings ist bekannt, dass Arten ihre klimatischen Präferenzen auf unterschiedlichen Zeitskalen verändern - sowohl über kurze (ökologische) als auch evolutionäre Zeiträume. Dies ist ein wichtiger, aber oft missachteter Faktor für die Nischenquantifizierung. Ein gutes Beispiel für solche ökologische Dynamiken sind Zugvögel, die etwa 20% aller Vogelarten ausmachen. Sie stellen eine interessante, aber auch herausfordernde Artengruppe für die Untersuchung klimatischer Nischen dar. Des Weiteren ist es wichtig klimatische Nischen über evolutionäre Zeiträume zu untersuchen, um die Prozesse zu verstehen, die Evolution, Diversifikation und Extinktion unterliegen, da sich Klimanischen mit der Anpassung einzelner Arten an neue klimatische Gegebenheiten ebenfalls wandeln. Bislang hat ein Mangel an geographisch expliziten Daten über terrestrische Umwelt-bedingungen durch evolutionäre Zeiträume eine explizite Überprüfung dieser Zusammenhänge verhindert.
Das übergeordnete Ziel dieser Dissertation war es, die ökologische (d.h. saisonale) und evolutionäre Dynamik klimatischer Nischen von Vögeln zu untersuchen. Dazu wurde ein Ansatz gewählt der makroökologische, und evolutionsbiologische Methoden vereint, um ein breites taxonomisches und zeitliches Spektrum abzudecken. Das erste Kapitel bearbeitet die Frage wie klimatische Nischen am besten zu quantifizieren sind, wenn man die Dynamik des Vogelzuges in Betracht zieht. Dazu wurde eine Datenbank erstellt, die das Zugverhalten aller 10.443 lebenden Vogelarten katalogisiert. Des Weiteren wurde eine Übersicht über die Methoden zur Quantifizierung klimatischer Nischen in der makroökologischen Literatur erstellt. Das Ergebnis derselben ist, dass die überwiegende Mehrzahl der Veröffentlichungen saisonalen Zugbewegungen nicht ausreichend berücksichtigt. Zuletzt habe ich anhand der Avifauna Australiens die Vor- und Nachteile der Verwendung von Verbreitungskarten gegenüber Punktverbreitungsdaten zur Erfassung saisonaler geographischer Muster der Artenvielfalt bewertet. Damit bietet dieses Kapitel Rahmenempfehlungen für die Datenanforderungen und Methoden, die je nach Zugverhalten einer Art, und dem geographischen, bzw. zeitlichen Fokus einer Studie für eine optimale Nischenquantifizierung notwendig sind.
Im zweiten Kapitel untersuchte ich die saisonale Dynamik klimatischer Nischen von Zugvögeln. Dabei überprüfte ich die Hypothese, dass Zugvögel in ihrem Jahreszyklus durch die Zugbewegung eine gewisse Klimanische verfolgen. Zu diesem Zweck habe ich mit Brut- und Überwinterungsarealkarten saisonale Klima-nischen für 437 Zug- und Standvogelarten aus acht Kladen der Sperlingsvögel (Passeriformes) charakterisiert. Mit Ordinationsmethoden wurde dann der innerartliche saisonale Nischenüberlapp quantifiziert. Der Beweis für die Verfolgung einer klimatischen Nische in einer Art war von mehreren Faktoren, z.B. der geographischen Verortung des Brutareals und der Zugrichtung, abhängig. Dies lässt darauf schließen, dass sich die Ursachen für den Vogelzug sowohl geographisch als auch saisonal (d.h. abhängig von der Zugrichtung) unterscheiden.
Im dritten Kapitel untersuchte ich die evolutionäre Dynamik klimatischer Nischen in Steinschmätzern (Gattung Oenanthe), um explizit zu untersuchen ob es einen Zusammenhang zwischen den Raten klimatischer Nischen-evolution und den Veränderungen paläoklimatischer Bedingungen gibt. Methoden der Klimanischen-quantifizierung wurden mit datierten molekularen Phylogenien verknüpft, um die Raten klimatischer Nischen-evolution mit einem variablen Ratenmodell abzuschätzen. Paläoklimatische Umweltbedingungen wurden mit paläobiologischen Methoden aus dem Fossilbericht altweltlicher Säugetiere der vergangenen 20 Millionen Jahre erschlossen. Die Fallstudie konnte keinen Zusammenhang zwischen Nischenevolution und Umwelt-bedingungen feststellen. Dies legt nahe, dass Vögel als überaus mobile Organismen, auf Klimaveränderungen eher durch Arealverschiebungen reagieren, als durch eine Anpassung ihrer klimatischen Nische. Die Klimanischen der Steinschmätzer waren allerdings an sich nicht statisch, so dass andere Faktoren wie z.B. biologische Wechselbeziehungen für die Nischenevolution dieser Gattung verantwortlich sein müssen.
Meine Dissertation beleuchtet die zentrale Bedeutung zeitlicher Dynamiken für den Nischenraum, den Arten über ökologische (d.h. saisonale) und evolutionäre Zeiträume einnehmen. Aus ihr ergeben sich methodische Konsequenzen für zukünftige Studien klimatischer Nischen. Der Befund, dass die klimatischen Nischen von Zugvögeln nicht saisonal konstant sind, zeigt dass es für mobile Kladen wie Vögel notwendig ist die klimatischen Bedingungen über den gesamten Jahreszyklus und das gesamte Verbreitungsgebiet in Betracht zu nehmen, um die jeweiligen klimatischen Nischen voll charakterisieren zu können.
Über diese methodischen Innovationen hinaus, hat meine Arbeit auch wichtige theoretische und praktische Schlussfolgerungen produziert. Zum einen zeigt die Betrachtung saisonaler Klimanischen, dass Zugvögel entgegen gängiger Annahmen nicht denselben Umweltbedingungen in ihren Brut- und Überwinterungsarealen ausgesetzt sind. Zum anderen zeigt meine Betrachtung von Klimanischen über evolutionäre Zeiträume, dass die Nischenevolution nicht von klimatischen Bedingungen angetrieben wird. Zusammengenommen zeigen diese Ergebnisse auf unterschiedlichen Zeitskalen, dass das Klima nicht der alleinige Faktor ist, der die Artverbreitung von Vögeln bestimmt. Während dieser Befund Raum für Optimismus schafft, was die Auswirkungen des aktuellen Klimawandels auf Vögel angeht, zeigt er auch auf, dass Faktoren wie wechselseitige Artbeziehungen und das Mobilitätspotential von Arten einen wichtigen Einfluss auf Artverbreitungen ausüben. Diese Faktoren könnten jedoch an sich vom Klimawandel beeinflusst sein, und Untersuchungen dieses Zusammenspiels zwischen Klima und anderen Faktoren und die daraus resultierenden Einflüsse auf Artareale bieten ein vielversprechendes Arbeitsfeld für zukünftige Studien.
Die vorliegende Dissertation mit dem Titel: Ecophysiological monitoring of Oaks in Central Europe, introduced in the framework of proactive climate change mitigation beschäftigt sich mit der Anwendung zerstörungsfreier, radiometrischer Methoden zur Bestimmung von Pigment- und Stickstoffkonzentrationen und der photosynthetischen Funktionalität in Blättern von heimischen und gebietsfremden Eichen und ihre Beeinflussung durch Trocken-, Hitze- und Kältestress.
Die Eichenarten Quercus robur L. (Stieleiche), Q. pubescens Willd. (Flaumeiche), Q. frainetto Ten. (Ungarische Eiche), Q. ilex L. (immergrüne Steineiche) und Q. rubra L. (amerikanische Roteiche) wurden im Frühjahr 2011 auf einer Versuchsfläche im Frankfurter Stadtwald gepflanzt, um ihre Nutzung als potentielle Waldbäume in einem sich ändernden Klima zu untersuchen. Über eine Dauer von zwei Jahren wurden diese Arten mit einem hohen Maß an blattspezifischer Merkmalsvariabilität beobachtet und beprobt. Ziel war es, die interspezifischen Unterschiede und die jahreszeitliche Dynamik von morphologischen und chemischen Blattmerkmalen sowie die Beeinflussung der radiometrischen Bestimmung des Chlorophyllgehaltes (und damit assoziierten Komponenten wie z.B. Blattstickstoffgehalt und Karotinoiden) und der photosynthetischen Funktionalität durch klimatische Umweltbelastungen in Eichen zu untersuchen. Die Analyse der Blattproben zielte neben der Bestimmung der Beziehung zwischen absoluten und optisch ermittelten Pigmentgehalten auf die Ermittlung des Einflusses der Blattstruktur auf die Lichttransmission im roten und infrarotem Bereich des Elektromagnetischen Spektrums ab, sowie auf die artspezifische Korrelation von Blattstickstoff zu Blattchlorophyll zu dessen indirekte Quantifizierung. Des Weiteren wurden Versuche zur Trocken- und Hitzestressanpassung durchgeführt, um eine potentiell artspezifische Stressantwort, sowie eine mögliche Beeinflussung der aufgenommenen radiometrischen Messwerte zu ermitteln. Ein zusätzliches Monitoringprogramm im Winter 2012/2013 mit einer Dauer von sechs Monaten ermöglichte die Überprüfung der Anpassungsfähigkeit der immergrünen Steineiche (Q. ilex) an mitteleuropäische Winterbedingungen und die Veränderung der photosynthetischen Funktionalität unter Kältestress. Messungen im Zusammenhang mit der praktischen Anwendbarkeit der zerstörungsfreien, optischen Methode und zur Bereitstellung von Referenzdaten für zukünftige Evaluierungen komplementieren die Untersuchungen.
Signifikante, artspezifische Unterschiede wurden in den blattmorphologischen Schlüsselmerkmalen in den Quercus-Arten ermittelt. Die artspezifischen Unterschiede in den morphologischen Blattmerkmalen beeinflussten auf signifikante Weise die Beziehung zwischen absoluten, massebasierten Pigment- und Stickstoffgehalten und deren radiometrischen Bestimmung. Wurden die Pigmentgehalte hingegen auf die Blattfläche bezogen und die Stickstoffgehalte mittels des Verhältnisses von Blattfläche zu Trockenmasse korrigiert, zeichnete sich eine Beziehung zwischen absoluten und optisch ermittelten Werten ab, der jegliche jahreszeitliche oder artspezifisch morphologische Variabilität fehlte und die somit für alle Quercus-taxa anwendbar ist. Koeffizienten für die Berechnung von flächenbezogenen Gehalten von Gesamtchlorophyll, Chl a, Chl b und Carotinoiden für die jeweiligen Quercus-taxa, wie auch für ein artübergreifendes Modell wurden ermittelt, um die Bestimmung dieser Gehalte während aller Entwicklungsstufen zu ermöglich. Aus der jahreszeitlichen Entwicklung der Pigmentgehalte konnten drei deutliche Phasen abgeleitet werden: Die Phase der Blattentwicklung im Frühling, einer Plateauphase mit geringen Veränderungen (“core vegetation time”) und die Phase des Pigmentabbaus während der Herbstlaubfärbung. Die Übergänge zwischen diesen Phasen variierten zum Teil erheblich zwischen einzelnen Individuen einer Art sowie zwischen den Arten, was Unterschiede in der potentiellen, jährlichen Kohlenstoffaufnahme nach sich zieht. Stressbedingungen, wie Hitze- Kälte- oder Trockenstress, können zu Veränderung von Fluoreszenzparametern ohne gleichzeitige Änderung des Pigmentgehaltes führen, wie auch die indirekte Bestimmung von mit Chl assoziierten Komponenten (Carotinoide, Chl a, Chl b) mittels optischer Bestimmung (durch die Veränderungen von Pigmentverhältnissen) beeinflussen.
Im Rahmen des Forschungsprojektes konnten, Modelle zur Berechnung von Blattpigmenten und Blattstickstoff aus optischem Messdaten, Veränderungen der photosynthetischen Funktionalität, sowie Referenzdaten für die zukünftig nutzbaren Eichenarten hinsichtlich artspezifischer und jahreszeitlicher Variabilität unter mitteleuropäischen Umweltbedingungen ermittelt werden, die eine Nutzung und Einordnung von zerstörungsfreien, optischen Messwerten zur Ermittlung von Vitalitätsunterschieden in Eichen ermöglichen.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
Clean water is fundamental to human health and ecosystem integrity. However, water quality deteriorates due to novel anthropogenic pollutants present at microgram per liter concentrations in urban water cycles (termed micropollutants). Wastewater treatment plants (WWTP) have been identified as major point sources for aquatic (micro-)pollutants. Chemical and ecotoxicological analyses have shown that conventional biological WWTPs do not fully remove micropollutants and associated toxicities, which is often because of mobile, polar and/or recalcitrant compounds and transformation products (TPs). To minimize possible environmental risks, advanced wastewater treatment (AWWT) technologies could be a promising mitigation measure. Multiple processes are therefore being developed and evaluated such as ozonation and ozonation followed by granulated activated carbon (GAC) or biological filtration. Assessing the performance of these combined AWWTs was the focus the TransRisk project. Within this project, this thesis accomplished four major goals.
Firstly, the preparation of (waste)water samples was optimised for in vitro bioassays. Acidification, filtration and solid phase extraction (SPE) were tested for their impact on environmentally relevant in vitro endocrine activities, mutagenicity, genotoxicity and cytotoxicity. Significantly different outcomes of these assays were detected comparing neutral and acidified samples. Sample filtration had a lesser impact, but in some cases retention of particle-bound compounds could have caused significant toxicity losses. Out of three SPE sorbents the Telos C18/ENV at sample pH 2.5 extracted highest toxicity, some undetected in aqueous samples. These results indicate that sample preparation needs to be optimised for specific sample matrices and bioassays to avoid false-positive or -negative detects in effect-based analyses.
Secondly, the above listed in vitro toxicities were monitored in a protected region for drinking water production in South-West Germany (2012-2015). Out of 30 sampling sites surface water and groundwater were the least polluted. Nonetheless, a few groundwater samples induced high anti-estrogenic activity that prompted further monitoring. The latter included a waterworks in which no toxicity was detected. Hospital wastewater also had elevated in vitro toxicities and hospitals are, thus, relevant intervention points for source control. The biological WWTPs were effective in removing most of the detected toxicity, and the selected bioassays proved to be pertinent tools for water quality assessment and prioritisation of pollution hotspots.
Thirdly, the in vivo bioassay ISO10872 based on Caenorhabditis elegans (C. elegans) was adapted for this thesis. Using this model, a median effect concentration (EC50) for reproductive toxicity of the polycyclic aromatic hydrocarbon β-naphthoflavone (β- NF) of 114 µg/L was computed which is slightly lower than reported in the scientific literature. β-NF induced cyp-35A3::GFP (a biomarker in transgenic animals) in a time and concentration dependent manner (≤ 21.3–24 fold above controls). β-NF spiked wastewater samples supported earlier hypotheses on particle-bound pollutants. Reproductive toxicity (96 h) and cyp-35A3 induction (24 h) of biologically treated and/or ozonated wastewater extracts and growth promoting effects of GAC/biologically filtered ozonated wastewater extracts were observed. This suggested the presence of residual bioactive/toxic chemicals not included in the targeted chemical analysis. It also highlighted the importance of integrating multiple (apical and molecular) endpoints in wastewater assessments.
Fourthly, five in vitro and the adapted C. elegans bioassay were integrated into a wastewater quality evaluation (developed within TransRisk). Out of the five AWWT options, ozonation (at 1 g O3,applied/g DOC, HRT ~ 18 min) combined with nonaerated GAC filtration was rated most effective for toxicity removal. All five AWWTs largely removed estrogenic and (anti-)androgenic activities, but not anti-estrogenic activity and mutagenicity, which even increased during ozonation. This has been observed in related studies and points towards toxic TPs. These results also emphasized the need for implementing an effective post-treatment for ozonation. The results from a parallel in vivo study with Lumbriculus variegatus and Potamopyrgus antipodarum conducted on site at the WWTP (using flow through systems) were in accordance with the C. elegans results. In this context, it is suggested to further implement C. elegans as sensitive, feasible and ecologically relevant model.
In conclusion, this thesis shows how optimised sample preparation, long-term (in vitro) environmental monitoring, sensitive and ecologically relevant (in vivo) bioassays as well as innovative evaluation concepts, are pivotal in improving the removal of micropollutants and their toxicities with AWWTs. Future research should further develop and evaluate measures at sewer systems, conventional biological, tertiary and other advanced treatment technologies, as well as sociopolitical strategies (e.g., source control or natural conservation) and restoration projects. The effect-based tools optimised in this thesis will support assessing their success.
The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
Biodiversity is threatened worldwide because of ongoing habitat loss and fragmentation, overexploitation, pollution, biological invasions and a changing global climate. Due to the major importance of biological diversity for modern human living, efficient conservation and management strategies are required to protect endangered habitats and species. For this purpose, ambitious multilateral agreements on regional and global scale were declared to prevent biodiversity loss.
Efficient biomonitoring methods are required to adequately implement these biodiversity conventions. Species monitoring as a core activity in biodiversity research is an effective tool to assess the status of species and trends within habitats. Data collection can be obtained with visual, electronic or genetic surveys. Still, these monitoring programs can be expensive, laborious and inefficient for accurate species assessments. New techniques based on environmental DNA (eDNA) allows for the detection of DNA traces in environmental samples (soil, sediment, water and air samples) and open up new possibilities for species monitoring. The eDNA methodology enables detection of single species in a qualitative (presence/absence) or (semi-) quantitative way. eDNA metabarcoding approaches can be an effective community structure assessment method.
This thesis, located at the interface between experimental and applied research, illustrates the suitability of the eDNA methodology in applied biomonitoring using the example of the water-borne crayfish plague pathogen Aphanomyces astaci (Schikora 1906). The obtained results provide new insights into A. astaci sporulation dynamics in natural water courses. A. astaci sporulation is influenced by seasonal variation of water temperatures and life history traits (molting, activity, mating) of infected crayfish. The results also imply a high transmission risk of A. astaci spores during the complete year. This thesis compares two eDNA methods, which are successfully and consistently detecting A. astaci spores. Each approach is suitable for different biomonitoring tasks due to the method-specific requirements. The obtained results also reveal spatial variation in A. astaci occurance in the tested water bodies. A. astaci spore estimates are positively correlated with population density and pathogen loads of captured A. astaci- positive crayfish. eDNA results show a downstream zoospore transport of up to three kilometres distance from a distribution hot spot area of A. astaci-infected crayfish. The eDNA methodology is helpful in gaining reliable information on A. astaci occurrence in large water bodies. This information is urgently needed to initiate efficient management decisions for the conservation of European crayfish species.
eDNA-based methods such as for A. astaci detection are a useful complement for conventional monitoring and should have a strong impact on conservation policy. eDNA methodology will be helpful for the practical implementation of the main aims of key conservation agreements and thus will make important contributions to biodiversity protection.
Angesichts heutiger Umweltprobleme ist die Stärkung positiver Mensch-Natur-Beziehungen wichtiger denn je. Zeitgenössische Umweltbildung zielt darauf ab, Motivation und Einstellungen zu fördern sowie eine grundlegende Wissensbasis zu schaffen (IUCN, UNEP, & WWF, 1991; Potter, 2010), um einen selbstbestimmten, verantwortungsvollen Umgang mit der Natur zu ermöglichen. Positiver Naturbezug und Umwelteinstellungen gelten als Basis für aktiven Umweltschutz. Direkte Naturerfahrungen gelten dabei als didaktische Möglichkeit, die Motivation für Umweltschutz zu festigen (Kaiser, Roczen, & Bogner, 2008). Einstellungen verändern sich im Laufe des Lebens und so kann das Alter eine wichtige Rolle bezüglich der Effektivität von Umweltbildungsprogrammen spielen (Ernst & Theimer, 2011). Auch Umweltwissen gilt als Grundlage von Umwelthandeln. Denn sinnliche Erfahrungen allein führen nicht zum Verständnis ökologischer Zusammenhänge (Frick, Kaiser, & Wilson, 2004; Liefländer, Bogner, Kibbe, & Kaiser, 2015). Die biologiedidaktische Forschung sieht Fakten-, Handlungs- und Effektivitäts-wissen als zentral für die Genese von Umwelthandeln (Frick, Kaiser, & Wilson, 2004). Isoliertes Fachwissen wiederum führt nach aktueller Erkenntnis auch nicht zur Entwicklung von Haltungen und Wertvorstellungen, welche unser Handeln beeinflussen (Barr, 2003; Finger, 2010; Leiserowitz, Kates, & Parris, 2005).
Bis heute sind altersbasierte Unterschiede bei Schülerinnen und Schülern bezüglich ihrer Naturverbundenheit und Umwelteinstellungen nicht hinreichend untersucht. Auch ist die nötige Dauer der Naturerfahrungen noch nicht nachgewiesen. Es gibt bislang keine Studie, die Umwelteinstellungen, -wissen und –handeln von Kindern verschiedener Regionen der Erde untersucht und Daten auf internationaler Ebene erhoben und ausgewertet hat. Die gezielte Integration der drei Umweltwissensarten in ein solch globales Umweltbildungsprojekt stellt eine zusätzliche bislang nicht angegangene Aufgabe dar. Die vorliegende Arbeit schließt diese Forschungslücken, indem sie auf internationaler Ebene jene Variablen mit einbezieht, die einen nahezu vollständigen Eindruck der Effektivität von Umweltbildung in verschiedenen Regionen, Sozialisationen und Altersklassen zulässt. So wird der Einfluss eines umfassenden Umweltbildungsprogramms auf Naturverbundenheit, Umwelteinstellungen und -wissen der verschiedenen Typen untersucht und ein Bezug zur eventuellen Veränderung des Umwelthandelns hergestellt. Dabei stehen sowohl traditionelle als noch unerforschte mögliche Einflussfaktoren im Fokus. Die Studie umfasst insgesamt 1454 Schülerinnen und Schüler aus Bangladesch, Malaysia, Deutschland und Singapur, die alle an dem Umweltbildungsprojekt „Global denken, lokal handeln – wir schützen unsere Umwelt!“ bzw. “Think global, act local – we protect our environment!“ teilgenommen haben.
