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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.
Inhibition of midbrain dopamine (DA) neurons codes for negative reward prediction errors, and causally affects conditioning learning. DA neurons located in the ventral tegmental area (VTA) display two-fold longer rebound delays from hyperpolarizing inhibition in comparison to those in the substantia nigra (SN). This difference has been linked to the slow inactivation of Kv4.3-mediated A-type currents (IA). One known suppressor of Kv4.3 inactivation is a splice variant of potassium channel interacting protein 4 (KChIP4), KChIP4a, which has a unique potassium channel inactivation suppressor domain (KISD) that is coded within exon 3 of the KChIP4 gene. Previous ex vivo experiments from our lab showed that the constitutive knockout of KChIP4 (KChIP4 KO) removes the slow inactivation of IA in VTA DA neurons, with marginal effects on SN DA neurons. KChIP4 KO also increased firing pauses in response to phasic hyperpolarization in these neurons. Here I show, using extracellular recordings combined with juxtacellular labeling in anesthetized mice, that KChIP4 KO also selectively changes the number and duration spontaneous firing pauses by VTA DA neurons in vivo. Pauses were quantified with two different statistical methods, including one developed in house. No other firing parameter was affected, including mean frequency and bursting, and the activity of SN DA neurons was untouched, suggesting that KChIP4 gene products have a highly specific effect on VTA DA neuron responses to inhibitory input.
Following up on this result, I developed a new mouse line (KChIP4 Ex3d) where the KISD-coding exon 3 of KChIP4 is selectively excised by cre-recombinase expressed under the dopamine transporter (DAT) promoter, therefore disrupting the expression of KChIP4a only in midbrain DA neurons. I show that these mice have a highly selective behavioral phenotype, displaying a drastic acceleration in extinction learning, but no changes in acquisition learning, in comparison to control littermates. Computational fitting of the behavioral data with a modified Rescorla-Wagner model confirmed that this phenotype is congruent with a selective increase in learning from negative prediction errors. KChIP4 Ex3d also had normal open field exploration, novel object preference, hole board exploration and spontaneous alternation in a plus maze, indicating that exploratory drive, responses to novelty, anxiety, locomotion and working memory were not affected by the genetic manipulation. Furthermore semi-quantitative IHC revealed that KChIP4 Ex3d mice have increased Kv4.3 expression in TH+ neurons, suggesting that the absence of KChIP4a increases the binding of other KChIP variants, which known to increase surface expression of Kv4 channels.
Furthermore, in the course of my experimental study I identified that the most used mouse line where cre-recombinase is expressed under the DAT promoter (DAT-cre KI) has a different behavioral phenotype during conditioning in relation to WT littermate controls. These animals displayed increased responding during the initial trials of acquisition and delayed response latency extinction, consistent with an increase in motivation, which is in line with a decrease in DAT function.
I propose a working model where the disruption of KChIP4a expression in DA neurons leads to an increase in binding of other KChIP variants to Kv4.3 subunits, promoting their increased surface expression and increasing IA current density; this then increases firing pauses in response to synaptic inhibition, which in behaving animals translates to an increase in negative prediction error-based learning.
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.
Cells within a tissue form highly complex, cellular interactions. This architecture is lost in two-dimensional (2D) cell cultures. To close the gap between 2D cell cultures and in vivo tissues, three-dimensional (3D) cell cultures such as spheroids or embryoid bodies were developed. To fully take advantage of the third dimension, imaging techniques are essential. The emerging field of "image-based systems biology" exploits the information in images and builds a connection between experimental and theoretical investigation of biological processes. Such interdisciplinary approaches strongly depend on the development of protocols to establish 3D cell cultures, innovations in sample preparation, well-suited imaging techniques and quantitative segmentation methods.
Although 3D cell cultures and image-based systems biology provide a great potential, 2D methods are still not completely replaced by 3D methods. This is mainly due to methodical and technical hurdles. Therefore, this thesis provides a significant contribution to overcome these hurdles and to further develop 3D cell cultures. I established computational and experimental methods related to 3D aggregates and investigated fundamental, cellular processes such as adhesion, growth and differentiation.
The automatic segmentation method "PAS" and "LoS" were developed in the context of this thesis. They extract essential biological properties such as the projected area or features of cell nuclei from 2D or 3D images of 3D aggregates. Both algorithms show their accuracy robustly over image data from different samples and different microscopes. In addition, the superior performance of PAS and LoS was proven in a comparison with state-of-the-art methods.