Zur Messung der Naturverbundenheit diente Schulz’ INS-Skala (Inclusion of Nature in Self) (2002). Umwelteinstellungen wurden mit dem 2-MEV-Modell (Two Major Environmental Values) gemessen (Johnson & Manoli, 2011). Eine Skala zur Erhebung von Umweltwissen wurde eigens erstellt und hinsichtlich der drei Wissenstypen nochmals modelliert. Eine Skala zur Ermittlung von Umwelthandeln wurde auf Grundlage von Bögeholz (1999) erstellt. Alle Skalen waren Teil eines Fragebogens, welcher in Form eines Pre-, Post- und Follow-up-Test eingesetzt wurde. Kinder aus Parallelklassen, die nicht am Projekt teilnahmen, aber Klassenunterricht zu den jeweiligen Themen erhielten, dienten als Kontrollgruppen.
Die Ergebnisse bestätigen einen positiven Effekt außerschulischer Umweltbildung bezüglich der Entwicklung der untersuchten Variablen. So wurde nach der Teilnahme am eintägigen und auch nach dem fünftägigen Umweltbildungsprogramm eine signifikante Verstärkung des Naturbezugs gemessen, wohingegen die Kontrollgruppen keine messbare Veränderung zeigten. Jedoch nur die fünftägige Intervention führte auch zu nachhaltigen Veränderungen. Hierbei am stärksten beeinflusst wurden Kinder zwischen sieben und neun Jahren.
Bei der Untersuchung demographischer Einflussfaktoren auf Umwelteinstellung, -wissen und –handeln stellten sich das Wohnsitzland sowie die städtische bzw. ländliche Prägung der Wohngegend als entscheidend heraus. So waren dies die einflussreichsten Determinanten zur Vorhersage des Grundvorhandenseins sowie Veränderungen der untersuchten Variablen in Folge der Bildungsmaßnahme. Einzig bei der Entwicklung des Umwelthandelns schien die direkte Naturerfahrung unwesentlich, zeigten die Kontrollgruppen ähnlichen Wandel in ihrem aktiven Einsatz für die Umwelt. Im internationalen Vergleich scheint die komplexe Verkettung diverser einflussnehmender Faktoren, wie der Wohlstand des jeweiligen Staates, das generelle politische System sowie spezifische bildungspolitische Begebenheiten, den Erfolg von Umweltbildungsprogrammen mit zu bestimmen.
Die Daten zeigen, dass Faktenwissen Grundlage für Handlungs- und Effektivitätswissen ist. Alle Dimensionen wurden durch die Intervention signifikant gesteigert. Effektivitätswissen wuchs am stärksten. Auch das Umweltverhalten wurde positiv verstärkt. Jedoch ließen sich nur schwache Korrelationen zwischen den einzelnen Wissenstypen und Handeln feststellen. Zusammenfassend war das durchgeführte Bildungsprojekt erfolgreich in der Förderung von Naturverbundenheit sowie Umwelteinstellungen, -wissen und- handeln. Die Ergebnisse werden im Rahmen dieser Arbeit im Hinblick auf ihre Bedeutung für die schulische Umweltbildung sowie die didaktische Forschung erörtert.
Eine qualitative und quantitative Studie zum Einsatz der virtuellen Mikroskopie in der Schule
(2019)
Das Mikroskop stellt in der Alltagswelt ein Sinnbild für naturwissenschaftliches Arbeiten dar (Coleman 2009, Paulsen 2010). Im Bereich der Lehre eröffnet dieses Laborgerät das Eintauchen in die mikroskopische Dimension und besitzt eine wesentliche Rolle bei der damit verbundenen Erkenntnisgewinnung, insbesondere von funktionsmorphologischen Konzepten (Gropengießer & Kattmann 2008, Kremer 2002). Jedoch wird die Durchführung der klassischen Mikroskopie und damit die aktive Auseinandersetzung mit mikroskopischen Präparaten im schulischen (Biologie-)Unterricht durch verschiedene Faktoren erschwert. Zu den Limitierungen gehören beispielsweise die Verfügbarkeit geeigneter Mikroskope und Dauerpräparate, die aufwendige Vor- und Nachbereitungszeit sowie der zeitliche Aufwand bei der Herstellung hochwertiger mikroskopischer Frischpräparate. Die virtuelle Mikroskopie könnte diese Schwierigkeiten umgehen. Das virtuelle Mikroskop kann als eine Simulation verstanden werden, bei der die bildanalytischen Vorgehensweisen bei mikroskopischen Präparaten analog zur klassischen Mikroskopie nachvollzogen werden können (Gu & Oglivie 2005, Hentschel 2009). Hierbei umfasst das virtuelle Mikroskop ein Akquisitionssystem zum Einscannen und Digitalisieren mikroskopischer Präparate, einen Server zum Speichern und Bereitstellen der entstandenen virtuellen hochauflösenden Aufnahmen (WSI) sowie eine Bildbetrachtungssoftware auf einem Anwendungsrechner (Kalinski et al. 2006). Basierend auf einer Nutzerbefragung wurde eine Betrachtungssoftware programmiert, die hinsicht¬lich ihrer Benutzerfreundlichkeit und ihren Eigenschaften auf den schulischen Einsatz angepasst wurde. Um die Relevanz in diesem Anwendungsfeld zu testen, wurden die Untersuchungen der vorliegenden Arbeit sowohl im Schülerlabor Goethe BioLab als auch in der universitären Lehre der Abteilung für Didaktik der Biowissen¬schaften der Goethe–Universität Frankfurt am Main durchgeführt. Der Schülerlabortag „Blut und das virtuelle Mikroskop“ wurde entwickelt, um die computerbasierte virtuelle Mikroskopie mit Schülern ergänzend zur klassischen Mikroskopie in einem fachlichen Kontext anzuwenden und zu erforschen.
Beruhend auf der Vergleichbarkeit beider Mikroskopiemethoden (Paulsen et al. 2010) lagen die Forschungsschwerpunkte neben der Nutzung der Software durch Schülerinnen und Schülern auf einer gegenüberstellenden Beurteilung beider mikroskopischer Verfahren von Schülern und Lehramtsstudierenden. Es wurden in diesem Zusammenhang drei zentrale Forschungsfragen formuliert.
Die erste Forschungsfrage untersucht das Nutzerverhalten der Schüler (n = 123) bei der virtuellen Mikroskopie mittels automatisch generierter Datensätze während der Anwendung der Bildbetrachtungssoftware. Die Analyse der Anwendungsdaten zeigt, dass das mikroskopische Sehen, insbesondere das Fokussieren auf relevante Bildbereiche, im virtuellen Humanblutausstrich angewandt wurde.
Die zweite Forschungsfrage untersuchte das aktuelle Interesses bei Schülern (n = 293) im direkten Vergleich zwischen virtueller und klassischer Mikroskopie. Dabei wurde das aktuelle Interesse aufgrund des engen Zusammenhangs zum Lernen (vgl. Krapp 1992a) als Indikator der Lernwirksamkeit gewählt. Die Erhebung erfolgte mittels eines Fragebogens. Die Ergebnisse dieser Untersuchung zeigen, dass der Einsatz beider mikroskopischer Verfahren das aktuelle Interesse fördert, das emotionale und das wertbezogene Merkmal sich jedoch zugunsten der klassischen Mikroskopie signifikant unterscheiden.
Im Rahmen der dritten Forschungsfrage erfolgte eine Beurteilung der Vorteile virtueller Mikroskopie gegenüber der klassischen Mikroskopie von Schülern (n = 504) sowie Lehramtsstudierenden (n = 247). Hierbei diente ebenfalls ein Fragebogen als Grundlage der Erhebung. Die Auswertung zeigt, dass sowohl die Schüler als auch die Studierenden die Vorteile der virtuellen Mikroskopie klar erkennen. Es liegen jedoch signifikante Unterschiede zwischen den Versuchsgruppen vor. Die Schüler bewerten die Vorteile betreffend der Förderung von Lernprozessen, des Erkennens von Strukturen und des mikroskopischen Zeichnens höher.
Zusammenfassend bestärken die Ergebnisse dieser Studie die Ansicht, dass das virtuelle Mikroskop nicht als Ersatz, sondern als sinnvolle Ergänzung zu der klassischen Lichtmikroskopie angesehen werden sollte (Bloodgood et al. 2006, Berg et al. 2016, Braun & Kearns 2008, Hufnagl et al. 2012, Mione et al. 2013, Santiago 2018, Scoville & Buskirk 2007). Dabei sollte die vorliegende Arbeit als Einstieg verstanden werden, um bestehende Forschungslücken zu verkleinern, damit ein Transfer der virtuellen Mikroskopie in den schulischen Kontext möglichst lernwirksam erfolgen kann.
Die Endometriose ist eine gynäkologische Erkrankung, bei der epitheliale und stromale Zellen des Endometriums Läsionen außerhalb des Uterus bilden, die in ihrem Aufbau dem Endometrium gleichen. Diese Läsionen, sowie deren zyklische Proliferation, führen zu Schmerzen bei betroffenen Frauen. In isolierten, invasiven Epithelzellen (EEC145T) einer Endometriose-Läsion konnte die Expression von Shrew-1 gezeigt werden. Auch in anderen zellulären Zusammenhängen fördert die Expression von Shrew-1 den invasiven Phänotyp. Shrew-1 ist ein Transmembranprotein, das in Epithelzellen mit den Adhärenzverbindungen assoziiert ist und Interaktionen mit β-Catenin und E-Cadherin eingeht. In MCF7-Zellen fördert die Expression von Shrew-1 die EGF-induzierte Internalisierung von E-Cadherin, welche zur Verminderung der Zell-Zell-Adhäsion führt. In 12Z- und HT1080-Zellen konnte eine Interaktion mit CD147 gezeigt werden. CD147 fördert die Aktivität von MMPs und in Shrew-1-überexprimierenden HT1080-Zellen konnte eine erhöhte Aktivität der MMP9 gezeigt werden. Shrew-1 wirkt somit auf die Invasivität von Zellen und ist gleichzeitig Teil der Adhärenzverbindung. Aus diesem Grund wird Shrew-1 eine modulatorische Rolle in diesem Kontext zugeschrieben.
In immunhistologischen Färbungen von Shrew-1 und E-Cadherin konnte in Adenomyose-Läsionen eine inverse Expression der beiden Proteine in einigen epithelialen Zellen gezeigt werden, die im Endometrium nicht detektiert werden konnten. In den epithelialen Endometriose-Zelllinien 12Z und 49Z, die kein E-Cadherin exprimieren und äquivalent zu der Zelllinie EEC145T sind, führte die Herunterregulation von Shrew-1 (Shrew-1 KD) zur Reexpression von E-Cadherin. E-Cadherin ist in den 12Z Shrew-1 KD-Zellen an der Plasmamembran lokalisiert und interagiert mit β-Catenin, wodurch seine Assoziation mit den Adhärenzverbindungen wahrscheinlich ist. Die Herunterregulation von Shrew-1 führt zu einer verminderten Motilität und Invasivität der 12Z-Zellen, wobei die reduzierte Invasivität nicht alleine auf die Reexpression von E-Cadherin zurückgeführt werden kann. Es ist zu vermuten, dass das verminderte invasive Verhalten mit der ausbleibenden Interaktion von Shrew-1 mit CD147 zusammenhängt, welches die Aktivität von MMPs fördert.
Da Shrew-1 eine direkte Interaktion mit β-Catenin eingehen kann, ist es möglich, dass die Herunterregulation von Shrew-1 zu Veränderungen in der Lokalisation von β-Catenin und weiteren Proteinen, die mit den Adhärenzverbindungen assoziiert sind (p120 Catenin und Aktin), führen. Dies konnte jedoch nicht beobachtet werden. Eine verstärkte Lokalisation von Vinculin an den Enden von Aktin-Stressfasern sowohl in Zellausstülpungen als auch an Zell-Zell-Kontakten konnte in 12Z-Zellen nach der Herunterregulation von Shrew-1 beobachtet werden. Dies könnte eine Folge der E-Cadherin-Reexpression oder entscheidend für die Lokalisation von E-Cadherin an der Membran sein.
Die Reexpression von E-Cadherin, die in den 12Z Shrew-1 KD-Zellen auf mRNA- und Protein-Ebene nachgewiesen werden kann, erfolgt in den 12Z-Zellen vermutlich hauptsächlich über Veränderungen von Histon-Acetylierungen, da die Behandlung mit dem HDAC-Inhibitor TSA die Expression von E-Cadherin in den 12Z-Zellen induziert. Eine verstärkte H3K9-Acetylierung am CDH1-Promotor konnte in ChIP-Analysen in den 12Z Shrew-1 KD-Zellen gezeigt werden. Die gesteigerte Acetylierung resultiert vermutlich aus der verminderten Assoziation von HDAC1 und HDAC2 mit dem CDH1-Promotor in diesen Zellen. Eine Beteiligung der Repressoren Snail, Slug, Twist und ZEB1 an der Reexpression von E-Cadherin in den 12Z Shrew-1 KD-Zellen konnte nicht gezeigt werden. Ebenso scheinen Veränderungen am Methylierungsstatus des CDH1-Promotors nach der Herunterregulation von Shrew-1 nicht zu erfolgen.
TSA induziert auch in weiteren epithelialen Endometriose-Zelllinien (10Z und 49Z) die Expression von E-Cadherin. In stromalen Zellen führt hingegen weder TSA noch die Herunterregulation von Shrew-1 zur Expression von E-Cadherin (17B, 18B und 22B). Dies weist darauf hin, dass die Herunterregulation von Shrew-1 über die Veränderungen von Histon-Acetylierungen wirkt und dass dieser Mechanismus in epithelialen Endometriose-Zellen entscheidend ist. In den stromalen Zellen muss die Expression von E-Cadherin über einen anderen und/oder weitere Mechanismen blockiert sein.
Auch der Wnt-Signalweg scheint an der Reexpression von E-Cadherin in 12Z-Zellen beteiligt zu sein. Die Inhibierung der GSK3β (LiCl und SB216763) führt zur Expression von geringen Mengen an E-Cadherin. In 12Z Shrew-1 KD-Zellen führt die Stabilisierung von Axin (XAV939) zur verminderten Expression von E-Cadherin. Dies lässt darauf schließen, dass Shrew-1 auch einen Einfluss auf den Wnt-Signalweg hat, was vor allem durch dessen Interaktion mit β-Catenin wahrscheinlich ist.
Im Kindes- und Jugendalter gehoert das Rhabdomyosarkom zu den haeufigsten Weichteilsarkomen. Bisher belaeuft sich das Therapieverfahren auf chirurgische Entfernung, gefolgt von Chemotherapie, bzw. bei nicht-operablen Faellen auf Radiotherapie und Chemotherapie, jedoch haben sich die Ueberlebenschancen fuer Patienten mit einer Erkrankung in metastasiertem oder rezidiviertem Stadium trotz intensiver Forschung ueber mehrere Jahrzehnte hinweg kaum gebessert und bleiben bei unter 30%. Neue therapeutische Strategien versuchen das Immunsystem des Patienten zu modulieren und dieses gezielter oder aggressiver gegen Tumorzellen zu machen. Nebst direkter Injektion von Zytokinen oder Antikoerpern bietet die adoptive Immunzelltherapie einen vielversprechenden Ansatz. In der vorliegenden Arbeit lag der Fokus auf Natuerlichen Killer- (NK) Zellen, da diese ein hohes zytotoxisches Potential gegenueber Tumorzellen aufweisen. Eine der groessten Herausforderungen der NK-Zellforschung ist die Breitstellung ausreichender Mengen an NK-Zellen mit optimaler antitumoraler Funktion fuer den klinischen Einsatz. Viele aktuell erprobte NK-Zellexpansionsstrategien basieren auf der Verwendung von Hilfs- oder Feeder-Zellen (Versorgerzellen), die jedoch vor der Applikation in Patienten aus dem finalen Produkt entfernt werden muessen. In der vorliegenden Arbeit sollten Feeder-zellfreie NK-Zellexpansionsprotokolle unter Verwendung von Gammakettenzytokinen getestet werden.
Interleukin (IL-) 15 erwies sich dabei vor allem fuer die Vermehrung der NK-Zellen als besonders foerderlich. Im Vergleich dazu fielen die Expansionsraten mit IL-2 oder IL-21 geringer aus. Interessanterweise wurde der expansionsfoerdernde Effekt von IL-15 durch dauerhafte Anwesenheit von IL-21 im Kulturmedium gehemmt. Ein kurzer, dreitaegiger IL-21-Boost am Ende der Expansionsphase wirkte sich wiederum positiv auf die NK-Zellexpansionsraten aus. Zudem zeigte sich durch IL-21 ein vermehrtes Auftreten von NK-Zellen des reiferen CD16posCD56dim Phaenotyps, der die zytotoxische Funktion vermittelt. Bei Degranulationsuntersuchungen wurden eine IL-21-induzierte Exozytoseaktivitaet und die vermehrte Ausschuettung von Perforin und Granzym B, welche Apoptose in den Zielzellen ausloesen, beobachtet. Vor allem der dreitaegige Boost mit IL-21 bewirkte eine gesteigerte Zytotoxizitaet gegenueber Tumorzellen, insbesondere gegenueber Rhabdomyosarkomzellen.
Auf dieser Grundlage bot es sich an fuer die NK-Zellexpansion ein Zwei-Phasen-Protokoll anzuwenden, bestehend aus einer initialen Proliferationsphase mit IL-15 und einem anschliessendem IL-21-Boost, durch den die antitumorale Funktionalitaet der NK-Zellen gesteigert wurde. Dieses IL-15+21boost-Protokoll wurde mit anderen Kombinationen aus den Gammakettenzytokinen IL-2, IL-15 und IL-21 verglichen und stellte sich hinsichtlich der NK-Zellexpansionsraten, der Degranulationskapazitaet und der damit verbundenen Zytotoxizitaet als den anderen Protokollen ueberlegen heraus.
Zytokinexpandierte NK-Zellen zeigten eine hoehere Rezeptorexpression an ihren Oberflaechen als unstimulierte Zellen. Die Expansion mit dem IL-15+21boost-Protokoll bewirkte die hoechste Dichte des Todesrezeptors TRAIL, jedoch auch der inhibitorischen KIR2D-Rezeptorfamilie. Fuer andere Oberflaechenmarker ergab sich jeweils eine mittlere Expressionsdichte verglichen mit dem IL-15- bzw. dem IL-15+21-Expansionsprotokoll. Die Sekretion von proinflammatorischen Zytokinen wie Interferon-gamma (IFN-g) und Tumor-Nekrose-Faktor-alpha (TNF-a) wurde zudem verstaerkt durch IL-21 angeregt, aber ebenso die Sekretion des immunsupprimierenden IL-10.
Weiter wurden die zytoinexpandierten NK-Zellen zur UEberpruefung ihrer in vivo Funktionalitaet anhand eines praeklinischen Xenograftmodells unter Verwendung von NOD SCID IL-2-Rgamma-/- (NSG) Maeusen und der Technologie der in-vivo-Biolumineszenzbildgebung getestet. Dabei konnte beobachtet werden, dass die NK-Zellen das Wachstum luciferaseexprimierender humaner Rhabdomyosarkome verlangsamten. Die Wirksamkeit der IL-15+21boost-expandierten NK-Zellen zeigte sich vor allem in einem kombinierten Ansatz, bei dem die Tumore zunaechst mit ionisierender Strahlung behandelt wurden und residuale Rhabdomyosarkomzellen anschliessend durch den adoptiven Transfer von humanen NK-Zellen in ihrem Wachstum gehemmt waren, solange die NK-Zelltherapie andauerte. Somit stellte sich die Kombination aus Bestrahlung und NK-Zelltransfer als wirksamer im Einsatz gegen Rhabdomyosarkome heraus als die alleinige Behandlung der Tumore durch Radiotherapie.
Zusammengefasst konnte in dieser Arbeit ein NK-Zellexpansionsprotokoll entwickelt werden, dass durch den ausschliesslichen Einsatz von Gammakettenzytokinen zu einem funktionalen NK-Zellprodukt fuehrte, welches auch in vivo lytische Aktivitaet gegenueber Rhabdomyosarkomzellen aufwies.
Hämophilie A ist eine X-chromosomal rezessiv vererbte Krankheit, die aufgrund von Mutationen innerhalb des Gens von Gerinnungsfaktor VIII (FVIII) zum funktionellen Defekt oder zum Fehlen des körpereigenen FVIII führt. FVIII zirkuliert als Heterodimer und besteht aus einer schweren Kette mit der Domänenstruktur A1-A2-B und einer leichten Kette mit der Domänenstruktur A3-C1-C2. Bei Patienten unter Prophylaxe wird durch regelmäßige Substitution mit rekombinanten oder aus Plasma gewonnenen FVIII-Präparaten die Hämostase wiederhergestellt. Allerdings entwickeln hierbei etwa 30% der Patienten mit einer schweren Hämophilie eine FVIII-spezifische Immunantwort in Form von neutralisierenden Antikörpern (Inhibitoren). Die sogenannte Immuntoleranz-Therapie (engl. immune tolerance induction therapy, ITI) ist bisher die einzige etablierte Therapie, die zu einer dauerhaften Eradikation der FVIII-Inhibitoren und Induktion von Toleranz gegenüber FVIII führen kann. Die Therapie beruht auf einer meist täglichen Gabe hoher FVIII-Dosen, welche sich, je nach Behandlungsdauer, über Wochen bis hin zu Jahren erstrecken kann. Bei etwa 30% der Patienten ist diese Therapie nicht erfolgreich. Für solche Patienten besteht die Gefahr lebensbedrohlicher, unkontrollierbarer Blutungen und erheblicher Gelenkschäden.
Die spezifische Ansteuerung des Membran-gebundenen Immunglobulin G (mIg) des B-Zellrezeptors (BZR) mithilfe von Immuntoxinen ist eine mögliche Option zur selektiven Eliminierung FVIII-spezifischer B-Zellen und somit zur Eradikation von FVIII-Inhibitoren. Solche Immuntoxine bestehen aus einer zellbindenden und einer zytotoxischen Domäne, welche nach Internalisierung zur Apoptose der Zielzelle führen soll. Da FVIII aufgrund der Größe als zellbindende Domäne ungeeignet ist, beschäftigt sich die vorliegende Arbeit mit der Entwicklung und Evaluierung alternativer Immuntoxine zur selektiven Eliminierung FVIII-spezifischer B-Zellen. Die FVIII-spezifische Immunantwort ist zwar polyklonal, jedoch vor allem gegen A2- und die C2-Domäne gerichtet. Aus diesem Grund wurden die humane A2- und C2-Domäne (hA2, hC2) als zellbindende Domäne verwendet und jeweils genetisch an eine verkürzte Version des Exotoxin A (ETA) aus Pseudomonas aeruginosa fusioniert, bei welcher die natürliche zellbindende Domäne entfernt wurde. Die rekombinanten Proteine wurden bakteriell produziert und im Anschluss an die Aufreinigung biochemisch charakterisiert. Während das bakterielle Expressionssystem für hA2-ETA nicht geeignet war, konnte hC2-ETA neben weiteren Kontrollproteinen mit korrekter Konformation der hC2-Domäne hergestellt und aufgereinigt werden.
Die Fähigkeit zur selektiven Eliminierung hC2-spezifischer B-Zellen wurde im weiteren Verlauf sowohl in vitro mithilfe einer hC2-spezifischen Hybridomazelllinie als auch ex vivo und in vivo mithilfe von Splenozyten aus FVIII-immunisierten FVIII-knockout Mäusen untersucht.
Durch Inkubation der hC2-spezifischen Hybridomazelllinie mit hC2-ETA konnten ca. 38 % der Zellen eliminiert werden. Weitere Untersuchungen der Zelllinie ergaben, dass diese keinen vollständigen funktionalen B-Zellrezeptor auf der Oberfläche exprimierte, welcher für die Bindung und die korrekte Internalisierung des Immuntoxins notwendig ist. Aufgrund dessen eignet sich diese Zelllinie nicht als Modell für eine genauere Analyse der in vitro Eliminierungseffizienz von hC2-ETA.
Weitere Analysen mithilfe von Splenozyten aus FVIII-immunisierten FVIII-knockout Mäusen haben jedoch gezeigt, dass durch ex vivo Inkubation der Splenozyten mit hC2-ETA, alle hC2-spezifischen B-Zellen vollständig, selektiv und konzentrations-abhängig eliminiert werden konnten. Auch die mehrfache Applikation von hC2-ETA in FVIII-immunisierten FVIII-knockout Mäusen führte bei der Hälfte der Tiere zur vollständigen Eliminierung aller hC2-spezifischen B-Zellen. Eine Reduktion des hC2-spezifischen Antikörpersignals konnte nach Gabe von hC2-ETA in allen behandelten Tieren beobachtet werden. Die unvollständige Eliminierung in der Hälfte der Tiere ist vermutlich auf die Präsenz hC2-spezifischer Antikörper zurückzuführen, die einen Teil des applizierten Immuntoxins neutralisiert haben, sodass nicht alle hC2-spezifischen Gedächtnis-B-Zellen erreicht und eliminiert werden konnten. Um die Eliminierungseffizienz von hC2-ETA weiter zu erhöhen, müsste das Behandlungsprotokoll geändert werden. Sowohl eine Verlängerung des Behandlungszeitraums als auch eine kombinierte Therapie aus FVIII und hC2-ETA sollte zu einer erhöhten Bioverfügbarkeit des Toxins und dadurch zu einer gesteigerten Eliminierungseffizienz führen.
Die Ausweitung des hier vorgestellten Ansatzes auf weitere FVIII-Domänen ist generell möglich, jedoch muss hierzu ein alternatives Expressionssystem aufgrund des eukaryotischen Ursprungs von FVIII in Betracht gezogen werden. Die hier vorgestellten Ergebnisse zeigen dennoch, dass FVIII-Domänen-Immuntoxine ein wirkungsvolles Mittel sind, um FVIII-spezifische B-Zellen selektiv zu eliminieren. Die Anpassung der Gabe von FVIII-Domänen-Immuntoxinen an die individuelle Immunantwort des Patienten könnte das Auftreten von Nebenwirkungen minimieren. Außerdem könnte eine kombinierte Therapie aus ITI und FVIII-Domänen-Immuntoxinen die Zeit bis zur Induktion von Toleranz verkürzen und die Chancen für den generellen Therapieerfolg erhöhen.
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.
To date, chemicals are used ubiquitous in everyday life and an increasing consumption of pharmaceuticals and personal care products and industrial chemicals results in an increased water pollution. Conventional wastewater treatment plants are not able to completely remove the variety of (polar) organic compounds from today’s wastewater and thus serve as constant key point sources for the unintentional release of (micro-)pollutants into the aquatic environment. Anthropogenic micropollutants are detectable in very low concentrations in almost every aquatic compartment and may cause adverse effects on aquatic organisms. Considering the current situation of water pollution and to enhance water quality with regard to environmental and human health, the implementation of advanced wastewater treatment technologies, such as ozonation and activated carbon filtration was extensively discussed and investigated in recent years. Yet, besides their advantages regarding the efficient removal of a variety of recalcitrant, organic compounds as well as pathogens from the wastewater, it is known that especially the treatment with ozone may lead to the formation of largely unknown ozonation by-products with often unknown toxicity and unknown threats to human and the environment. To address these topics the joint research project TransRisk aimed at the “characterization, communication and minimization of risks originating from emerging contaminants and pathogens in the water cycle”. Within this research project the present thesis focuses on the ecotoxicological investigation of emerging waterborne contaminants, including their potential transformation products (TPs). Additionally, focus was laid on the investigation of combined effects of anthropogenic contaminants and pathogens with effects especially on aquatic invertebrate organisms.
The potential ecotoxicological effects of the antiviral drug acyclovir and two of its structurally identified TPs, were investigated on three aquatic organisms (Raphidocelis subcapitata, Daphnia magna and embryos of Danio rerio). While the parent compound acyclovir caused no acute toxicity up to a tested concentration of 100 mg/l on any of the investigated organisms, both TPs were shown to exhibit an increased aquatic toxicity. Carboxy-acyclovir, the biodegradation product of acyclovir, significantly reduced reproduction of D. magna by 40% at 102 mg/l, and the ozonation product COFA significantly inhibited growth of green algae R. subcapitata (EC10 = 14.1 mg/l). In the present case, advanced wastewater treatment was shown to lead to the formation of TPs, that reveal a higher toxicity towards investigated organisms, than the parent compound. Results highlight the necessity of further research related to the topic of identification and characterization of TPs, formed during advanced wastewater treatment processes.
To investigate the potential reduction or enhancement of toxic effects of nine differently treated wastewater effluents, selected bioassays with Daphnia magna, Lumbriculus variegatus and Lemna minor were conducted in flow-through test systems on a pilot treatment plant. The different treatment processes included ozonation of conventional biological treatment, with subsequent filtration processes as well as membrane bioreactor treatment in combination with ozonation. While exposure to the conventionally treated wastewater did not result in significant impairing effects on D. magna and L. minor, a reduced abundance of L. variegatus (by up to 46%) was observed compared to the medium control. Subsequent ozonation and additional filtration of the wastewater enhanced water quality, visible in an improved performance of L. variegatus. In general, direct evidence for the formation of toxic TPs due to the advanced wastewater treatments was not found, at least not in concentrations high enough to cause measurable effects in the investigated test systems. Additionally, no evidence for immunotoxic effects of the investigated wastewater effluents were observed. Yet, study-site- and species-specific effects hindered the definite interpretation of results. That underline the importance of a suitable test battery consisting of representatives of different taxonomic groups and trophic levels, to ensure a comprehensive evaluation of the complex matrix of wastewater and to avoid false-negative or false-positive results.
With aim to improve knowledge regarding immunotoxicity in invertebrates, the potential immunotoxic effects of the immunosuppressive pharmaceutical cyclosporine A (CsA) were investigated by applying the host-parasite model system Daphnia magna – Pasteuria ramosa in an adapted host resistance assay. Co-exposure to CsA and Pasteuria synergistically affected long-term survival of D. magna. Additionally, the enhanced virulence of the pathogen upon chemical co-exposure was expressed in synergistically increased infection rates and an increased speed of Pasteuria-induced host sterilization. In conclusion, results provide evidence for a suppressed disease resistance in a chemically stressed invertebrate host, highlighting the importance of investigating the conjunction of environmental pollutants and pathogens in the environmental risk assessment of anthropogenic pollutants.
Cardiovascular diseases are still regarded as the main cause of death in the modern world. However, the generic term "cardiovascular diseases" is not uniformly defined. It essentially describes diseases of the cardiovascular system and includes diseases such as hypertension, arteriosclerosis, myocardial infarctions, heart failure, coronary heart diseases, rheumatic heart diseases and heart valve defects. In addition to the well-known risk factors such as obesity, smoking, hypercholesterolemia and lack of exercise, age is a further risk factor that plays an important role in the development of cardiovascular diseases. As the modern societies age; this becomes an increasing problem.
But why does the prevalence of cardiovascular diseases increase with age? In gen-eral, age-dependent changes at the cellular level are assumed to be responsible for the pathological changes in the cardiac and vascular tissues. Important mechanisms such as autophagy, oxidative stress, mitochondrial dysfunctions, genomic instability, cellular senescence and disturbances in signaling pathways of growth factors play a decisive role. In old age, myocardial hypertrophy occurs, which results in cardiac wall thickening and an altered geometry of the ventricle. Chronic inflammations, paracrine and age-dependent cell-intrinsic factors further lead to activation of cardiac fibro-blasts with increase cell proliferation, collagen secretion and matrix cross-linking. The consequences are interstitial and perivascular fibrosis, which stiffen the heart and blood vessels. Oxidative stress and inflammations additionally attack the blood ves-sels and impair endothelial function, which is further aggravated by possible pre-existing conditions such as diabetes mellitus and hypertension.
In the past decades, the main focus has therefore been on researching these age-dependent changes in the hope of better understanding cardiovascular ageing and developing possible regenerative interventions. By studying the repair mechanisms of other organs such as the lungs and the bone marrow, the endothelium in particular showed a high regenerative capacity, which influences the proliferation and cell func-tion of the surrounding cells.
For a long time, the general opinion was that the endothelium is only the internal lin-ing of blood and lymphatic vessels, as well as the heart chambers, which as a single-layer barrier guarantees the integrity of the blood vessels. However, endothelial cells are very heterogeneous, depending on the type of blood vessel and the type of tis-sue they serve. In addition to their barrier function, endothelial cells also regulate the exchange of substances between blood and tissue, stimulate the formation of new blood vessels and re-model existing vascular networks. They are also able to re-structure the extracellular matrix that surrounds them. They release not only matrix proteins, but also cytokines and growth factors into the extracellular space. On de-mand, these factors are then released and stimulate angiogenesis or cell prolifera-tion. In addition, the secretion of various matrix proteins not only stabilizes the cellu-lar neighborhood, but also regulates various cell functions.
By modelling the endothelial environment - the so-called vascular niche - endothelial cells are able to communicate with the surrounding cells. As a result, a regenerative effect of the vascular niche has already been described in various organs. In the liv-er, for example, it has been shown that increased concentrations of endothelial Ang2 and decreased endothelial activin A after partial hepatectomy stimulate the prolifera-tion of hepatocytes and thus liver regeneration. In the bone marrow, endothelial cells mobilize stem cells via nitric oxide and in the lungs, endothelial MMP14 releases growth factors from the extracellular matrix, which stimulate epithelial cell prolifera-tion after partial pneumectomy. Whether such a regenerative effect of the vascular niche also plays a role in the heart is largely unknown.
Since both the regenerative capacity of the heart and endothelial function decrease with age, the aim of this dissertation was to investigate the role of the vascular niche and endothelial cell communication in the aged heart. Human cell lines as well as mouse and artificial rat models were used for these investigations. Since this thesis is a cumulative dissertation with partially published papers, it is divided into three parts.
In the first part of this thesis, the transcriptional signature of secretory genes in the aged cardiac endothelium was studied. Perfused endothelial cells from hearts of young (12-week-old animals) and old mice (20-month-old animals) were isolated and used for bulk RNA sequencing. The two matrix proteins laminin β1 and β2 were among the top-regulated genes. While laminin β2 was particularly expressed in the young cardiac endothelium, laminin β1 was predominantly found in the old endotheli-um. This change in laminin expression was confirmed histologically at protein level and its autocrine function was investigated in vitro. To mimic the in vivo situation in vitro, cell culture dishes were coated with human recombinant laminin 421 or laminin 411 and sutured with human endothelial cells from the umbilical vein (HUVEC). Di-verse functional investigations showed that endothelial cells migrated and adhered poorly in the presence of laminin 411, while in Matrigel tube formation assays HU-VEC formed reduced endothelial networks when cultured on LM 411.
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The growing number of infections with multi-resistant bacteria or the current COVID-19 pandemic put compounds with therapeutic properties into the public focus. Non-ribosomal peptides (NRPs) are natural products that are already marketed as antibiotics, cytotoxic agents or immunosuppressants. Their biological activities rely on the structural diversity including non-proteinogenic amino acids (AAs), heterocycles or modifications like methylation or acylation.
The biosynthesis of NRPs is carried out by non-ribosomal peptide synthetases (NRPSs). These multifunctional megaenzymes show a modular architecture like in an assembly-line. Each module is thereby responsible for the incorporation and modification of one AA and therefore contains different catalytic domains. The adenylation (A) domain recognizes and activates its specific substrate in an ATP-dependent manner which is transferred to a 4’-phosphopantetheine cofactor post-translationally attached to the thiolation (T) domain. Peptide bond formation between two T domain bound substrates catalysed by the condensation (C) domain transfers the growing peptide chain to the following module. Such a C-A-T module can be extended with optional domains to integrate structural diversity and a terminal thioesterase (TE) domain usually releases the peptide via hydrolysis or intramolecular attack of nucleophiles. Inspired by the modular architecture, NRPS engineering deals with the modification of NRPs in order to increase biological activities, circumvent bacterial resistances or create de novo peptides. This can be achieved by mutasynthesis or modification of the substrate binding pocket as well as single and multiple domain substitution. However, the few successful approaches led to impaired enzymes and did not establish a general applicable guideline. In the first publication as part of this work, the development of such a guideline comprising three rules is addressed. First, the A-T-C tridomain named exchange unit (XU) is seen as a catalytic unit instead of a module. When using them as building blocks, the C domain’s specificity for the AA of the following XU has to be considered as second rule. Third, a conserved WNATE motif within the C-A linker depicts the fusion point of the XUs. Upon heterologous expression of the cloned plasmids in E. coli and high performance liquid chromatography coupled mass spectrometry-based analysis of the extracts, the ambactin-producing NRPS from Xenorhabdus was reprogrammed with one and two XUs. This only leads to a moderate loss of production titre or an even higher one when the AA configuration was changed by introducing a dual condensation/epimerization (C/E) domain. The pentamodular GameXPeptide-producing NRPS was reconstructed using up to five XUs of four different NRPSs and even completely de novo synthetases were created. The second publication describes the exchange unit condensation domain (XUC) concept and relies on a fusion point between the two subdomains (N-terminal CDsub and C-terminal CAsub) of the C domain’s V-shaped pseudodimeric structure which generates A-T didomains with flanking CAsub and CDsub. These hybrid C domain-forming building blocks depict an improvement to the XU concept by avoiding the drawback of C domain specificity. This allows a more flexible NRPS engineering that can e.g. enable peptide library design. Furthermore, beside a combination of both concepts within one NRPS and a transfer to Bacillus NRPSs, the use of XUC with relaxed A domain specificity allowed further peptide modifications by introducing non-natural AAs. The third publication deals with aldehyde and alcohol-generating reductase (R) domains which depict an alternative for peptide release in NRPSs. A promoter exchange in X. indica identified a pyrazine-producing NRPS with a minimal architecture of an A, T and R domain and was therefore termed ATRed. R domains were additionally used in engineered NRPSs to produce pyrazinones and derivatives thereof by XU substitution although most constructs failed to show production. Beyond that, an R domain has been shown to replace a TE domain in wild type synthetases leading to slightly modified NRPs and the postulated biosynthesis was incidentally revised. Furthermore, an NRPS with terminal R domain was engineered to produce a free peptide aldehyde, which are known to be potent proteasome inhibitors. For the above mentioned ATReds, the presence of up to three coding regions was further identified in 20 different Xenorhabdus strains but only six of them were verified to produce pyrazines. All ATReds share variable sequence similarities among each other and were subsequently divided into three subtypes. One subtype is supposed to perform the pyrazine biosynthesis via a non-canonical catalytic triad.
Auf der Oberfläche von Erythrozyten, Thrombozyten und Neutrophilen befinden sich mehrere hundert verschiedene polymorphe, ungekoppelt vererbte Blutgruppenantigene. Dementsprechend birgt jede Bluttransfusion das Risiko einer Immunisierung gegen fremde Blutgruppenmerkmale. Auch während der Schwangerschaft können aufgrund väterlich vererbter Antigene Alloantikörper induziert werden. Deshalb muss das Blut vor jeder Transfusion oder während einer Schwangerschaft auf das Vorhandensein irregulärer erythrozytärer Antikörper untersucht werden. Dabei greifen die aktuellen diagnostischen Verfahren auf primäre, stabilisierte Testerythrozyten von Blutspendern zurück, deren relevante Blutgruppenantigene bekannt sind. Antikörperspezifitäten können anhand von Agglutinationsreaktionen der Testzellen mit dem zu untersuchenden Patientenplasma auf ein oder mehrere Antigene zurückgeführt werden. Ist jedoch ein Antikörper gegen ein häufiges, ein hochfrequentes oder ein nicht-polymorphes, ubiquitäres Antigen gerichtet, kann in Ermangelung Antigen-negativer Testzellen keine adäquate Diagnostik gewährleistet, die Verträglichkeit der Transfusion also nicht definitiv sichergestellt werden. Auch der medizinische Einsatz therapeutischer Antikörper, welche Antigene adressieren, die auch auf Erythrozyten exprimiert werden, führt zunehmend zu Problemen. Tests auf granulozytäre Antikörper sind mangelhaft bezüglich ihrer Robustheit, besitzen eine unzureichende Auflösung und sind zudem meist zeitaufwändig und daher teuer. Antikörper gegen humane Plättchenantigene spielen insbesondere in der Schwangerschaft eine Rolle; sie vermögen bei Neugeborenen thrombozytopenische Blutungen bis hin zu massiven Hirnblutungen zu verursachen, die zu schweren Entwicklungsstörungen führen können. Bisher erfolgt jedoch mangels geeigneter Reagenzien keine standardisierte pränatale Untersuchung auf thrombozytäre Antikörper. In dieser Arbeit wurde ein neuartiges Verfahren für die Identifikation und Differenzierung irregulärer Blutgruppenantikörper etabliert, welches auf gentechnisch hergestellten, xenogenen Testzellen basiert, die einzelne definierte humane Blutgruppenantigene auf ihrer Oberfläche präsentieren. Die nicht humanen Zellen co exprimieren Fluorochrome, anhand derer Antikörper-markierte Testzellen durchflusszytometrisch voneinander unterscheidbar sind. Weiterhin können die generierten Testzellen zur Depletion von Antikörpern aus polyagglutinierenden Plasmen unter Erhalt der anderen Antikörperspezifitäten verwendet werden. Diese Technologie könnte die konventionelle Diagnostik erheblich erleichtern und bietet zudem die Möglichkeit, therapeutische Antikörper (wie z. B. anti-CD38, anti CD47, etc.), die häufig zu Interferenzen mit der Routinediagnostik führen, spezifisch prädiagnostisch aus Patientenproben zu entfernen.
This dissertation aimed to shed light on changes of the epigenetic landscape in heart and skeletal muscle tissue of the turquoise Killifish N. furzeri, a novel, short-lived animal model for aging research. The following results could be obtained:
1. A global trend towards closed chromatin conformation could be observed; histone markers for H3K27me3, H3K9me3 and H4K20me3 accumulated in skeletal muscle tissue from old N. furzeri. Markers for open chromatin conformation such as H3K4me3, H3K9ac and H4K16ac decreased in old skeletal muscle tissue. In old hearts from N. furzeri an accumulation of H3K27me3 could be detected while H3K9ac was found to increase with age as well. mRNA expression levels of methylating enzymes were higher in skeletal muscle tissue from old N. furzeri when compared to expression levels in skeletal muscle tissue from young N. furzeri.
2. The shift of epigenetic pattern was accompanied by a change of gene expression. Via mRNA sequencing in collaboration with the MPI, Bad Nauheim it could be shown that genes associated with cell cycle and DNA repair were lower expressed in skeletal muscle tissue from old N. furzeri than in tissue from young N. furzeri. Genes, associated with inflammatory signaling and glycolysis, displayed increased mRNA levels in skeletal muscle tissue from old N. furzeri. These results could be confirmed by Western blot and qRT-PCR analyses.
3. Markers for DNA damage and senescence increased in skeletal muscle tissue from old N. furzeri.
4. Cells derived from young and old N. furzeri skeletal muscle could be isolated and cultured for many passages. These cells were a mix of different cell types with properties and features of the native tissue. They could be used for treatment with drugs and/small compounds modulating the epigenetic landscape via specific interference with methylating enzymes.
5. DNA methylation and hydroxy-methylation were found to go in different directions in skeletal muscle and heart tissue from N. furzeri: while increasing in skeletal muscle tissue, a both DNA modifications declined in heart tissue with age.
6. In the heart of N. furzeri microRNA expression changes with age were assed with sequencing in collaboration with the FLI, Jena. It could be demonstrated that miRNA expression is age-dependent. Particular focus was on miR-29 and its target genes: miR-29 was highly upregulated in heart and skeletal muscle tissue, while target genes such as collagens and dnmts were reduced with age in the heart of N. furzeri.
7. Cardiac function remained stable with age and no accumulation of collagens could be found when comparing hearts of young and old N. furzeri despite the increase of markers for oxidative stress.
8. Cell culture experiments with human cardiac fibroblasts revealed that miR-29 is upregulated with increasing age of the donor. In addition to that, it could be shown that miR-29 is positively regulated by oxidative stress.
9. A zebrafish mutant with modified expression of miR-29 that was created in collaboration with the SNS, Pisa, presented a severe hypoxic phenotype and an altered mRNA expression profile compared to wild type control zebrafish. Cardiac dysfunction and hypertrophy were observed as well as an increase in DNA methylation and collagens.
Taken together, it could be shown that the aging process in skeletal muscle and heart tissue from N. furzeri leads to a series of changes on epigenetic levels. It remains to be elucidated whether these changes are result or cause for further changes of mRNA expression, protein levels and pathophysiology, yet the N. furzeri represents a promising research model for further aging studies.
European pea crabs - taxonomy, morphology, and host-ecology (Crustacea: Brachyura: Pinnotheridae)
(2010)
Pinnotherids are small crabs symbiotic to a variety of invertebrates. The European species infest bivalves and sea squirts. Their way of life is parasitic and poses a threat to commercially exploited bivalves. While juveniles of both sexes still look very similar - being agile swimmers and partially free living - a metamorphosis takes place in the female after mating and results in a conspicuous sexual dimorphism. Thereafter, the female settles in its host definitely and is morphologically strongly adapted to the parasitic life phase. A very high reproductive output was demonstrated among several pea crab species infesting bivalves. Despite from that, hardly any information is present in the literature on the pinnotherids’ reproductive biology and the underlying morphology.
Due to their cryptic way of life, the sexual dimorphism, and the different morphotypes of the female, the taxonomy of the Pinnotheridae is a serious challenge. Two widely accepted species are recognized on European coasts: Pinnotheres pisum and Nepinnotheres pinnotheres. Pinnotheres pectunculi was so far only known from the bivalve Glycymeris glycymeris in its type locality Roscoff (France), while Pinnotheres ascidicola and Pinnotheres marioni were described as living exclusively in ascidians without careful comparison with the previously described species. In order to produce standardized comparative descriptions, pea crabs were collected and studied from different hosts and localities in the Northeast Atlantic and in the Mediterranean. Nepinnotheres pinnotheres and Pinnotheres pisum were redescribed with consideration to characters of female and male. According to our morphological analysis, Pinnotheres ascidicola and Pinnotheres marioni are junior synonyms of Nepinnotheres pinnotheres, whereas the status of Pinnotheres pectunculi as a valid species was ascertained. Important characters are the mouthparts, the male gonopods, and especially chelipeds that showed consistent characteristics among different crab stages of both sexes.
Based on our sampling, we estimated the host-range of the European species. Nepinnotheres pinnotheres lives in ascidians and in the pen shell Pinna nobilis. Pinnotheres pisum infests numerous bivalve species - Pinna nobilis included. For Pinnotheres pectunculi novel host records are presented, all from the bivalve family Veneridae. Furthermore, feeding of the Pinnotheres-species was observed. They use a setae comb ventrally on the claw to brush mucus (and the accumulated food particles) from the bivalve gills. Feeding strategies and host-ecology will be thoroughly discussed in consideration to other Pinnotheridae.
We investigated the reproductive systems of European pinnotherids by histological methods, scanning and transmission electron microscopy, and confocal laser scanning microscopy.
The Eubrachyura have internal fertilization: paired vaginas enlarge into storage structures, the spermathecae, which are connected to the ovaries by oviducts. Sperm is stored until the oocytes are mature and transported into the spermathecae, where fertilization takes place. In the investigated pinnotherids, the vagina is of the ‘concave pattern’. Musculature is attached alongside flexible parts of the vagina-wall to control the dimension of its lumen. The genital opening is closed by a muscular mobile operculum.
The spermatheca can be divided into two distinct regions by function and morphology. The ventral part includes the connection with vagina and oviduct and is regarded as the zone where fertilization takes place. It is lined with cuticle except where the oviduct enters the spermatheca by the ‘holocrine transfer tissue’. At ovulation, the oocytes have to pass through this multi-layered glandular epithelium, which has a holocrine mode secretion. The dorsal part of the spermatheca is lined by a highly secretory apocrine glandular epithelium, which was to date only found in fiddler crabs of the genus Uca.
The male internal reproductive system consists of paired testes and corresponding vasa deferentia. The sperm morphology of pinnotherids conforms to other thoracotremes, with slight differences between Nepinnotheres pinnotheres and Pinnotheres pisum. Spermatozoa become enveloped into spermatophores in the secretory proximal vas deferens. The medial vas deferens is strongly enlarged and stores spermatophores embedded in seminal plasma. The distal vas deferens holds tubular appendices, which extend into the ventral cephalothorax and slightly into the pleon. These appendices produce and store vast quantities of seminal plasma. The copulatory system of the Brachyura is formed by paired penes and two pairs of gonopods, which function in sperm transfer. In pinnotherids, the long first gonopods transfers the sperm mass to the female. It holds the ejaculatory canal inside, which opens proximally and distally. The second gonopod is solid, short and conical. During copulation, the penis and the second gonopod are inserted into the base of the tubular first gonopod. The second gonopod functions in the transport of the sperm mass inside the ejaculatory canal towards its distal opening. The specific shape of the second gonopod is strongly adapted for a sealing of the tubular first gonopod with longitudinal cuticle foldings that interlock inside the first gonopod. The presented results are discussed concerning their function in reproduction and in respect of the systematic account.
The role of secretion in sperm transfer, storage and fertilization among the Brachyura is still under debate. It is notable that structure and function of secretion are more complex in pinnotherids and probably more efficient than in other brachyuran crabs, which will be discussed, in view of the parasitic way of life and the high fecundity of pinnotherids.
Introduction:
The evolutionary patterns of symbiotic organisms are inferred using cophylogenetic methods. Congruent phylogenies indicate cospeciation or host-switches to closely-related hosts, whereas incongruent topologies indicate independent speciation. Recent studies suggest that coordinated speciation is a rare event, and may not occur even in the highly specialized associations. The cospeciation hypothesis was mainly tested for free-living mutualistic associations, such as plant-pollinator interactions, and host-parasitic systems but was rarely tested on obligate, mutualistic associations involving intimate physiological interactions. Symbionts with lower partner selectivity may not experience coordinated speciation due to frequent switching of partners. On the other hand, symbionts with high partner selectivity may influence each other’s evolution owing to the highly interdependent lifestyles. Symbiont association patterns are also influenced by habitat and it has been proposed that symbiotic interactions are stronger in warm regions as compared to cooler regions (also referred as latitudinal gradient of biotic specialization). This hypothesis however, has recently been challenged and it has been suggested that a gradient of biotic specialization may not exist at all. Reliable species concepts are a prerequisite for understanding the association and evolutionary patterns of symbiotic organisms. The species concepts of many groups traditionally relied on the morphological species concept, which may not be adequate for distinguishing species due to the: i) homoplasious nature of morphological characters, an due to the inability to distinguish cryptic species. Thus phylogenetic species concept along with coalescent-based species delimitation approaches, which utilize molecular data for inferring species boundaries have been used widely for resolving taxonomic relationships. Lichens are obligatory symbiotic associations consisting of a fungal partner (mycobiont) and one or more photosynthetic partners, algae, and/or cyanobacteria (photobionts). I used the lichen forming fungal genus Protoparmelia as my study system, which consists of ~25-30 previously described species inhabiting different habitats, from the arctic to the tropics. This makes Protoparmelia an ideal system to explore the association and evolutionary patterns across different macrohabitats.
Objectives:
The objectives of this thesis were to 1. Elucidate the phylogenetic position of Protoparmelia within Lecanorales, and infer the monophyly of Protoparmelia; 2. Understand species diversity within Protoparmelia s.str. using coalescent-based species delimitation approaches; and 3. To identify the Trebouxia species associated with Protoparmelia using phylogenetic and species delimitation approaches and to infer the association and cophylogenetic patterns Protoparmelia and Trebouxia in different macrohabitats.
Results and discussion:
Chapter 1: Taxonomic position of Protoparmelia
In the first part of this study I explored the taxonomic position of Protoparmelia within the order Lecanorales. Overall this study included 54 taxa from four families, sequenced at five loci (178 sequences). I found Protoparmelia to be polyphyletic and sister to Parmeliaceae.
Chapter 2: Multilocus phylogeny and species delimitation of Protoparmelia spp.
In this part of the study, I identified and delimited the Protoparmelia species forming a monophyletic clade sister to Parmeliaceae i.e., Protoparmelia sensu stricto group, based on the multilocus phylogeny and coalescent-based species delimitation approaches. I included 18 previously described and three unidentified Protoparmelia species, which represents ~70% of the total described species, and 73 other taxa, sequenced at six loci. I found that the sensu stricto group comprised of 25 supported clades instead of 12 previously described Protoparmelia species. I tested the speciation probabilities of these 25 clades using species delimitation softwares BP&P and spedeSTEM. I found nine previously unrecognized lineages in Protoparmelia and I propose the presence of at least 23 species for Protoparmelia s.str., in contrast to the 12 described species included in the study.
Chapter 3: Association and cophylogenetic patterns of Protoparmelia and its symbiotic partner Trebouxia
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Die CXCR4/CXCL12-Achse ist von entscheidender Bedeutung für die Entstehung und Aufrechterhaltung einer gesunden, reifen Hämatopoese. Erstmals beschrieben wurde der später als CXCR4 bezeichnete Rezeptor 1996 allerdings als Co-Rezeptor für den Eintritt humaner HI-Viren in Lymphozyten. Ein großes Interesse bestand daraufhin darin, sowohl natürliche Inhibitoren des G-Protein gekoppelten Rezeptors zu identifizieren, als auch synthetische herzustellen, um einen Eintritt des Virus in den menschlichen Organismus zu verhindern bzw. seine Ausbreitung zu unterbinden. Ein natürlich vorkommender CXCR4-Ligand, der 2015 von Zirafi und Kollegen erstmals beschrieben wurde, fand sich im Hämofiltrat von Dialysepatienten. Der im weiteren Verlauf als EPI-X4 bezeichnete CXCR4-Antagonist wurde als Spaltprodukt von Albumin identifiziert, welches über viele Spezies hochkonserviert ist. Diese Eigenschaft interpretieren wir als Hinweis auf eine relevante physiologische Funktion des Peptids. Da die Halbwertszeit von natürlich vorkommendem EPI-X4 beim Menschen vermutlich sehr kurz ist, sind in vivo- und darauffolgende in vitro-Analysen schwierig durchzuführen. In-vitro-Spike-Analysen von synthetischem EPI-X4 in humanem Plasma ergaben eine Halbwertszeit von nur 17 Minuten. Die geringen auftretenden Konzentrationen erschweren die Problematik zusätzlich. In dieser Arbeit sollen deshalb im Mausmodell in vivo-Analysen durchgeführt werden, um die Effekte von potentiell entstehendem EPI-X4 in verschiedenen experimentellen Ansätzen aufzudecken. Ein probates, hier verwendetes Mittel, ist die Analyse einer Knock-out (KO)-Maus. Die für die Bindung an CXCR4 entscheidende Aminosäure von EPI-X4, das am N-Terminus gelegene Leucin, wurde durch Alanin ersetzt, welches die Entstehung von EPI-X4 unterbindet und zusätzlich dessen Bindung an CXCR4 verhindert. Mit Hilfe zweier Mausmodelle können nun Analysen im EPI-X4-defizienten Modell durchgeführt werden, die im Umkehrschluss Informationen über die organismische Wirkung von EPI-X4 beinhalten. Zunächst wurde in beiden Modellen die physiologisch normale reife und unreife Hämatopoese charakterisiert. Hierbei zeigte sich kein signifikanter systematischer Einfluss von EPI-X4 auf reife Leukozyten (WBC), lediglich eine leichte Lymphozytose in der HR-Ala-Variante. Im weiteren Verlauf der homöostatischen Analyse der Hämatopoese der Ala-EPI-X4-Mäuse zeigten sich keine signifikanten Unterschiede zu wildtypischen Mäusen. Sowohl reife als auch unreife Zellen zeigten, außer in der T- und B-Zelllinie, keine zahlenmäßigen oder funktionalen Auffälligkeiten, weder im Blut, noch in der Milz oder im Knochenmark. Analysen der Zellzyklusaktivität unterschiedlicher Unreifestufen wiesen ebenfalls keine Auffälligkeiten auf. Diese Daten einer normalen, von einer C57Bl/6-Maus zu erwartenden Ergebnisse dienten als Grundlage zur Bewertung und Analyse von durchgeführten hämatopoetischen Stressmodellen. Hierfür wurden
zunächst hämatopoetische Stamm- und Vorläuferzellen (HSPC) mobilisiert. In den angewandten Mobilisierungsmodellen fanden sich lediglich unter G-CSF-Behandlung im Knochenmark eine größere Anzahl Granulozyten, was auf einen Einfluss von EPI-X4 auf HSPC schließen lässt. Um potentielle Auswirkungen von EPI-X4 im Knochenmark weiter zu untersuchen, wurde ein weiteres Stressmodell gewählt, welches ebenfalls mutmaßlich die Bedingungen zur EPI-X4-Generierung schafft: Subletale Bestrahlung der Mäuse sorgt für Schäden an allen Zellarten im Knochenmark, es wird ein steriles entzündliches Milieu kreiert. Unter diesen Umständen wurde die Regeneration von Blutzellen analysiert. Es zeigten sich keine nennenswerten Unterschiede sowohl in der akuten Phase des Schadens als auch in regelmäßigen Blutentnahmen während der Regenerierung.
Die Beschreibung von natürlich vorkommendem EPI-X4 in Vaginal- und Rektalschleimhaut zeigt seine Entstehung an Schleimhautbarrieren auf. Ala-EPI-X4-Muse werden deshalb auf deren Durchlässigkeit untersucht: LPS-Konzentrationen als Marker für eindringende pathogene Bakterien wurden im Plasma untersucht. Hierbei zeigten sich keine Unterschiede zwischen den Gruppen, eine Störung scheint hier nicht vorzuliegen. Zusätzlich wurde die Zusammensetzung des Mikrobioms im Darm untersucht, da beschrieben wurde, dass sich Mikrobiom und die Integrität der Darmschleimhaut gegenseitig beeinflussen. Im Falle der EPI-X4-defizienten Mäuse liegt zwar keine offensichtliche pathologische Veränderung vor, dennoch konnte in männlichen HR-Ala-Mäusen die Abwesenheit des Proteobakteriums Parasutterella nachgewiesen werden. Um eine mögliche Defizienz der Barrierefunktion weiter zu testen, wurden zwei Stressmodelle gewählt: Zunächst wurde den Mäusen eine akute, sterile Peritonitis zugefügt, woraufhin die Anzahl und Zusammensetzung der ins Peritoneum einströmenden Leukozyten analysiert wird. Die Reaktion auf diesen Entzündungsprozess war nicht verändert. Ähnliche Ergebnisse zeigten sich auch in einem akuten Colitis-Stressmodell.
Insgesamt konnte in dieser Arbeit mithilfe zweier KO-Mausmodelle die Rolle von EPI-X4 in der Hämatopoese und der Immunologie von Mäusen beginnend charakterisiert werden. Die homöostatische Hämatopoese scheint kaum von EPI-X4 abhängig zu sein, lediglich die Zahl der B- und T-Zellen, insbesondere der regulatorischen T-Zellen, scheint beeinflusst. Damit einhergehend konnten Veränderungen in Zytokinlevels bei inflammatorischen Ereignissen gezeigt werden. Experimente zur beeinflussten, eventuell gestörten Barrierefunktion von Ala-EPI-X4-Mäusen zeigten vielversprechende Ansätze und sollten in Zukunft weiter analysiert werden.
The objectives of this thesis were to understand how distinct classes of cell types interact to shape oscillatory activity in cortical circuits of the turtle. We chose the turtle cortex as a model system for cortical computations for two reasons. One is that the phylogenetic position of turtles makes their cortex functionally and anatomically particularly interesting. The second is that reptilian brains present several unique experimental advantages. Turtles have a three-layered cortex that forms the dorsalmost part of their pallium and receives direct input from visual thalamus. Thus turtle cortex, while sharing several features with mammalian cortices, constitutes a simpler system for studying cortical computations and dynamics. Freshwater turtles are semiaquatic species, that dive for hours and hibernate for months without breathing. Their brains are adapted to these behaviors so that they can operate under severe anoxia. This property allows for ex vivo wholebrain and whole-cortex (”cortical slab”) preparations in vitro, enabling the use of many sophisticated techniques for monitoring activity in parallel.
I thus set out to utilize the advantages of our model system, by using optogenetic methods to reliably evoke oscillations in an ex vivo whole-cortex preparation while observing activity in parallel with planar multi-electrode arrays (MEA), linear silicon depth-electrodes and patch-clamp recording techniques. This required several technical aspects to be solved. Prior work in turtle cortex (Prechtl, 1994; Prechtl et al., 1997; Senseman and Robbins, 2002) indicated that visual stimuli evoke complex activity patterns (e. g. wave patterns) in dorsal cortex. The goal was to examine these dynamics in detail and to provide mechanistic explanations for them whenever possible. The recent advent of optogenetics, the development of microelectrode arrays, and the possibility to combine these techniques with classical electrophysiological approaches on a resistant, accessible and stable preparation led me to explore a number of technical avenues.
First I had to establish gene delivery methods in reptiles. I settled on recombinant viruses, and show results from several serotypes of adeno-associated virus (AAV), i lentivirus and rabies virus. I report successful gene expression of genes of interest with several subtypes of AAV, including the commonly used AAV2/1 and AAV2/5 serotypes. Second I had to find promoters enabling global and cell-type specific gene expression in reptiles. Ubiquitous high-yield promoters such as CAG/CB7 or CMV drive high levels of expression in turtles; cell-type specific promoters such as hSyn (expression limited to neurons) and CaMKIIa (expression limited exclusively o mostly to excitatory neurons) appear similarly biased in turtles. Other cell-type specific promoters reported in the literature (fNPY, fPV, fSST) failed to express in turtles.
A second major aspect of my work focused on electrophysiological recordings using microelectrode arrays and the interpretation of extracellular signals recorded from cortex in ex vivo preparations. We observed that spike signals produced by pyramidal and inhibitory neurons were very often followed by a slower potential. We identified these slower potentials as reflections of synaptic currents, and thus of the axonal projections of the neurons, at least within the deep layers of cortex. This also resulted in a means to classify neurons as excitatory or inhibitory with much higher reliability than classical methods (e. g. spike width). The final aspect of my work concerns the use of optogenetics to dissect the mechanisms of cortical oscillations and wave propagation. I show that oscillations can be induced by light in turtle cortex after transfection with AAV2/1 carrying the gene for channelrhodopsin 2 (ChR2). By using the CaMKIIa promoter, ChR2 induced currents are limited to LII/III excitatory cells; we can therefore control excitatory drive to cortical networks. If this drive is strong enough, layer III inhibitory interneurons are recruited and fire in a concerted fashion, silencing the excitatory population. The visually evoked 20 Hz oscillations observed in chronically recorded animals (Schneider, 2015) or in anaesthetized animals (Fournier et al., in press) thus appear to result from a feedback loop between E and I cells within layers II & III. Details of these interactions are being investigated but - layer I interneurons, by contrast, do not seem to be involved. By pulsing light I could control the frequency of the oscillations within a range of several Hz around the natural oscillation frequency. Above this range, cortex could only follow the stimulus at a fraction (1/2, 1/3,...) of the light pulse frequency. Using a digital micromirror device, I limited activation of the cortical networks spatially, enabling the study of wave propagation in this system.
Reptilian cortex offers a relatively simple model system for a reductionist and comparative strategy on understanding cortical computations and dynamics. Turtle dorsal cortex could thus give fundamental insights to the primordial organization tional, computational and functional principles of cortical networks. These insights are relevant to our understanding of mammalian brains and may prove valuable to decipher fundamental questions of modern neuroscience.
Exploring strategies to improve the reverse beta-oxidation pathway in Saccharomyces cerevisiae
(2024)
Microbes are the most diverse living organisms on Earth, with various metabolic adaptations that allow them to live in different conditions and produce compounds with different chemical complexity. Microbial biotechnology exploits the metabolic diversity of microorganisms to manufacture products for different industries. Today, the chemical industry is a significant energy consumer and carbon dioxide emitter, with processes that harm natural ecosystems, like the extraction of medium-chain fatty acids (MCFAs). MCFAs are used as precursors for biofuels, volatile esters, surfactants, or polymers in materials with enhanced properties.
However, their current extraction process uses large, non-sustainable monocultures of coconut and palm trees. Therefore, the microbial production of MCFAs can help reduce the current environmental impact of obtaining these products and their derivatives.
In nature, fatty acids are mostly produced via fatty acid biosynthesis (FAB). However, the reverse β-oxidation (rBOX) is a more energy-efficient pathway compared to FAB. The rBOX pathway consists of four reactions, which result in the elongation of an acyl-CoA molecule by two carbon units from acetyl-CoA in each cycle. In this work we used Saccharomyces cerevisiae, an organism with a high tolerance towards toxic compounds, as the expression host of the rBOX pathway to produce MCFAs and medium-chain fatty alcohols (MCFOHs).
In the first part of this work, we expanded the length of the products from expressing the rBOX in the cytosol and increased the MCFAs titres. First, we deleted the major glycerol-3-phosphate dehydrogenase (GPD2). This resulted in a platform strain with significantly reduced glycerol fermentation and increased rBOX pathway activity, probably due to an increased availability of NADH. Then, we tested different combinations of rBOX enzymes to increase the length and titres of MCFA. Expressing the thiolase CnbktB and β-hydroxyacyl-CoA dehydrogenase CnpaaH1 from Cupriavidus necator, Cacrt from Clostridium acetobutylicum and the trans-enoyl-CoA reductase Tdter (Treponema denticola) resulted in hexanoic acid as the main product.
Expressing Cncrt2 (C. necator) or YlECH (Y. lipolytica) as enoyl-CoA hydratases resulted in octanoic acid as the main product. Then, we integrated the octanoic (Cncrt2 or YlECH) and the hexanoic acid (Cacrt)-producing variants in the genome of the platform strain and we achieved titers of ≈75 mg/L (hexanoic acid) and ≈ 60 mg/L (octanoic acid) when growing these strains in a complex, highly buffered medium. These are the highest titers of octanoic and hexanoic acid obtained in S. cerevisiae with the rBOX. Additionally, we deleted TES1 and FAA2 to prevent competition for butyryl-CoA and degradation of the produced fatty acids, respectively.
However, these deletions did not improve MCFA titers. In addition, we tested two dual acyl-CoA reductase/alcohol dehydrogenases (ACR/ADH), CaadhE2 from C. acetobutylicum and the putative ACR/ADH EceutE from Escherichia coli, in an octanoyl-CoA-producing strain to produce MCFOH. As a result, we produced 1-hexanol and 1-octanol for the first time in S. cerevisiae with these two enzymes. Nonetheless, the titres were low (<10 mg/L and <2 mg/L, respectively), and four-carbon 1-butanol was the main product in both cases (>80 mg/L). This showed the preference of these two enzymes for butyryl-CoA.
In the second part of this work, we expressed the rBOX in the mitochondria of S. cerevisiae to benefit from the high levels of acetyl-CoA and the reducing environment in that organelle. First, in an adh-deficient strain, we mutated MTH1, a transcription factor regulating the expression of hexose transporters, and deleted GPD2. This resulted in a strain with a reduced Crabtree effect and, therefore, an increased carbon flux to the mitochondria. We partially validated the increased flux to the mitochondria by expressing the ethanol-acetyltransferase EAT1 from Kluyveromyces marxianus in this organelle. This resulted in a higher isoamyl acetate production in the MTH1-mutant strain. Isoamyl acetate is synthesised by Eat1 from acetyl-CoA and isoamyl alcohol, a product of the metabolism of amino acids in the mitochondria. Then, we targeted different butyryl-CoA-producing rBOX variants to the mitochondria, and we used the production of 1-butanol and butyric acid as a proof-of-concept. The strong expression of all the enzymes was toxic for the cell, and the highest butyric acid titres (≈ 50 mg/L) in the mitochondria from the rBOX were obtained from the weak expression of the pathway. The highest 1-butanol titers (≈ 5 mg/L) were obtained with the downregulation of the mitochondrial NADH-oxidase NDI1. However, this downregulation led to a non-desirable petite phenotype.
In summary, we produced hexanoic and octanoic acid for the first time in S. cerevisiae using the rBOX and achieved the highest reported titers of hexanoic and octanoic acid so far using this pathway in S. cerevisiae. In addition, we successfully compartmentalised the rBOX in the mitochondria. However, competing reactions, some of them essential for the viability of the cell, limit the use of this organelle for the rBOX.
Exploring the in vivo subthreshold membrane activity of phasic firing in midbrain dopamine neurons
(2021)
Dopamine is a key neurotransmitter that serves several essential functions in daily behaviors such as locomotion, motivation, stimulus coding, and learning. Disrupted dopamine circuits can result in altered functions of these behaviors which can lead to motor and psychiatric symptoms and diseases. In the central nervous system, dopamine is primarily released by dopamine neurons located in the substantia nigra pars compacta (SNc) and ventral tegmental area (VTA) within the midbrain, where they signal behaviorally-relevant information to downstream structures by altering their firing patterns. Their “pacemaker” firing maintains baseline dopamine levels at projection sites, whereas phasic “burst” firing transiently elevates dopamine concentrations. Firing activity of dopamine neurons projecting to different brain regions controls the activation of distinct dopamine pathways and circuits. Therefore, characterization of how distinct firing patterns are generated in dopamine neuron populations will be necessary to further advance our understanding of dopamine circuits that encode environmental information and facilitate a behavior.
However, there is currently a large gap in the knowledge of biophysical mechanisms of phasic firing in dopamine neurons, as spontaneous burst firing is only observed in the intact brain, where access to intrinsic neuronal activity remains a challenge. So far, a series of highly-influential studies published in the 1980s by Grace and Bunney is the only available source of information on the intrinsic activity of midbrain dopamine neurons in vivo, in which sharp electrodes were used to penetrate dopamine neurons to record their intracellular activity. A novel approach is thus needed to fill in the gap. In vivo whole-cell patch-clamp method is a tool that enables access to a neuron’s intrinsic activity and subthreshold membrane potential dynamics in the intact brain. It has been used to record from neurons in superficial brain regions such as the cortex and hippocampus, and more recently in deeper regions such as the amygdala and brainstem, but has not yet been performed on midbrain dopamine neurons. Thus, the deep brain in vivo patch-clamp recording method was established in the lab in an attempt to investigate the subthreshold membrane potential dynamics of tonic and phasic firing in dopamine neurons in vivo.
The use of this method allowed the first in-depth examination of burst firing and its subthreshold membrane potential activity of in vivo midbrain dopamine neurons, which illuminated that firing activity and subthreshold membrane activity of dopamine neurons are very closely related. Furthermore, systematic characterization of subthreshold membrane patterns revealed that tonic and phasic firing patterns of in vivo dopamine neurons can be classified based on three distinct subthreshold membrane signatures: 1) tonic firing, characterized by stable, non-fluctuating subthreshold membrane potentials; 2) rebound bursting, characterized by prominent hyperpolarizations that initiate bursting; and 3) plateau bursting, characterized by transient, depolarized plateaus on which bursting terminates. The results thus demonstrated that different types of phasic firing are driven by distinct patterns of subthreshold membrane activity, which may potentially signal distinct types of information. Taken together, the deep brain in vivo patch-clamp technique can be used for the investigation of firing mechanisms of dopamine neurons in the intact brain and will help address open questions in the dopamine field, particularly regarding the biophysical mechanisms of burst firing in dopamine neurons that control behavior.
Humans and other primates are highly visual animals. Our daily visual activities such as recognizing familiar faces, interacting with objects, or reading, are supported by an extensive system of interacting brain areas. The interactions between the many individual nerve cells both within and between brain areas need to be coordinated. One possible solution to achieve flexible coordination between cells in the network is rhythmic activity, or oscillations. The focus of the thesis will be activity in the largest visual area, V1, in non-human primates. In V1, high-frequency activity, so-called gamma-band activity (“gamma”, ca. 30-90 Hz) can be frequently observed and has been suggested to play a role in coordinating activity in the visual system. In Chapter 1, the coordination problem, the primate visual system and gamma-band oscillations are introduced in detail. The following chapters explore the dependence of gamma on contextual influences. Does V1 use contextual information to optimize co-ordination? In the first part, the short-term consequences of repeated encounters with visual stimuli on V1 responses are explored (Chapters 2 and 3). Inspired by results from colored, naturalistic images in the first part, the second part tests the dependence of gamma on spatial and chromatic stimulus aspects (Chapters 4 and 5).
Stimulus repetition is a simple yet powerful way to tap into our brains’ ability to learn and adapt to our environment. Repeated presentation of a visual stimulus tends to decrease responses to this stimulus. Is this accompanied by changes in the coordination of brain activity? In Chapter 2, the stimulus-specificity of repetition effects on gamma was tested using naturalistic stimuli. V1 is most typically studied using black-and-white, artificial stimuli that are very familiar to the animals. Here, colored natural images were repeatedly presented that were initially novel to the animals, to provide a wider and more naturalistic range of stimulation. Both multi-unit spiking activity (MUA) and gamma showed stimulus-specific repetition effects. MUA responses de-creased most strongly for initial repetitions and less for later repetitions. In contrast, gamma could increase or decrease for initial repetitions, but tended to increase for later repetitions. This points to the operation of multiple plasticity mechanisms. One process may rapidly decrease MUA and gamma and be related to initial novelty or adaptation. The other increases gamma, is active for more repetitions, and could constitute a form of refinement of coordination over time. Moreover, based on the spacing of stimulus repetitions, stimulus memory in V1 persisted for tens of seconds.
In the following Chapter 3, the stimulus location specificity and persistence of the repetition effects for longer timescales were tested. To this end, the observation that the increase in gamma with repetition was strongest for the first tens of repetitions was used to test for location specificity and memory. Using simple artificial stimuli that were repeated many times at two alternating locations, both location specificity and memory on the order of minutes was observed. Due to the structure of the primate visual system, location specificity suggests that the repetition effects involve early to mid-level visual areas such as V1. Memory for previous stimulus presentations on the order of minutes has not been previously reported for V1 gamma. Taken together, these experiments demonstrate short-term plasticity of gamma that is stimulus- and location specific and persists on the timescale of minutes.
In Chapter 2, the average gamma-band response to the large, naturalistic stimuli was highly stimulus dependent. Relative increases in gamma-band activity scaled between tens and thousands of percent change depending on the stimulus. Particularly the color of the stimuli appeared to play a strong role, although the stimulus set was too limited and uncontrolled to draw strong conclusions. In Chapters 4 and 5, underlying mechanisms for the stimulus specificity of gamma were explored using more well-controlled, artificial stimuli that varied in color and spatial structure.
Much of vision relies on the analysis of spatial structure. Each nerve cell in V1 only responds to visual stimuli in a particular, small part of the visual field, its so-called “receptive field” (RF). Compared to isolated RF stimulation, nearby cells that are stimulated by a similar structure from different parts of visual space can show response decreases, commonly known as “surround suppression”, and may show coordinated activity in the gamma band. In Chapter 3, responses to large, uniformly colored disks are contrasted with responses to black or white (achromatic) disks. A first experiment showed that gamma-band responses were stronger for colored than achromatic stimuli, whereas MUA responses could decrease below baseline for colored stimuli. To test whether these phenomena were related to surround suppression, stimulus size was manipulated in a second experiment. When stimuli were of sufficient size to induce surround suppression, clear gamma-band responses emerged. Surround suppression and gamma were stronger for chromatic stimuli. However, the change of stimulus size could have changed not only surround suppression but also stimulus saliency. Therefore, in a third experiment, the overall size of the stimulus was kept constant, and the spatial structure of the stimulus was manipulated. In comparison to uniform, predictable stimulus structure, mismatches between the center of the stimulus and the surrounding visual space led to strong increases in MUA responses and strong de-creases in gamma-band activity. These effects were restricted to the recording sites with RFs at the mismatch location. These experiments underpin the strong role of both spatial structure and color for gamma in V1.
In Chapter 4, responses to different color hues are studied in more detail. Gamma response strength depended on hue, being strongest for red compared to blue and green stimuli when measured with a gray background. To better understand the underlying mechanisms of the differential responses, the spatio-temporal context in the form of the background color was manipulated. Background color had a strong influence on gamma strength. Using differently colored backgrounds, different parts of the color signaling pathways could be adapted. Response differences to different color hues could be explained well with a model that incorporates differences in adaptation between pathways involving long- compared to medium-wavelength cone signals.
Taken together, these experiments indicate a strong role of both spatial context (stimulus size and structure) and temporal context and drive (repetition, adaptation) for the generation of gamma-band activity in V1. Functional implications of these dependencies are considered in the final Chapter 6, and a role for gamma-band syn-chronization in a coding regime for visual inputs that generate strong drive and high predictability is suggested.
Fatty acids in oomycetes
(2021)
Es gibt für die Orientierung von Vögel ein allgemeingültiges Konzept, das Karte-Kompass-Prinzip (Kramer 1953, 1957): Der Karten-Schritt besteht darin, den eigenen Standort zu ermitteln und mit dem Ziel in Beziehung zu setzten. Damit wird die geografische Richtung bestimmt, die im Kompass-Schritt in eine konkrete Richtung umgesetzt wird. Für Beides nutzen Vögel auch das Magnetfeld der Erde; in der Karte als einen Faktor den Verlauf der Intensität, im Magnetkompass die Achse der Feldlinien. Der Magnetrezeptor, der die Karte mit Informationen versorgt, ist im Schnabel lokalisiert, der des Kompasses im Auge. Ich habe mich in meiner Arbeit darauf konzentriert, die zwei potenziellen Magnetrezeptoren der Vögel feinstrukturell und immunhistologisch weiter zu charakterisieren.
Für den Magnetkompass wird auf Grund des Radikalpaar-Modells angenommen, dass Cryptochrome die Rezeptormoleküle sein könnten (Ritz et al. 2000). Bei Vögeln sind vier Cryptochrome bekannt, allerdings muss das Rezeptormolekül des Magnetkompasses auch in seiner Lokalisation bestimmte Kriterien erfüllen. Die für meine Arbeit bedeutsamen Kriterien sind: (1) die gleiche Ausrichtung der Proteine in einer Rezeptorzelle und (2), dass die einzelnen Rezeptorzellen alle Raumrichtungen abdecken. Ich habe in meiner Arbeit Cryptochrom 1a (Cry1a) und Cryptochrom 1b (Cry1b) auf ihr Vorkommen in der Retina von Rotkehlchen (Erithacus rubecula) und Hühnern (Gallus gallus) untersucht. Cry1b befindet sich bei Rotkehlchen während der Zugzeit in den Ganglienzellen, in denen es teilweise an Membranen gebunden vorliegt, die jedoch keine bevorzugte Richtung haben. Somit erscheint mir Cry1b als Rezeptormolekül für den Magnetkompass als eher ungeeignet. Cry1b könnte, wie viele Cryptochrome, an der Steuerung von circadianen Rhythmen beteiligt sein. Cry1a hingegen ist bei beiden untersuchten Vogelarten in den UV/V-Zapfen an die Diskmembranen gebunden, was eine Ausrichtung ermöglicht. Die UV/V-Zapfen sind über die gesamte Retina gleichmäßig verteilt, und durch die sphärische Form des Auges decken die einzelnen Rezeptoren jede Raumrichtung ab. Somit erfüllt Cry1a die Bedingungen des Radikalpaar-Modells, und ich schließe daraus, dass es sich hierbei um das Rezeptormolekül des Magnetkompasses handeln könnte. Cry1a ändert nach Lichtabsorption wie viele Cryptochrome seine Konformation. Der von mir verwendete Antikörper bindet nur die lichtaktivierte Form des Proteins. In Versuchen, in denen Hühner verschiedenen monochromatischen Lichtern ausgesetzt wurden, zeigt sich, dass sich Cry1a in UV bis Gelb in lichtaktiviertem Zustand befindet. Dies stimmt sowohl mit der spektralen Empfindlichkeit des Magnetkompasses der Vögel als auch mit der des Flavins, des lichtsensitiven Teils des Cryptochroms, überein. Versuche mit grünem Licht lassen vorsichtige Rückschlüsse auf das für den Magnetkompass relevante Radikalpaar zu: so ist das Flavin erst im zweiten Oxidationsschritt grünlicht-sensitiv, und Cry1a ist nur nachweisbar, also lichtaktiviert, wenn der erste Schritt bereits im Hellen abgelaufen ist. Versuche in denen die Tiere vorab im Dunkeln waren, führen nicht zur erneuten Lichtaktivierung unter grünem Licht. Dies macht nur eines der beiden im Flavinzyklus entstehenden Radikalpaare wahrscheinlich, nämlich das in der Reoxidation entstehende, da das Radikalpaar im ersten Schritt der Oxidation unter Grün nicht entsteht.
In Bezug auf den Magnetrezeptor im Schnabel konnte bereits bei Tauben eine detaillierte Struktur beschrieben werden, die als Magnetrezeptor geeignet ist, nämlich Magnetit- bzw. Maghemit-Teilchen in Dendriten der Nerven (Fleissner et al. 2003). Auch Hühner haben eisenhaltige Strukturen im Oberschnabel, die in ihrer Eisenoxid-Zusammensetzung denen der Tauben entsprechen (Falkenberg et al. 2010). Ich konnte in meiner Arbeit zeigen, dass die eisenhaltigen Strukturen im Oberschnabel der adulten Hühner an oder in Nervenfasern liegen. Elektronenoptisch bestehen diese eisenhaltigen Strukturen im Nervengewebe bei Hühnern, wie bei Tauben beschrieben, aus einem 3-5 µm großen Vesikel, der von eisenhaltigen ‘Schuppen’ besetzt ist, aus circa 1 µm langen Plättchen und Kugeln mit einem Durchmesser von etwa 1 µm. Sie sind in Feldern angeordnet, in denen diese Zellstrukturen gleich ausgerichtet sind. In der Anzahl und Lokalisation der Felder der eisenhaltigen Dendriten gibt es Unterschiede zwischen Hühnern und Tauben, allerdings ist unklar, inwie¬weit dies zu Unterschieden in der Verarbeitung im Gehirn führt. Die Entwicklung der eisenhaltigen Dendriten der Hühner beginnt erst nach dem Schlupf, am Tag des Schlupfes haben Küken noch keine eisenhaltigen Strukturen, abgesehen von roten Blutkörperchen. In den ersten 5 Tagen werden eisenhaltige Makrophagen im frontalen Bereich des Schnabels gebildet, die anschließend wieder reduziert werden. Bei 12 Tage alten Hühnern werden diese auch im lateralen Bereich des Oberschnabels angelegt und ebenfalls dort bis Tag 21 wieder reduziert. 21 Tage alte Hühner haben nur noch wenige eisenhaltige Makrophagen, allerdings ein erstes Feld von eisenhaltigen Dendriten. Die Röntgenabsorption zeigt einen Unterschied in der Eisenoxid-Zusammensetzung zwischen eisenhaltigen Makrophagen und eisenhaltigen Dendriten. Es könnte sein, dass die eisenhaltigen Makrophagen an der Synthese der eisenhaltigen Dendriten beteiligt sind, da sie Eisen aufnehmen, aber auch wieder abgeben können und in demselben Zeitraum reduziert werden, wie die eisenhaltigen Dendriten aufgebaut werden.
Sowohl Tauben als auch Rotkehlchen haben sich phylogenetisch bereits vor 95 Millionen Jahren von den Hühnern abgespalten. Es gibt sowohl in der Lokalisation von Cry1a als auch in der Struktur der einzelnen eisenhaltigen Dendriten keine Unterschiede, so dass es sich bei den beiden Magnetrezeptoren der Vögel vermutlich um sehr alte Mechanismen handelt, die sich in der Evolution kaum verändert haben. Vermutlich sind sie vogelspezifisch, da es in dieser Hinsicht keine erkennbare Gemeinsamkeit mit anderen Wirbeltieren gibt.
Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
Alzheimer’s disease (AD) is a common, age associated neurodegenerative disease that manifests as progressive dementia and is characterized by accumulation of the amyloid beta (Aβ) peptide which is a processing product of a transmembrane protein termed Alzheimer Amyloid Precursor Protein (APP). The Aβ peptide is generated by a sequential proteolytic processing of APP by two distinct proteases that are termed β- and γ-secretase. The β-secretase, also called BACE-1 or memapsin 2, belongs to the family of aspartyl proteases. BACE-1 evidently cleaves APP in an acidic endosomal compartment after endocytosis of APP, thereby facilitating Aβ peptide generation.
Sorting of transmembrane proteins is generally controlled by sorting signals in the cytoplasmic domains of the cargo proteins. The short cytoplasmic tail of BACE-1 with 23 amino acids contains a sorting signal of the acidic cluster, di-leucine (ACDL) type. The two Leu residues in this determinant are important for the clathrin mediated endocytosis of BACE-1, whereas the acidic residues together with the Leu are required for the endosomal sorting and recycling of BACE-1 back to the plasma membrane. The ACDL motif binds to the members of the GGA (Golgi-localized γ ear-containg ARF- binding proteins) family (GGA1-GGA3) that are involved in the sorting of BACE-1.
One of the major aims of this study was to address the role of flotillins in the intracellular sorting of BACE-1. This study shows that flotillin-1 directly binds to the di-leucine motif in the cytoplasmic tail of BACE-1, whereas flotillin-2 only shows an association mediated by flotillin-1. Flotillin-1 competes with GGA2 for the binding to BACE-1 tail, and thus influences the endosomal sorting of BACE-1. Importantly, depletion of flotillins results in an altered localization of the wildtype BACE-1, whereas the plasma membrane resident Leu to Ala (LLAA) mutant is not affected. Flotillin knockdown results in an accumulation of BACE-1, implicating reduced degradation and enhanced stability of this protease. Thus, flotillins appear to be important for the cellular targeting of BACE-1 and also influence the amyloidogenic processing of APP, as demonstrated by an increase in the amyloidogenic C-99 processing fragments.
When flotillin depleted cells were subjected to apoptotic stresses including Aβ25-35 synthetic peptide (inducer of the extrinsic apoptosis pathway) or several chemotherapeutic agents (staurosporine, brefeldin A, doxorubicin, carboplatin and paclitaxel: intrinsic apoptosis pathway) and cytotoxicity was determined, various apoptotic markers were activated in flotillin depleted cells. Caspase-3 and GGA3 are well accepted apoptosis markers and an enhanced caspase-3 cleavage was detected upon STS induced apoptosis in SH-SY5Y, HeLa, and HaCaT cell lines and increased GGA3 cleavage was observed in MCF7 cell line.
One of the major reasons for the apoptotic sensitivity in the absence of flotillins was a PI3K/Akt signaling defect. Neuroblastoma cells depleted of flotillins showed diminished levels of total Akt, phospho-Akt and phospho-ERK upon STS induced apoptosis. Since PI3K/Akt was the primary survival pathway affected upon STS induced apoptosis, ectopic expression of Akt in neuroblastoma cell line reduced caspase-3 cleavage and retarded apoptosis.
The direct downstream target of Akt is FOXO3a, whose localization was investigated in flotillin depleted cells. A major proportion of FOXO3a was localized in the nucleus of flotillin knockdown cells, implicating that FOXOs are active in these cells and subsequently trigger the transcription of death genes. Strikingly, an essential anti-apoptotic molecule and a major cancer target, Mcl-1, was inherently downregulated in flotillin knockdown cells. Mcl-1 is a chief member of the Bcl-2 family as it plays a pivotal role in cell survival and it is a critical protein in cancer therapeutics as suppression of Mcl-1 protein can curtail the survival and growth of tumorous cells.
Neuroblastoma cells were rescued from undergoing permanent damage due to STS induced apoptosis by overexpression of anti-apoptotic Bcl-2. Phorbol esters are well known PKC activators, and pre-treatment of neuroblastoma cells with phorbol esters along with staurosporine reduced caspase-3 cleavage.
These results demonstrate that absence of flotillins can sensitize cellular systems to apoptosis induction. The two main characteristics of cancer cells include resistance to apoptosis and unresponsiveness to chemotherapeutic agents. It is a well established fact that impaired apoptosis is central to tumour development. This study implicates that the downregulation of flotillin function can trigger cellular susceptibility and enhances apoptosis in response to conventional chemotherapeutic agents. Therefore, flotillins can serve as vital regulators in providing a more rational approach in molecular-targeted therapies for receding cancer growth and survival.
Cellular communication is a concept that can be explained as the transfer of signals or material (such as cytokines, ions, small molecules) between cells from the same or different type, across either short or long distances. Once this signal or material is received, it will, as a rule, promote a functional effect. Several routes, involved in this transfer, are well described and are of global importance for organ/tissue communication in an organism.
The brain interacts dynamically with the immune system, and the main route known to mediate this communication, is via the release of cytokines (by peripheral blood cells), which can then activate certain brain cell types, such as microglia, directly, or activate the vagus nerve transferring signals to neuronal populations in the brain. The communication between these two systems plays a key role in the pathophysiology of neurodegenerative diseases, and the mechanisms involved in this interaction are of central importance for understanding disease initiation and progression and search for therapeutic models.
The Momma lab previously addressed the mechanisms of interaction between the peripheral immune system and the brain by investigating cellular fusion of haematopoietic cells with neurons after inflammation. They addressed the question of whether this phenomenon also occurs under non-invasive conditions. To approach this problem, a genetic tracing model that relies on the Cre-Lox recombination system was used. Transgenic mice expressing Cre recombinase specifically in the haematopoietic lineage were crossed into a Cre-reporter background, thus all haematopoietic cells irreversibly express the reporter marker-gene EYFP. Using this model, EYFP was detected in non-haematopoietic tissues, suggesting the existence of a communication mechanism never described before. As cells containing two nuclei were never detected, fusion as a mechanism was excluded, suggesting that Cre reaches non-haematopoietic cells via a different signalling pathway. The Momma lab investigated whether the transfer of material through extracellular vesicles (EVs) could be behind this periphery-to-brain communication. Using the genetic mouse model, they were able to trace the transfer of Cre RNA via EVs between cells in vivo, generating the first in vivo evidence of functional RNA transfer by EVs between blood and brain.
The last decade has witnessed a rapid expansion of the field of EVs. Initially considered as waste disposal material, recent evidence has challenged this view. EVs are currently considered as a widespread intercellular communication system that can transport and transfer all types of biomolecules, from nucleic acids to lipids and proteins. However, several important questions are still under investigation. One of them is whether EVs are involved in brain pathophysiology, as inflammation plays an important role in onset and progression of neurodegenerative diseases and is well described in Parkinson Disease (PD). Based on preliminary data in a mouse, peripherally injected with a low dose of Lipopolysaccharide (LPS, an endotoxin found in the outer-membrane of Gram-negative bacteria, which causes an immune response), neurons and other cell population in the brain take up EVs from the periphery. Particularly, dopaminergic neurons from Substantia Nigra and Ventral Tegmental Area have been shown to receive functional RNA, transported through EVs, which can lead up to 20% of recombination. Furthermore, different neuronal populations from Hippocampus, Cortex and Cerebellum exhibit recombination, indicating a widespread signalling from blood to the brain. Therefore, the goal of my PhD thesis was to investigate the mechanisms of this transfer and the triggers that lead to EV uptake by neural cells in vivo both in pathological and physiological conditions.
In this project, the extent and function of EV-mediated signalling from blood to brain is explored in the context of peripheral inflammation and neurodegenerative diseases. Firstly, EVs isolated from WT mice were further characterized using size-exclusion chromatography (SEC), Western Blot (WB) and electron microscopy in order to extend the knowledge from previous work done in the Momma lab. Secondly, to expand on the biological relevance of the fact that inflammation is correlated with an increase in EV uptake, different approaches using the genetic murine tracing model were used. Recombination events from haematopoietic cells to the brain have been followed after peripheral injection of LPS. Peripheral inflammation caused by LPS injection led to widespread recombination events in the brain, specifically in microglia and neurons, including dopaminergic (DA) neurons. In contrast, astrocytes, oligodendrocytes and endothelial cells were never or very rarely recombined. Additionally, peripheral LPS injection in a murine model, where Cre is expressed only in erythrocytes, led to recombination events only in microglia, suggesting that the type of EV-secreting cell plays a role in the targeting of EVs to a specific cell population.
The genus Giraffa likely evolved around seven million years ago in Indo-Asia and spread over the Arabian-African land bridge into Eastern Africa. The oldest fossil of the African lineage was found in Kenya and dated to 7-5.4 Mya. Beside modern giraffe, four additional African species have likely existed (G. gracilis, G. pygmaea, G. stillei, and G. jumae). Based on their morphological similarities, G. gracilis is often considered to be the closest relative of the modern giraffe. Nevertheless, the phylogeny within the genus Giraffa is largely unresolved.
Modern giraffe (Giraffa sp.) have been neglected by the scientific community for a long time and still very little is known about their biology. Traditionally, present-day giraffe have been considered a single species (G. camelopardalis) which is divided into six to eleven subspecies, with nine subspecies being the most accepted classification. This classification was based on morphological differences and geographic ranges. However, recent genetic analyses found hidden diversity within Giraffa and proposed four genetically distinct giraffe species (G. camelopardalis, G. reticulata, G. tippelskirchi, G. giraffa) with presumably little gene flow among them.
Gene flow on a population level is the exchange of genetic information among populations facilitated by the migration of individuals between populations. Additionally, it is an important criterion to delineate species, because many species concepts, especially the Biological Species Concept, rely on the concept of reproductive isolation. Yet, new genetic methods are identifying an increasing number of species that show signs of introgressive hybridization or gene flow among them. Therefore, strict reproductive isolation cannot always be applied to delineate species, especially in young, probably still diverging, species such as giraffe.
Therefore, giraffe are ideal study organisms to investigate the level of gene flow in recently diverged species with adjacent or potentially overlapping ranges. Furthermore, their recent classification as “Vulnerable” by the IUCN and their unreliable distribution maps require the genetic evaluation of their population structure, distribution and conservation status.
In Publication 1 (Winter et al. (2018a), Ecological Genetics and Genomics, 7–8, 1–5), I studied the distribution and matrilineal population structure of Angolan giraffe (G. giraffa angolensis) using sequences from the cytochrome b gene (1,140 bp) and the mitochondrial control region for individuals from across their known range and beyond, and additionally including individuals from all known giraffe species and subspecies. The reconstruction of a phylogenetic tree and a mitochondrial haplotype network allowed to identify the most easterly known natural population of Angolan giraffe, a population that was previously assigned to their sister-subspecies South African giraffe (G. giraffa giraffa), indicating the limit of classification by morphology and geography. Furthermore, the analyses show that Namibia’s iconic desert-dwelling giraffe population is genetically distinct, even from the nearest population at Etosha National Park, suggesting very limited, if any, natural exchange of matrilines. Yet, no geographic barriers are known for this region that would prevent genetic exchange. Therefore, the two populations are likely on different evolutionary trajectories. Limited individuals with an Etosha haplotype further suggest that translocation of Etosha giraffe into the desert population had only a minor impact on the local population. Two separate haplogroups within Etosha National Park suggest an “out of Etosha” radiation of Angolan giraffe to the East followed by a later back-migration.
In Publication 2 (Winter et al. (2018b), Ecology and Evolution, 8(20), 10156–10165), I investigated the genetic population structure of giraffe across their range (n = 137) with focus on the amount of gene flow among the proposed giraffe species with a 3-fold increased set of nuclear introns (n = 21). Limited gene flow of less than one effective migrant per generation, even between the closely related northern (G. camelopardalis) and reticulated giraffe (G. reticulata) further supports the existence of four giraffe species by a different methodology, gene flow. This is significant because most species concepts build on reproductive isolation. Furthermore, this result is corroborated by four distinct major clades in a phylogenetic tree analysis, and distinct clusters in Principal Component Analysis and STRUCTURE analysis. All these analyses suggest a low level of genetic exchange among the four giraffe species and, therefore, a high degree of reproductive isolation in accordance with the Biological Species Concept (BSC). In Addition, only a single individual in 137 was identified as being potential of natural hybrid origin, which promotes the four-species concept further. ...
Fungal organisms, including the most common human pathogens Candida spp., are commensal organisms that are widely present as part of the human flora. Fungal infections are, most frequently, local infections that do not compromise the life of patients. However, mycotic diseases can be life-threatening if they become systemic infections. Systemic fungal infections have risen over the last three decades in parallel to the increased immune-compromised population as a consequence of diseases (e.g. HIV/AIDS) or therapeutic interventions that affect the immune system (e.g. chemotherapy for cancer treatment and immunosuppressors used for patients with organ transplants). This has resulted in the demand of new antifungal drugs that can eradicate the new infections caused by these opportunistic fungal pathogens. However, most of the current compounds have poor pharmaceutical properties such as narrow spectrum of activity, susceptibility to be extruded by efflux pumps or lack of specificity, which make them not suitable for human clinical applications. The treatment of fungal and parasitic infections has been traditionally difficult because the infective organisms are eukaryotic cells that share most of the pathways and enzymes with human cells. To avoid side effects and to develop a targeted therapy, the research has traditionally been centered on the very few enzymes and pathways existing in the infectious organism but absent in humans. Until now, antifungal therapeutic options are limited and are almost dominated by azole class of sterol biosynthesis inhibitors affecting the synthesis of ergosterol, a major constituent of the fungal cell membrane. Because human cells do not have a cell wall, the development of effective and safe antifungal agents has also been directed to enzymes required for the synthesis of the cell wall. Alternatively, it is theoretically possible to target enzymes that are present in fungal organisms and in humans, when: 1) sufficient selectivity can be achieved, and 2) inhibition of the fungal enzyme is lethal to the fungus but does not produce major side effects to humans. In this line, it would be ideal to evaluate the development of selective inhibitors of enzymes which are already known to be drug targets, like protein kinases.
In order to form an organ, cells need to take up specialized functions and tasks. Cellular specialization is guided by an interplay of chemical signals and physical forces, where one influences the other. One aspect in cellular identity is its shape, which e.g. defines how susceptible the cell may be to intercellular signaling or in which section of the cell cycle it is and therefore can tell us about its current state. Shape changes are introduced by motor proteins that are controlled and activated in a locally confined manner. For my thesis, I was interested to understand better how cellular shape and geometry impacts downstream cell and organ development. What happens if a cell cant transition to a specific shape? How does it affect tissue structure? How does it affect further development?
One regulator of motor proteins like non-muscle myosin is Shroom3, which recently has been been shown to be expressed and involved in the development of the zebrafish lateral line organ (1 ). Development of the lateral line occurs through a migrating cluster of initially about 150 cells, the posterior lateral line primordium (pLLP), which migrates from the anterior (head) to the posterior (tail) while depositing cell clusters in a regular pattern. Literature on development of the lateral line suggests that in order for a cell cluster to be deposited from the pLLP, rosette formation is a key requirement. Therefore our expectation from the shroom3 mutant was that the number of clusters deposited was significantly reduced. To our surprise, when we first inspected the end of migration lateral line phenotype we found many individuals with a significant increase in cell clusters deposited.
This made us re-think the role of Shroom3 during rosette assembly and the processes its involved in.
To study the effects of Shroom3 on lateral line development, a mutant line was generated and crossed with various transgenic lines which express fluorescently labeled proteins that locate to organelles such as the plasmamembrane or the nucleus. Following, the mutant with its fluorescent labels was microscopically imaged under different conditions to quantify and analyze various cell-morphometric features. Even though the zebrafish is a popular model organism and its perfectly suited for developmental biology and advanced microscopy, there were no methods that would allow for a standardized and more automated pipeline of data acquisition and processing.
Therefore, in order to accurately quantify the morphogenic processes Shroom3 is involved in, I developed a new toolset that significantly improved and facilitated my research. The toolset consists of (1) a new sample mounting method that is based on a 3D agarose gel that increases the number of embryos that can be mounted and imaged at once and speeds up the imaging process significantly (2) for subseqent image analysis I developed four programs that automate the process and therefore make the results much more reproducible and the analysis much more efficient. The first program is used for end of migration analyses, to deduce the pattern, count and size of Lateral Line cell clusters. The second is used not for end of migration, but for migration analyses (on timelapse recordings). Besides this it also prepares the images for more advanced downstream migration analyses and allows to analyse fluorescence signal on a second channel. The third program is used to analyse the pLLP only at high spatial resolution and to deduce the cell count, 3D cell morphometrics (like the volume) and cell orientation. The fourth program finally is used downstream of the second and third program and is capable of detecting and comparing them with the look of wildtype rosettes.
Here I show that in absence of Shroom3 rosette formation in the migrating pLLP is destabilized leading to facilitated cell cluster deposition and I show how this might be related to traction forces due to a possible interdependence of pLLP acceleration and speed of migration. Furthermore I show that apical constriction and rosette formation is not blocked in Shroom3 deficient embryos, but that larger rosettes are fragmented into many smaller ones. Finally, I give an outlook on how the absense of Shroom3 and hence the absense of morphological changes may deregulate gene transcription by elevating the levels Atoh1a, a transcription factor necessary for hair cell development.
My results and methodology demonstrate the importance of morphology in guiding developmental processes and how rather small morphological changes on the cellular level can impact further development significantly. My work also shows how powerful modern genetics, imaging and image analysis are and how diverse they are in terms of range of questions they are capable of answering. The methods and tools I developed prepare the ground for at least three quarters of the analyses I carried out and together with the documentation and data I provide, they are highly reproducible. In that regard I am especially happy that one of my developments, an improved sample preparation method, is already used by many different labs all over the world helping them to make their results more reproducible.
Genetic and genomic tools have provided researchers with the opportunity to address fundamental questions regarding the reintroduction of species into their historical range with greater precision than ever before. Reintroduction has been employed as a conservation method to return locally extinct species to their native range for decades. However, it remains unknown how genetic factors may impact population establishment and persistence at the population and metapopulation level in the short- and long-term. Genetic methods are capable of producing datasets from many individuals, even when only low quality DNA can be collected. These methods offer an avenue to investigate unanswered questions in reintroduction biology, which is vital to provide evidence based management strategies for future projects. The Eurasian lynx (Lynx lynx) and European wildcat (Felis silvestris) are elusive carnivores native to Eurasia and have been the subject of multiple reintroduction attempts into their native range. During the 19th and 20th century, the Eurasian lynx was extirpated from West and Central Europe due to increasing habitat fragmentation and persecution. Similarly, the European wildcat was the subject of human persecution, residing in a few refugia in West and Central Europe. After legal protection in the 1950s, subsequent reintroduction projects of both species began in the 1970s and 1980s and continue to the present. Despite this large focus on species conservation, little attention has been given to the consequences these reintroductions have on the genetic composition of the reintroduced populations and if the populations have a chance of persisting in the long term. These species have not yet benefited from the large range of genetic and genomic techniques currently available to non-model organisms, leaving many fundamental aspects of their reintroduction poorly understood. In my dissertation, I investigate demography, population structure, genetic diversity and inbreeding at the population and metapopulation level in both species. In the introduction, which lays the foundation for the subsequent chapters of this PHD, I provide background on reintroduction, its role in conservation and the genetic consequences on populations, especially populations of apex and mesocarnivores. In Publication I, I investigated the reemergence of the European wildcat in a low mountain region in Germany using fine-scale spatial analysis. I found that the reintroduced population has persisted and merged with an expanding natural population. The reintroduced population showed no genetic differentiation from the natural population suggesting there is a good chance this population has retained sufficient genetic diversity despite reintroduction. In Publication II, I tracked population development and genetic diversity over 15 years in a reintroduced lynx population to determine the genetic ramifications on a temporal scale. I found slow genetic erosion after a period of outbreeding, which fits in line with other reintroduced taxa sharing similar demographic histories. I also found the number of genetic founders to be a fraction of the total released individuals, indicating that reintroduced populations of elusive carnivores may have fewer founder individuals than previously thought. In Publication III, I sampled all surviving lynx reintroductions in West and Central Europe as well as 11 natural populations to compare levels of genetic diversity and inbreeding across the species distribution. I found that all reintroduced populations have lower genetic variability and higher inbreeding than natural populations, which urgently requires further translocations to mitigate possible negative consequences. These translocations could stem from other reintroduced populations or from surrounding natural populations. The results contribute to a growing body of evidence indicating that inbreeding is likely to be more prevalent in wild populations than previously understood. Finally, in the discussion I explore how genetic methods can be applied to post-reintroduction monitoring of felid species to illuminate questions relating to genetic composition after release. The methods employed in these studies and in future work will be highly dependent on the research questions posed. Additionally, I investigate the drivers of the observed genetic patterns including founder size, source population, environmental factors, and population growth. I found that genetic diversity loss patterns across these two felid species are not clearly defined, however, management actions can be taken to mitigate the negative effects of reintroductions. These management actions include further translocation, introducing a sufficient number of released individuals and situating reintroductions adjacent to natural populations. All of these actions can minimize genetic drift and inbreeding, two factors which negatively impact small populations. This thesis further supports mounting evidence that genetic considerations should be assessed before releasing individuals, which allows for incorporation of scientific evidence into the planning process thereby increasing the overall success of reintroduction projects. Ultimately, the resources developed during this dissertation provide a solid baseline and foundation for future work regarding the consequences of reintroductions. This is especially important as an increasing number of species are at risk of extinction and reintroductions of both the European wildcat and Eurasian lynx, as well as many others, are planned in the coming years.
The existence of all living organisms depends on their multidimensional adjustment to the conditions of the environment in which they live. Organisms must constantly deal with not only abiotic stress factors (such as water availability or extreme temperatures), but also with various biotic interactions (the competition between different organisms, both intraspecific and interspecies). When there is a consensus between an organism and the environment it means that this organism is well adjusted and increases its probability of survival.
Symbiotic organisms possess the ability to establish an intimate interaction with another species (symbiont) that provides benefits for survival. Organisms that are involved in obligate symbiosis may adapt to a new environment by switching to another symbiotic partner that is locally better adapted; or by reshuffling symbiont communities present in the holobiont. This ability potentially gives them the opportunity to flexibly react to changing environmental conditions.
In this thesis I studied the genetic diversity and geographic distribution of symbiont lineages in a lichen symbiosis to better understand environmental adaptation in symbiotic systems. Lichens are symbiotic associations of photobionts (one or several green-algal species or cyanobacteria), filamentous mycobionts (lichen-forming fungi) and co-inhabiting symbiotic microorganisms (lichen-associated bacteria, endolichenic fungi, and basidiomycete yeast). The coccoid green algae of the genus Trebouxia are the most common and the most studied lichen photobionts. However, the lack of formal Trebouxia taxonomy impedes our understanding of this photobiont diversity.
Different species of mycobionts may share the same photobionts and a single species of mycobiont may associate with multiple, genetically different photobionts. Interactions among symbionts are not random and are constrained by evolutionary and environmental processes. The ability to associate with specific symbiotic partner is considered as a lichen strategy to facilitate adaptation to the constantly changing environments.
The objectives of this thesis were to 1. Elucidate the intraspecific diversity of fungal and algal symbionts in the lichen Umbilicaria pustulata, given a range-wide (Europe-wide) sampling; 2. Evaluate species delimitation in trebouxioid photobionts based on molecular data, and 3. Quantify the climatic niches of photobiont lineages within U. pustulata, to establish whether the association with particular photobionts may modify the range and ecological niche of this lichen.
The main findings of this thesis are:
1. The genetic diversity within trebouxoid photobiont of U. pustulata is higher than within the mycobiont. The most variable photobiont loci are nrITS rDNA, psbJ-L, and COX2. RbcL is the least variable photobiont locus. The most variable mycobiont loci are MCM7 and TSR1. This study shows a lack of genetic variability in the mycobiont loci EF1, nrITS rDNA, RPB1, and RPB2.
2. U. pustulata shows a low level of selectivity and is associated with numerous (most likely six) putative algal species. All photobiont haplotypes found in U. pustulata are shared between other lichen-forming fungi species, showing different patterns of species-to-species and species-to-community interactions.
3. The geographic distribution of U. pustulata symbionts associations is strongly connected to changes in the climatic niches. The mycobiont-photobiont interactions change along latitudinal temperature gradients (cold-adapted hotspot) and in Mediterranean climate zones (warm-adapted hotspot). U. pustulata broadens its distribution range by switching between photobionts that posses specific environmental preferences.
Overall, this thesis contributes to the understanding of the symbiont diversity, fungal-algal association patterns and local adaptation linked to symbiont-mediated niche expansion in lichens. While identifying intraspecific diversity of both lichen symbionts is a key predisposition to understand symbiont interactions, population dynamics or co-evolution, my comparative study of the sequence-based molecular markers is relevant to reveal cryptic diversity in other lichen-forming fungi and their photobionts.
The determination of species boundaries in lichen symbionts is essential for the study of selectivity and specificity, co-distribution, and co-evolution. Whereas the phylogenetic relationships of Trebouxiophyceae are poorly understood, the application of a novel multifaceted approach based on phylogenetic relationships, coalescence methods and morphological traits presented in this thesis is a promising tool to address species boundaries within this heterogeneous genus.
This thesis provides evidence for symbiont-mediated niche expansion in lichens and highlights the preferential photobiont association from a niche-modeling perspective. My results shed light on symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation in the lichen symbiosis. The spatial genetic pattern found in U. pustulata symbionts supports the concept of ecological fitting and is consistent with patterns found in other lichen studies. Results presented here relate also to findings in different symbiotic systems, like reef-building corals, where different latitudinal patterns and symbiont switching has been reported as an adaptive response to severe bleaching events. Furthermore, this study is timely in light of global warming, because the identification of interaction hotspots among symbionts helps to understand how lichens or other symbiotic organisms adjust to the ongoing climate change. This knowledge will, in turn, facilitate the proper conservation of the most vulnerable lichen populations. My doctoral thesis provides a conceptual framework for analyzing symbiont diversity, interaction patterns, and symbiont-mediated niche expansion that could be applied to other types of lichen species as well as other organisms involved in facultative or obligate symbiosis.
Genetic engineering of Saccharomyces cerevisiae for improved cytosolic isobutanol biosynthesis
(2021)
The finite nature of fossil resources and the environmental problems caused by their excessive usage requires alternative approaches. The transformation from a fossil based economy to one based on renewable biomass is called a “bioeconomy”. To substitute fossil resources, various microorganisms have already been modified for the biosynthesis of valuable chemicals from biomass. However, the development of such efficient microorganisms at an industrial scale, remains a major challenge. The most prominent and robust microorganism for industrial production is the yeast Saccharomyces cerevisiae, which is known to produce ethanol that is used as renewable biofuel. However, S. cerevisiae is also naturally able to produce isobutanol in small amounts. Isobutanol is favoured as a biofuel compared to ethanol due to its higher octane number and lower hygroscopicity, which makes it more suitable for application in conventional combustion engines. In S. cerevisiae, the biosynthesis of isobutanol is permitted by the combination of mitochondrial valine synthesis (catalysed by Ilv2, Ilv5 and Ilv3) and its cytosolic degradation (catalysed by Aro10 and Adh2). The different compartmentalisation of the two pathways limit isobutanol biosynthesis. Thus, Brat et al. (2012) were able to increase the isobutanol yield up to 15 mg/gGlc by cytosolic re localisation of the enzymes Ilv2Δ54, Ilv5Δ48 and Ilv3Δ19 (cyt-ILV), with simultaneous deletion of ilv2. This corresponds to approximately 3.7% of the theoretical yield of 410 mg/gGlc, implying existing limitations in isobutanol biosynthesis, which have been investigated in this work.
For yet unknown reasons, isobutanol was only produced by S. cerevisiae in a valine free medium, according to Brat et al. (2012). This work shows that this can be attributed to the catalytic activity of Ilv2Δ54, which acted as growth inhibitor to S. cerevisiae. By this logic, a negative selection on the ILV2∆54 gene was exerted, which made the ilv2 deletion and simultaneous valine exclusion necessary to maintain the functional expression of toxic ILV2∆54. Furthermore, it was shown that valine exclusion is not mandatory due to the feedback regulation of Ilv2, permitted by Ilv6. Rather, increased isobutanol yield was observed when cytosolic Ilv6∆61 was expressed in the valine free medium, which is explained by the enhanced regulation of Ilv2Δ54 by Ilv6∆61 when BCAA are absent. Isobutanol biosynthesis is neither redox nor NAD(P)H co factor balanced. It was seen that co factor imbalance could be mitigated by the expression of an NADH oxidase (NOX), but not by expression of the NADH dependent ilvC6E6, since the latter showed low in vivo activity. Furthermore, it was seen that NAD(H) imbalance did already limit isobutanol biosynthesis, but the NADP(H) imbalance did not. Another limitation of cytosolic isobutanol biosynthesis is the secretion of the intermediate 2‑dihydroxyisovalerate, which then no longer is taken up by S. cerevisiae, causing a reduced isobutanol yield. This is attributed to insufficient Ilv3∆19 activity, due to poor iron sulphur cluster apo protein maturation. Therefore, it was aimed to replace Ilv3∆19 by heterologous dihydroxyacid dehydratases. Even though some of the enzymes were functionally expressed, none showed better in vivo activity than Ilv3∆19. Therefore, the Ilv3∆19 apo protein maturation was improved. This was achieved by the genomic deletion of fra2 or pim1 as well as by the cytosolic expression of Grx5∆29.
In addition to the isobutanol pathway, S. cerevisiae was optimised for isobutanol biosynthesis by rational and evolutionary engineering. For this purpose, the genes which are necessary for isobutanol production were integrated into the ilv2 locus, and the resulting strain was evolved in a medium containing the toxic amino acid analogue norvaline. Evolved single colonies were isolated, which presented improved growth and increased isobutanol yields (0.59 mg/gGlc) in a valine free medium, as compared to the initial strain. This is explained by a gene dosage effect which occurred during the evolutionary engineering experiment. In collaboration with Dr. Wess, the genes ilv2, bdh1/2, leu4/9, ecm31, ilv1, adh1, gpd1/2 and ald6 were cumulatively deleted in CEN.PK113 7D to block competing metabolic pathways. The resulting strain JWY23 achieved isobutanol yields up to 67.3 mg/gGlc, when expressing the cyt ILV enzymes from a multi copy vector. The most promising approaches of this work, namely the deletion of fra2 and the expression of Grx5∆29, Ilv6∆61, and NOX, were confirmed in this JWY23 strain. The highest isobutanol yield from this work was observed at 72 mg/gGlc for Ilv6∆61 and cyt ILV enzymes expressing JWY23, which corresponds to 17.6% of the theoretical isobutanol yield.
Isobutyric acid (IBA) is a by product of isobutanol biosynthesis, but it is also considered a valuable platform chemical. Therefore, the approaches that improved isobutanol biosynthesis were applied to the biosynthesis of IBA in S. cerevisiae. The highest IBA yield of 9.8 mg/gGlc was observed in a valine free medium by expression of cyt ILV enzymes, NOX and Ald6 in JWY04 (CEN.PK113 7D Δilv2; Δbdh1; Δbdh2; Δleu4; Δleu9; Δecm31; Δilv1). This corresponded to an 8.9 fold increase compared with the control and is, to our best knowledge, the highest IBA yield reported to date for S. cerevisiae.
Stechmücken (Dipteren: Culicidae) sind weltweit mit über 3500 Arten und mit Ausnahme der arktischen Regionen ubiquitär vertreten. Die medizinische Relevanz dieser Tiergruppe, begründet durch die hämatophage Lebensweise der Weibchen, erschloss sich bereits Ende des 19. Jh. und hat bis heute Bestand. Jedes Jahr sterben rund 600.000 Menschen an den Folgen der Malaria und fast 100 Mio. Menschen infizieren sich mit dem Denguefieber. Zwar beziehen sich diese Zahlen fast ausschließlich auf die Entwicklungsländer, aber im Zuge des Klimawandels und des immer stärkeren Welthandels kommt es auch in Europa und den USA immer wieder zu Ausbrüchen vorher nicht relevanter Krankheiten. So hat sich das West-Nil- Virus seit 1999 in Nordamerika rasant verbreitet. Im Jahr 2013 gab es dort rund 2500 Fälle, von denen 119 zum Tod führten. In Europa traten hingegen Krankheiten wie das Chikungunyafieber (Italien 2007) oder das Denguefieber (Frankreich 2010/2013) auf. Die Gründe für diese Ausbrüche sind vor allem in der Einschleppung neuer Vektorspezies und Krankheitserreger sowie in den veränderten Wirtspräferenzen einheimischer Stechmückenarten zu suchen. Das Wissen um das Vektorpotential der in Deutschland heimischen Stechmücken konnte vor allem durch die seit 2009 initiierten Monitoring-Programme stetig erweitert werden. Auch die Veränderung der heimischen Fauna durch invasive Arten wie Ochlerotatus japonicus japonicus oder Aedes albopictus wird intensiv erforscht. Dennoch ist hinsichtlich der Biologie, Ökologie sowie Genetik vieler Arten noch immer wenig bekannt.
Die vorliegende Dissertation, welche auf Basis von vier (ISI-) Einzelpublikationen kumulativ angefertigt wurde, beschäftigte sich mit der Analyse der genetischen Variabilität sowie der Zoogeographie der untersuchten Arten und der Etablierung einer schnellen und kostengünstigen Methode zur Artdiagnostik. Besonderes Augenmerk wurde bei den Analysen auf die beiden heimischen Arten Culex pipiens und Culex torrentium sowie die invasive Art Ochlerotatus japonicus japonicus gelegt. Ziel war es, die noch bestehenden Wissenslücken zu füllen, um zukünftige Monitoring-Programme besser koordinieren sowie Analysen zur Vektorkompetenz und Genetik dieser Arten gezielter durchführen zu können.
Es konnte gezeigt werden, dass Cx. pipiens und Cx. torrentium deutliche Unterschiede in ihren Populationsstrukturen aufwiesen welche auf verschiedene evolutive Prozesse hindeuten. Die geringere genetische Variabilität in Cx. pipiens lässt auf positive Selektion durch z.B. Insektizidresistenz im Zuge durchgeführter Bekämpfungsmaßnahmen oder die Infektion mit Wolbachien schließen. Die analysierte Populationsstruktur von Cx. torrentium spricht hingegen für eine geringe Ausbreitung, wodurch der genetische Austausch reduziert wurde und so die untersuchten Populationen genetisch stärker voneinander abwichen. Des Weiteren ließen die Analysen des Cytochrom c Oxidase Untereinheit 1-Fragmentes (cox1) Rückschlüsse auf die Zoogeographie dieser Arten in Deutschland zu - wobei beide Arten über das Untersuchungsgebiet verteilt waren, Cx. torrentium jedoch in den neuen Bundesländern weniger häufig nachgewiesen wurde als in den alten und eine geringere gefangene Individuenzahl aufwies. Basierend auf der ökologischen Nischenmodellierung konnten potentiell neue Verbreitungsgebiete für die Art Ochlerotatus japonicus japonicus identifiziert werden. Als klimatisch besonders günstig zeigten sich dabei Südhessen, das Saarland sowie nördliche Teile Nordrhein-Westfalens. Mit Hilfe der etablierten Methode der direct-PCR wird in Zukunft eine schnellere und kostengünstigere Identifizierung von Stechmücken erfolgen können, welche aufgrund bestimmungsrelevanter Merkmale nicht mehr morphologisch zu identifizieren sind.
Um das Wissen über die Stechmücken in Deutschland fortlaufend zu intensivieren, ist sowohl das Weiterführen der Monitoring-Programme als auch die molekularbiologische Aufarbeitung der Proben nötig. Durch die Anwendung neuer Techniken und weiterer molekularer Marker wird es möglich sein, weitere Krankheitserreger sowie genetische Besonderheiten der heimischen Stechmückenfauna nachzuweisen. Aber auch die Überwachung invasiver Stechmückenarten durch die Modellierung potentieller Verbreitungsgebiete und die Anwendung molekularbiologischer Analysemethoden zum Detektieren der Arten und möglicher Krankheitserreger wird ein wichtiger Bestandteil der weiteren Forschung sein.
Get3 in Arabidopsis
(2021)
Der guided entry of tail-anchored proteins (GET) Biogenese-Weg vermittelt den Transport und die Insertion von tail-anchor (TA) Proteinen in die Doppellipidschicht des Endoplasmatischen Retikulums (ER). TA Proteine sind dadurch gekennzeichnet, dass sie eine Transmembran Domäne (TMD) in den letzten 50 Aminosäuren ihrer Sequenz beherbergen. Diese TMD enthält die notwendigen Informationen, mit denen die Proteine an ihren jeweiligen subzellulären Zielort transportiert werden können. TA Proteine erfüllen eine Vielzahl von essentiellen biologischen Prozessen, sie fungieren zum Beispiel als Rezeptoren, sind maßgeblich an der Fusion von Vesikeln beteiligt sowie an der Initiation von Apoptose. Durch ihren modularen Aufbau können TA Proteine nicht mit dem Signalerkennungspartikel interagieren und müssen deshalb posttranslational zum ER geleitet werden. Im Modellorganismus Bäckerhefe (Saccharomyces cerevisiae) ist der GET Biogenese-Weg am besten beschrieben und läuft wie folgt ab: Nach der Termination der Translation bindet das Protein SgtA das TA Protein und händigt es über den Adapter-Komplex, bestehend aus Get4 und Get5, an die zytosolische ATPase Get3 aus. Get3 ist der zentrale Zielsteuerungsfaktor des GET Biogenese-Weges. Sobald sich ein Komplex aus Zeilsteuerungsfaktor und TA Protein gebildet hat, wird dieses zur Membran des ERs überführt. Dort wird das TA Protein an den Rezeptorkomplex bestehend aus Get1 und Get2 übergeben, welcher anschließend die Insertion des TA Proteins in die Doppellipidschicht des ERs initiiert.
Get3 hat im zellulären Kontext noch eine weitere Funktion. Unter oxidativem Stress oder Energie depletierenden Bedingungen wird Get3 zu spezifischen Foci rekrutiert, an denen sich noch weitere durch Stress -induzierbare Proteine, wie z.B. die der Familie der Hitze Stress Proteine (HSPs) versammeln. Analysen haben gezeigt, dass Get3 unter den oben genannten Bedingungen, Konformationsänderungen durchläuft und dann als ATP unabhängige Holdase fungiert. Diese kann die exponierten, hydrophoben Anteile von Proteinen binden, um dadurch die Proteostasis aufrechtzuhalten.
Durch die Bedeutsamkeit der TA Proteinen ist die zentrale ATPase Get3 in allen Domänen des Lebens hochgradig konserviert. Phylogenetische Analysen ergaben, dass sich Get3 im Allgemeinen in eine „A“ Gruppe sowie eine „BC“ Gruppe aufspaltet. Im Modellorganismus Arabidopsis thaliana (Ackerschmalwand) wurden drei Orthologe zu Get3 identifiziert. Eins davon gehört zu der „A“ Gruppe und befindet sich im Zytoplasma. Die anderen zwei Orthologe befinden sich in den Organellen endo-symbiotischen Ursprungs und gehören der „BC“ Gruppe an. Untersuchungen an verschiedenen Deletionsmutanten in A. thaliana haben gezeigt, dass die Mutationen einzelner GET Komponenten zu einer signifikanten Verkürzung der Haarwurzeln führen, obwohl der restliche Habitus der Pflanze unverändert bleibt. Diesbezüglich wurde SYP123 als einziges TA Proteine identifiziert, dessen Abundanz durch die Deletion von GET Komponenten beeinflusst werden kann. Von den anderen beiden Orthologen organellären Ursprungs ist, abgesehen von ihrer Lokalisation nichts weiter bekannt
Vier Orthologe Gruppen in Pflanzen
Da bislang nicht mehr als zehn Pflanzenarten für phylogenetische Analysen herangezogen wurden, wurden in dieser Arbeit die taxonomischen Beziehungen von Get3 zu einander in 50 Spezies der Viridiplantae auf Basis der Orthologie sowie Homologie untersucht. Dies führte zur Identifizierung einer zytolischen (AtGet3a), einer plastidären (AtGet3b), einer mitochondriellen (AtGet3c) sowie einer Monokotyledone spezifischen Gruppe (SBGet3). Die Lokalisation der ersten drei Gruppen wurde in selektierten Pflanzen, sowohl homolog als auch heterolog, der unterschiedlichen Spezies mittels saGFP untersucht, und es konnte gezeigt werden, dass mehrere Get3 Orthologe mit unterschiedlichen subzellulären Lokalisationen eine unter Pflanze häufig auftretende Eigenschaft ist. Das Weitern konnte gezeigt werden, dass manche Komponenten des Präzielsteuerungskomplexes (SgtA und Get4) sowie des Rezeptorkomplexes (Get1) in fast allen der 50 untersuchten Pflanzenarten vorhanden sind. Dies weist auf eine Konservierung des gesamten GET Biogenese-Weges in Pflanzen hin.
Get3a in Arabidopsis thaliana
Da die molekulare Zusammensetzung des Präzielsteuerungskomplexes für AtGet3a in A. thaliana nicht bekannt ist, habe ich Co-Immunpräzipitationen mit Zellextrakten aus weißer Zellkultur und einen von mir selbst aufgereinigten Antikörper gegen AtGet3a durchgeführt. Nach anschließender Gelelektrophorese und einer Anfärbung mit Coomassie Brilliant Blue ließ sich ein reproduzierbares Muster aus Proteinbanden erkennen, welche ausgeschnitten und mittels LC-MS/MS analysiert wurden. Dadurch wurde ein putativer Kandidat für Get5 identifiziert sowie eine Assoziation mit Chaperonen und proteasomalen Untereinheiten.
Um die Zielsteuerungseffizienz und Topologie von ER-Membranproteinen zu analysieren habe ich (i) die rekombinante Synthese eines Modell-TA Proteins mit glykosylierbarem opsin bovine glycosylation Tag (OPG) etabliert sowie (ii) eine Methode etabliert um in isolierten Protoplasten die Richtigkeit der Insertion zu überprüfen. Mit Hilfe dieser Methoden können nun verschiedene Mutanten auf ihre Insertions-Wirksamkeit untersucht werden. Desweitern können durch Mutationsanalysen die notwendigen physikochemischen Eigenschaften für die Erkennung des Substrates ermittelt werden.
Eine weit verbreitete Methode im GET Feld ist die tail-anchor translocation (TAT). Bei dieser Methode werden isolierte mikrosomale Fraktionen des rauen ERs mit rekombinanten Komplexen bestehend aus Zielsteuerungsfaktor und TA Protein inkubiert. Durch einen rekombinanten OPG, der im Lumen des ERs post-translational modifiziert werden kann, ist die Beobachtung einer zeitabhängigen Kinetik der Glykosylierung möglich. Dieses System wurde bislang nur für Komponenten aus Säugern oder Hefen benutzt, aber noch nie mit einem System auf pflanzlicher Basis. Um dies zu verwirklichen, habe ich die rekombinante Proteinexpression soweit optimiert, dass der Großteil des synthetisierten Proteins sich im löslichen Anteil des Lysats statt in den Inclusion Bodies befand. Mittels dieser Optimierung konnte ich die Ko-Expression von Zielsteuerungsfaktor mit TA Protein als löslichen Komplex etablieren. Ergänzend zu den löslichen Komplexen habe ich eine geeignete Methode etabliert um mittels Saccharosegradienten mikrosomale Fraktionen aufzutrennen in denen AtGet3a angereichert ist. Leider müssen noch die Parameter der Reaktion optimiert werden, aber die Akquirierung alle nötigen Bestandteile ist etabliert.
Ziel dieser Arbeit war es erstmals durch eine Kombination aus chemischer Mutagenese und gezielter genetischer Modifikation (hier: „metabolic engineering“) einen Phaffia-Stamm herzustellen, welcher über die Mutagenese hinaus über eine weiter verstärkte Astaxanthin-Synthese verfügt.
Die von „DSM Nutritional Products“ bereitgestellten chemischen Mutanten wurden analysiert und über einen Selektionsprozess auf Pigmentstabilität und Wachstum hin optimiert, da die Stämme aus cryogenisierter Dauerkultur starke Pigmentinstabilitäten und ein verzögertes Wachstum aufwiesen.
Über eine exploratorische Phase wurde die Carotinoidsynthese analysiert und festgestellt, dass in den Mutanten keine Einzelreaktionen betroffen sind, welche für die Heraufregulierung der Carotinoidsynthese in den Mutanten verantwortlich sind. Hierbei wurden Limitierungen identifiziert und diese durch Transformation von Expressionsplasmiden mit geeigneten Genen aufgehoben, um damit eine noch effizientere Metabolisierung von Astaxanthin-Vorstufen hin zu Astaxanthin zu erreichen. Eine Überexpression der Phytoensynthase/Lycopinzyklase crtYB resultierte in einem gesteigerten Carotinoidgehalt bei gleichbleibendem Astaxanthin- Anteil. Durch eine zweite Transformation mit einer Expressionskassette für die Astaxanthin-Synthase asy konnte der Carotinoidgehalt weiter gesteigert und zusätzlich eine Limitierung der Metabolisierung von Astaxanthin-Vorstufen behoben werden, sodass die Transformante nahezu alle Intermediate der Astaxanthinsynthese zu Astaxanthin metabolisieren konnte (Gassel et al. 2013). Es konnte gezeigt werden, dass auch in den Mutanten, aus Experimenten mit dem Wildtyp bekannte, Limitierungen identifiziert und ausgeglichen werden konnten.
Colorectal cancer (CRC) has the third highest incidence and the fourth highest mortality rate worldwide and represents a substantial health care burden and affects the life of millions of people. CRC is a genetic disease caused by the stepwise accumulation of genetic alterations. The initiating event in colorectal carcinogenesis is the aberrant activation of the WNT pathway, but other pathways are also commonly deregulated, including the PI3K/AKT pathway. A number of previous studies using genetically engineered mouse models aimed at dissecting the exact role of PI3K/AKT pathway in CRC, but have yielded in rather conflicting results. Despite the inconsistent results, these studies already put forward the idea that PI3K/AKT signaling in combination with other genetic events might substantially contribute to tumor progression. Since the PI3K/AKT pathway is frequently activated in CRC, it represents an ideal candidate for therapeutic intervention. Although extensive efforts had led to the development of numerous inhibitors targeting the PI3K/AKT pathway, the diversity of genetic alterations can challenge the identification of the most effective therapeutic targets. Therefore, the discovery of shared tumor-promoting mechanisms downstream of these genetic alterations might unravel new biomarkers and druggable targets. The aim of this study was to elucidate the precise role of PI3K/AKT pathway during the course of colorectal carcinogenesis and to decipher novel protumorigenic molecular mechanisms downstream of PI3K/AKT activation that can be used for therapeutic intervention.
To obtain a better insight into the role of the PI3K/AKT pathway during colorectal carcinogenesis, mice expressing an oncogenic variant of AKT1 (AktE17K) specifically in the intestinal epithelial cells (IEC) were used. At the age of 6 months untreated AktE17K mice showed clearly perturbed intestinal homeostasis, but no tumor formation. To induce colonic tumorigenesis, AktE17K mice were subjected to treatment with the colonic carcinogen azoxymethane (AOM). In response to AOM, AktE17K mice developed invasive but non-metastatic tumors, which showed strong nuclear accumulation of TP53. To investigate the role of PI3K/AKT signaling specifically in CRC progression, AktE17K mice were crossed to TP53-deficient mice (Tp53ΔIEC). Unlike AktE17K mice, untreated Tp53ΔIEC; AktE17K, developed highly invasive small
intestinal tumors by the age of 6 months. To investigate the role of AKT hyperactivation in colonic tumor progression, Tp53ΔIEC; AktE17K mice were subjected to AOM treatment. AKT hyperactivation significantly enhanced tumor progression and induced metastatic dissemination.
To get a better insight how AKT signaling can promote tumor progression, whole tumor tissues from AOM-treated Tp53ΔIEC and Tp53ΔIEC; AktE17K mice were subjected to next generation mRNA sequencing and phospho-proteomic analysis by mass spectrometry. Both analyses indicated that AKT hyperactivation expands the inflammatory tumor microenvironment and upregulates pathways associated with invasion and metastasis. Importantly, Gene Set Enrichment Analysis revealed that AOM-induced colon tumors of Tp53ΔIEC; AktE17K animals, are highly similar in their gene expression profile to the CMS4 subtype of human CRC, which is associated with worse overall- and relapse-free survival. Gene expression analysis also suggested elevated NOTCH signaling in the Tp53ΔIEC; AktE17K tumors. Interestingly, while the expression of Notch3 mRNA was increased in the tumors of Tp53ΔIEC; AktE17K mice, the expression of the other NOTCH receptors was unaffected by AKT hyperactivation. In vitro experiments using TP53-deficient mouse tumor organoids with hyperactive AKT signaling confirmed the direct, tumor cell-intrinsic link between AKT activation and increased Notch3 expression. Moreover, inhibition of EZH2 mimicked the effect of AKT hyperactivation on Notch3 expression, suggesting that AKT regulates Notch3 via an epigenetic mechanism.
Knock-down of Notch3 in TP53-deficient mouse tumor organoids with hyperactive AKT signaling resulted in differential regulation of several pathways with potential role in invasion and metastasis and in cell death and survival. Subsequent in vivo experiments confirmed the role of NOTCH3 signaling in CRC progression. Treatment of AOM-induced Tp53ΔIEC; AktE17K mice with a NOTCH3 antagonistic antibody or the γ-secretase inhibitor DAPT significantly reduced invasion and metastasis. Importantly, NOTCH3 expression was also found to be associated with human CRC progression, suggesting that NOTCH3 represent a valid target for the treatment of CRC. This work, using genetically engineered mouse models and advanced in vitro techniques, has demonstrated a strong tumor promoting role for PI3K/AKT signaling in CRC progression and has identified NOTCH3 signaling as a potential therapeutic target downstream of the PI3K/AKT pathway.
Colorectal cancer (CRC) has the third highest incidence and the fourth highest mortality rate worldwide and represents a substantial health care burden and affects the life of millions of people. CRC is a genetic disease caused by the stepwise accumulation of genetic alterations. The initiating event in colorectal carcinogenesis is the aberrant activation of the WNT pathway, but other pathways are also commonly deregulated, including the PI3K/AKT pathway. A number of previous studies using genetically engineered mouse models aimed at dissecting the exact role of PI3K/AKT pathway in CRC, but have yielded in rather conflicting results. Despite the inconsistent results, these studies already put forward the idea that PI3K/AKT signaling in combination with other genetic events might substantially contribute to tumor progression.
Since the PI3K/AKT pathway is frequently activated in CRC, it represents an ideal candidate for therapeutic intervention. Although extensive efforts had led to the development of numerous inhibitors targeting the PI3K/AKT pathway, the diversity of genetic alterations can challenge the identification of the most effective therapeutic targets. Therefore, the discovery of shared tumor-promoting mechanisms downstream of these genetic alterations might unravel new biomarkers and druggable targets. The aim of this study was to elucidate the precise role of PI3K/AKT pathway during the course of colorectal carcinogenesis and to decipher novel pro-tumorigenic molecular mechanisms downstream of PI3K/AKT activation that can be used for therapeutic intervention.
To obtain a better insight into the role of the PI3K/AKT pathway during colorectal carcinogenesis, mice expressing an oncogenic variant of AKT1 (AktE17K) specifically in the intestinal epithelial cells (IEC) were used. At the age of 6 months untreated AktE17K mice showed clearly perturbed intestinal homeostasis, but no tumor formation. To induce colonic tumorigenesis, AktE17K mice were subjected to treatment with the colonic carcinogen azoxymethane (AOM). In response to AOM, AktE17K mice developed invasive but nonmetastatic tumors, which showed strong nuclear accumulation of TP53. To investigate the role of PI3K/AKT signaling specifically in CRC progression, AktE17K mice were crossed to TP53- deficient mice (Tp53ΔIEC). Unlike AktE17K mice, untreated Tp53ΔIECAktE17K, developed highly invasive small intestinal tumors by the age of 6 months. To investigate the role of AKT hyperactivation in colonic tumor progression, Tp53ΔIECAktE17K mice were subjected to AOM treatment. AKT hyperactivation significantly enhanced tumor progression and induced metastatic dissemination.
To get a better insight how AKT signaling can promote tumor progression, whole tumor tissues from AOM-treated Tp53ΔIEC and Tp53ΔIECAktE17K mice were subjected to next generation mRNA sequencing and phospho-proteomic analysis by mass spectrometry. Both analyses indicated that AKT hyperactivation expands the inflammatory tumor microenvironment and upregulates pathways associated with invasion and metastasis. Importantly, Gene Set Enrichment Analysis revealed that AOM-induced colon tumors of Tp53ΔIECAktE17K animals, are highly similar in their gene expression profile to the CMS4 subtype of human CRC, which is associated with worse overall- and relapse-free survival7 . Gene expression analysis also suggested elevated NOTCH signaling in the Tp53ΔIECAktE17K tumors. Interestingly, while the expression of Notch3 mRNA was increased in the tumors of Tp53ΔIECAktE17K mice, the expression of the other NOTCH receptors was unaffected by AKT hyperactivation. In vitro experiments using TP53-deficient mouse tumor organoids with hyperactive AKT signaling confirmed the direct, tumor cell-intrinsic link between AKT activation and increased Notch3 expression. Moreover, inhibition of EZH2 mimicked the effect of AKT hyperactivation on Notch3 expression, suggesting that AKT regulates Notch3 via an epigenetic mechanism.
Knock-down of Notch3 in TP53-deficient mouse tumor organoids with hyperactive AKT signaling resulted in differential regulation of several pathways with potential role in invasion and metastasis and in cell death and survival. Subsequent in vivo experiments confirmed the role of NOTCH3 signaling in CRC progression. Treatment of AOM-induced Tp53ΔIECAkt E17K mice with a NOTCH3 antagonistic antibody or the γ-secretase inhibitor DAPT significantly reduced invasion and metastasis. Importantly, NOTCH3 expression was also found to be associated with human CRC progression, suggesting that NOTCH3 represent a valid target for the treatment of CRC. This work, using genetically engineered mouse models and advanced in vitro techniques, has demonstrated a strong tumor promoting role for PI3K/AKT signaling in CRC progression and has identified NOTCH3 signaling as a potential therapeutic target downstream of the PI3K/AKT pathway.
Hyperparasitic fungi on black mildews (Meliolales, Ascomycota) : hidden diversity in the tropics
(2023)
Meliolales (Sordariomycetes, Ascomycota) is a group of obligate plant parasitic microfungi mainly distributed in the tropics and subtropics. Meliolalean fungi are commonly known as “black mildews”, as they form black, superficial hyphae on the surface of vegetative and reproductive organs of vascular plants. They are considered biotrophic parasites, and the infections caused by black mildews can lead to a decrease in the photosynthetic activity of plants, as well as to an increase in the temperature and respiration rate of their leaves.
Meliolales are frequently parasitized by hyperparasitic fungi, i.e., parasitic fungi that have parasitic hosts. These hyperparasites are all Ascomycota and belong mainly to the Dothideomycetes and Sordariomycetes. Although hyperparasites represent a megadiverse group, species were only described by morphology until 1980, and the systematic position of more than 60 % of known species is still unclear. In addition, there are no DNA reference sequences available in public databases for any of the species of hyperparasites of Meliolales, and no ecological studies have been done up to now.
Before this study, no exact number of hyperparasitic fungi growing on colonies of black mildews existed. Here, we present a checklist including 189 species of fungi known to be hyperparasitic on Meliolales, but the number of existing species is likely to be even higher. The elaboration of this species checklist laid the foundations for this investigation, as it helped to understand the present state of knowledge of hyperparasitic fungi on Meliolales worldwide.
For the present study, fresh specimens of leaves infected with colonies of Meliolales and hyperparasites were opportunistically collected at 32 collection sites in Western Panama and Benin, West Africa, in 2020 and 2022, respectively. In total, 100 samples of plant specimens infected with black mildews were collected, of which 58 samples were parasitized by hyperparasitic fungi. 31 species and morphospecies of hyperparasitic fungi were identified. In addition, 35 historical specimens, including 12 type specimens, were examined for the present work.
DNA of hyperparasitic fungi was isolated directly from conidia, synnemata, apothecia, perithecia or pseudothecia of fresh and dried specimens. The main challenges faced by scientists in doing molecular studies of hyperparasitic fungi are related to the fact that the hyperparasitic fungi are intermingled with tissues of the meliolalean hosts and other organisms present in a given sample. This makes the isolation of DNA exclusively from the hyperparasite difficult. Moreover, hyperparasitic fungi on Meliolales are biotrophs and cannot be grown axenically. The hosts themselves are also biotrophic, further complicating DNA isolation from either partner. These factors have contributed to a lack of reference sequences in public databases. After more than 100 attempts, DNA of 20 specimens of hyperparasitic fungi, representing seven species, has been isolated in the context of the present investigation. Three partial nuclear gene regions were amplified and sequenced: nrLSU, nrSSU and nrITS. The datasets were assembled for phylogenetic analyses applying Maximum Likelihood (ML) and Bayesian inference (BI) methods. DNA sequences of hyperparasitic fungi on Meliolales were generated for the first time in the context of the present investigation.
Hyperparasitic fungi on Meliolales do not represent a single systematic group, but a polyphyletic ecological guild of fungi. Because of this huge diversity, only the systematics of species of perithecioid hyperparasites, as well as of the species of the genera Atractilina and Spiropes known to be hyperparasitic on black mildews was discussed in this thesis, as they represented the most common groups of fungi found in Benin and Panama. The results indicated, for example, the systematic position of Dimerosporiella cephalosporii and Paranectriella minuta in the Sordariomycetes and Dothideomycetes, respectively. In addition, the first record of a hyperparasitic fungus of black mildews in the Lecanoromycetes, namely Calloriopsis herpotricha, is reported here. The systematics of Atractilina parasitica and of some species of Spiropes is also discussed here.
In the context of the present investigation, four species new to science were described. They are presented with detailed descriptions, photos and scientific illustrations. Taxonomic studies of this thesis also generated seven new synonyms, nine new records for Benin, seven for Panama, one for Africa and two for mainland America, as well as the confirmation of one anamorph-teleomorph connection by molecular sequence data.
The ecology of hyperparasitic fungi on Meliolales is complex and far from being completely understood. The hypothesis of host specificity between hyperparasitic fungi, their meliolalean hosts and their plant hosts was tested for the first time, through a tritrophic network analysis. Results indicate that hyperparasites of Meliolales are generalists concerning genera of Meliolales, but apparently specialists at the level of order. In addition, hyperparasitic fungi tend to be found alongside their meliolalean hosts, suggesting a pantropical distribution.
Viele Gruppen der Lebewesen, insbesondere Insekten breiten sich durch steigende Temperaturen zunehmend in Gebieten aus, in denen sie ursprünglich nicht vorkommen(Novikov und Vaulin 2014; Bebber 2015). Hierbei ist die steigende Temperatur in
verschiedenen Gebieten der Hauptfaktor für Expansionen dieser Arten in Richtung des nördlichen Polarkreises. Einige dieser Arten sind sehr tolerant für verschiedene Variablen und können damit ihr Verbreitungsgebiet deutlich nach Norden hin ausdehnen. Aufgrund steigender Temperaturen werden jedoch andere Arten in ihrem Verbreitungsgebiet eingeschränkt oder ihre Verbreitung verschiebt sich in nördliche Richtung (Ogden und Lindsay 2016; Lawler et al. 2009). Auch für die Verbreitung von Krankheiten spielen Temperaturen, Ausbreitungen oder Verbreitungsverschiebungen eine wichtige Rolle (Mordecai et al. 2019).
So können, durch die Etablierung der passenden Vektoren, bisher nur in wärmeren Gebieten auftretende Krankheiten zukünftig auch in unseren Breitengraden eingeschleppt und
verbreitet werden. Bremsen, invasive Stechmücken aber auch einheimische Mücken tragen alle ein Potential,verschiedenste Krankheitserreger zu verbreiten, auch wenn die Eignung als
Vektor für jede Art unterschiedlich groß ausfällt und manche Arten daher kaum beobachtet und untersucht werden. Mit dem Augenmerk auf sich ändernde Verbreitungsgebiete hinsichtlich zukünftigen klimatischen Veränderungen und sich wandelnden anthropogenen Einflüssen sollten jedoch auch Arten mit bisher geringem Vektorpotential mit in Beobachtungsprogramme aufgenommen werden.
Wir untersuchten in Projekt I auf kontinentaler Skala die Verbreitung von sechs verschiedenen Bremsenarten und konnten sowohl Rückschlüsse auf eine mangelhafte Beobachtung der
Arten ziehen als auch Artpräferenzen hinsichtlich der Landschaftsnutzung, Auswirkungen des Klimas auf die Verbreitung der Art und bisher unbekannte Toleranzen hinsichtlich tiefen Temperaturen und äußerst verkürzten Wärmeperioden aufdecken. Eine Größenordnung niedriger wurde in Projekt II, basierend auf aktuellen und Vergangenen Klimadaten, die zukünftige und aktuelle Verbreitung einer invasiven, sich zukünftig ausbreitenden Stechmückenart innerhalb Deutschlands modelliert. Durch bisherig im Untersuchungsgebiet nur begrenztes Auftreten konnten noch keine Rückschlüsse auf die unterschiedlichen Präferenzen für das Habitat gezogen werden, es können jedoch für zukünftige Berechnungen Habitatpräferenzen aus anderen Gebieten hinzugezogen werden um die Art und ihre fortschreitende Ausbreitung genauer zu beobachten. Auf der kleinsten untersuchten Ebene konnten in Projekt III innerhalb eines Mikrohabitates verschiedenste Rückschlüsse auf limitierende oder förderliche abiotische Faktoren, die teilweise bisherig nicht oder nur geringfügig beobachtet wurden, gezogen werden. Ebenfalls konnten Auswirkungen der umgebenden Landschaft auf die Abundanzen der Tiere beobachtet werden. Mithilfe von verschiedenen Modellen und in Abhängigkeit von Klimakarten, Landbedeckungsdaten und Landnutzung sowie Eigenschaften und Toleranzen der untersuchten Arten lassen sich in verschiedenen Größenordnungen geeignete Habitate von einheimischen sowie invasiven Arten identifizieren und zukünftige Verbreitungen effizient vorhersagen.
Insgesamt können, basierend auf all diesen Daten, dadurch für alle untersuchten Faktoren Modelle auf andere Gebiete übertragen werden um somit potentielle Verbreitungen dort
vorherzusagen. Auf unseren Daten basierend können so zum Beispiel Modellierungen für die potentielle Ausbreitung der untersuchten Tabaniden innerhalb anderer Kontinente berechnet werden und Monitoringprogramme können die Ergebnisse unserer Studie als Startpunkt aufgreifen, um durch Beprobung an modellierten Standorten die Korrektheit unserer Modelle zu überprüfen und sowohl Landschaftstypen als auch Artzusammensetzung aufzunehmen um das Modell zu bestätigen oder zu verbessern. Die Modellierung der invasiven Art Aedes albopictus bietet die Möglichkeit, diese Art in Zukunft innerhalb der möglichen Ausbreitungskorridore genauer zu beobachten um ihre fortschreitende Verbreitung zu
verifizieren oder eventuelle Änderungen des klimatischen Verlaufes mit einzubinden und das Modell anzupassen. Die Untersuchung des Mikrohabitats von Culex pipiens pipiens und Culex torrentium bietet, auch hinsichtlich anderer Arten in diesem Habitat, eine potente Methode, Vorhersagen für Artvorkommen innerhalb anderer Unterirdischen Objekte zu berechnen. Hier können, bei ausreichend großer Datenlage, eine Vielzahl von Faktoren in die Auswertung mit einfließen.
Die durchgeführten Studien bestätigen die Notwendigkeit für verbesserte Monitoringkonzepte für alle vektorkompetenten Tiergruppen hinsichtlich der sich ändernden klimatischen Bedingungen, des globalen Handels und die sich wandelnde Nutzung der Landschaften durch den Menschen und darin begründete Veränderungen der Artenzusammensetzung eines Habitates, zeigen Möglichkeiten, diese Konzepte mit bisher
ungenutzten Daten aufzubauen und zu verbessern und können gleichzeitig zu deren Verbesserung herangezogen werden.
Despite all advancements in cancer research and clinical practice, cancer remains a life- threatening disease with an increasing incidence. According to a 2018 WHO forecast, cancer incidence will double to approximately 37 million new cancer cases by 2040. Today, clinical management of cancer is based on a "one-fits-all" strategy. Most cancers are still treated by surgical therapy followed by adjuvant or neoadjuvant chemotherapy based on rather strict guidelines (S3 guidelines in Europe) which are based on studies of large cohorts of patients with the same tumor entity. While this approach has led to substantial increases in progression-free survival and overall patient survival, most patients do not benefit from the administered treatment regimen. One reason for this is intra-tumor heterogeneity, which results from clonal evolution between cancer cells and their environment. This means that cancer patients may respond differently to a particular drug due to the different mutation patterns of their tumor cells. Therefore, patients should be screened in advance for reliable cancer biomarkers that definitively predict whether they will respond to a particular therapy. This would increase the probability of a successful treatment.
Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. The main cause of death in CRC is a metastatic disease, which is presented in 20 % of patients and eventually develops in more than 30 % of early-stage patients. Despite the significant increase (to more than 30 months) in median survival with the development of cytotoxic agents and the introduction of targeted therapy, the progression-free survival in the first-line setting has remained largely unchanged over the past decade.
The heterogeneity in CRC is characterized by alterations in multiple signaling pathways that affect cellular functions such as cell proliferation or apoptosis. Commonly affected signaling pathways include the mitogen-activated protein kinase (MAPK)- and the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP)-pathway. Alterations in the TGF-β/BMP pathway, due to mutations in the SMAD4 gene (mothers against decapentaplegic homolog 4), are associated with different drug response and promote resistance to chemotherapy. In addition, they are associated with a higher recurrence rate.
SMAD4 is one of the most common cancer driver genes, and mutations occur in up to 15 % of CRC cases. Therefore, there is an urgent need for therapeutic agents that can specifically target SMAD4-mutated tumors.
The aim of the present study was the identification of the clinical relevance of the SMAD4 gene and the investigation of its suitability as a potential biomarker in CRC.
For this purpose, I investigated sibling patient-derived organoids (PDOs) derived from different regions of a chemo-naïve CRC tumor. PDOs are 3D cell cultures that reliably recapitulate the architecture of the tissue of origin, as well as preserve the genomic background and intra-tumor heterogeneity. The sibling PDOs (R1R361H and R4wt) shared the most common CRC mutations, such as KRASG12D (kirsten rat sarcoma), PIK3CAH1047R (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and TP53C242F (tumor protein 53), but differed in a SMAD4R361H mutation and showed a different drug response. The single nucleotide variant R361H of the SMAD4 gene is among the most common pathogenic alterations in various cancers, including CRC.
The sibling PDOs showed significant differences in response to the MEK-inhibitors cobimetinib, trametinib, and selumetinib. MEK-inhibitors are antineoplastic agents that inhibit the function of MEK1 and MEK2, preventing phosphorylation of transcription factors, which leads to inhibition of tumor cell proliferation. MEK-inhibitors are approved for the treatment of malignant melanoma. Currently, they are in phase-III clinical trials for the treatment of patients with metastatic CRC.
To investigate whether SMAD4R361H is responsible for sensitivity to MEK-inhibitors, Iestablished three syngeneic PDOs harboring a SMAD4R361H mutation using the CRISPR/Cas9 genome editing system. All CRISPR-PDOs were significantly more sensitive to the MEK-inhibitors, compared to R4wt. I have shown that the SMAD4R361H mutation is responsible for sensitivity to MEK inhibition in CRC models and may be a predictive biomarker.
To test this hypothesis, I examined 62 CRC PDO models and treated them with the MEK-inhibitors cobimetinib, trametinib, and selumetinib. All models that had a pathogenic mutation or deletion in the SMAD4 gene (15 %) were sensitive to cobimetinib, 10 % of models were sensitive to trametinib, and 8 % were sensitive to selumetinib.
I performed transcriptome (RNA sequencing) and proteome analyses using the DigiWest® method to investigate the mechanism underlying MEK-inhibitor sensitivity.
DigiWest® is a Luminex® bead-based analysis that allows the simultaneous analysis of over 100 (phospho-)proteins. The transcriptome and proteome data support the observation that MEK inhibition primarily affects SMAD4R361H PDOs. Furthermore, I have shown that activation of the BMP signaling pathway in organoids with wild-type SMAD4 appears to be responsible for resistance to MEK-inhibitors. Thus, a genetic alteration in the BMP signaling pathway, beyond SMAD4, could lead to sensitivity to MEK-inhibitors.
I identified four genes involved in the TGF-β/BMP signaling pathway that are frequently mutated in CRC and grouped them into the so-called SFAB-signature (SMAD4, FBXW7 (F-box/WD repeat-containing protein 7), ARID1A (AT-rich interactive domain-containing protein 1A), or BMPR2 (Bone morphogenetic protein receptor type II). Clinical data show that approximately 36 % of CRC patients have at least one pathogenic mutation in these genes.
I tested all 62 CRC PDO models and found a significant positive prediction for sensitivity to cobimetinib (95 %) and selumetinib (70 %) for the SFAB-signature. Trametinib and the newly approved MEK-inhibitor binimetinib showed a similar trend. Therefore, the SFAB-signature has high predictive power for response to MEK-inhibitors and could be used as a predictive biomarker panel.
The current clinically used biomarkers for CRC are based on the mutation status of driver genes KRAS and BRAF, which are present in up to 50 % and 10 % of CRC, respectively. Investigation of molecular alterations in CRC revealed that mutations in the KRAS gene, which is downstream of EGFR (epidermal growth factor receptor) in the MAPK-pathway, interfere with an anti-EGFR-antibody therapy (e.g., cetuximab). Therefore, cetuximab is only relevant for RAS wild-type tumors. However, approximately 40 % of patients with RAS wild-type status do not respond to this treatment.
About 53 % of CRC PDO models carry a pathogenic RAS mutation, about 10 % harbor a pathogenic BRAF mutation. Both, the RAS and RAF status alone as well as the combination of RAS and RAF status with SFAB-signature did not provide a better prediction of sensitivity to MEK inhibition.