The PAS approach served as an essential basis for investigating cellular processes such as adhesion and growth which are tightly regulated to contribute to tissue integrity. These processes are involved in the formation of spheroids. The temporally resolved data of spheroid formation of three mammary epithelial cell lines revealed differences in their formation dynamics as well as in the onset of spheroid formation phases (aggregation, compaction and growth). Despite these differences, adhesion- and growth-associated proteins such as E-cadherin, actin, microtubules, and the focal adhesion kinase show similar importance in a particular phase. Notably, certain proteins (e.g. E-Cadherin) contribute differently to spheroid formation of cells from different cell types in terms of cell adhesion and growth. Overall, analyses of the individual phases of spheroid formation revealed the temporal coordination of fundamental tissue-specific processes. The results contribute to a better understanding of the maintenance and disruption of tissue integrity.
An important but yet unknown process is how cells accomplish to arrange themselves against the gravitational force to form a spheroid. Live imaging with light sheet-based microscopy provides the best solution for a temporally and in particular spatially resolved investigation of spheroid formation. Although the imaging possibilities increase with this particular microscopy technique, available sample preparation methods are rare. Therefore, I have significantly optimized "agarose beaker" as preparation method for 3D long-term imaging of spheroid formation. The data show that upward movement of the cells takes place early. This movement is initiated in the centre of the initially flat cell layer. Subsequently, the cells move from the periphery of the cell layer toward the centre. Cells rearrange within the spheroid which is followed by growth. It is very likely that 3D aggregates form by adopting an energetically favoured, spherical shape by increasing cell-cell or cell-matrix contacts.
Besides the knowledge gained from the examination of the self-assembly process in different contexts, fully formed cellular aggregates can serve as basis to investigate differentiation processes. Differentiation guide cell fate specification during early embryonic development (i.e. preimplantation) and is not fully understood yet. Due to the lack of an in vitro system for preimplantation, I have developed "blastoids". These are 3D multicellular aggregates of mouse embryonic stem cells which represent important phases of preimplantation and beyond. In qualitative and quantitative analyses, a strong similarity was proven between blastoids and the inner cell mass of in vivo mouse embryos. Further results strongly suggest that both, the cell number and the trophectoderm play a subordinate role for cell fate decision during preimplantation. Furthermore, 3D neighbourhood analyses have shown that both, blastoids and mouse embryos, do not show a random "salt-and-pepper" pattern during differentiation. Instead, they show a yet unknown local clustering of cells with identical fates, suggesting local cell interactions that influence cell fate decision. Furthermore, the data indicate that the maturation of the epiblast in the later stages of preimplantation is initiated by an interaction between cells of the epiblast and the primitive endoderm.
Using image-based systems biology, I have investigated fundamental cellular processes such as adhesion, growth and differentiation in the context of tissue integrity and early embryonic development using 3D cellular aggregates. This highly interdisciplinary work is a major contribution to 3D cell biology and demonstrates how cells bind and interact within a complex system. The main methods developed in this thesis as well as the biological findings can be used not only in further biological but also in medical and pharmacological studies. They have the potential to advance our understanding of complex biological systems and to provide new opportunities for practical applications.
In der vorliegenden dreiteiligen Studie werden Mongolische Wüstenrennmäuse untersucht, deren Hörspektren im tieffrequenten Bereich und deren Unterscheidungsfähigkeiten von Kommunikationsrufen denen des Menschen ähneln. Die extrazelluläre Aktivität im primären auditorischen Kortex (AI) der narkotisierten Versuchstiere, evoziert durch Reintöne und arteigene Kommunikationsrufe, wird in der linken (LH) und rechten Gehirnhemisphäre (RH) aufgenommen. Es werden Multikanalelektroden (16 Eingangskanäle) verwendet, welche eine simultane Aufnahme der neuronalen Aktivitäten aller kortikalen Schichten ermöglichen. Zur Analyse der neuronalen Mechanismen werden Wellenformen einzelner Elektrodenkanäle und Aktivitätsprofile, bestehend aus den Wellenformen aller Elektrodenkanäle in einem Zeitfenster von 600 ms, auf Ebene von Aktionspotentialen (MUA), lokalen Feldpotentialen (LFP) und Current-source-density (CSD) Analysen, untersucht. Während MUAs die neuronalen Aktionspotentiale im Nahfeld der Elektrode reflektieren, umfassen die LFPs die summierten Potentiale (inhibitorisch und exzitatorisch) von Neuronen eines größeren Areals. Die CSDs hingegen werden durch die Integration von LFP-Wellenformen benachbarter, linear angeordneter Elektrodenkanäle berechnet und ermöglichen so eine Lokalisation der Ursprünge geräuschspezifischer Aktivitätsflüsse.
Im ersten Teilprojekt werden CSD-Profile in Antwort auf unterschiedliche Reintöne untersucht, um die Aktivitätskomponenten, die so genannten Sinks, für weiterführende Analysen zu quantifizieren. Es können zwei primäre (s1 und s2), drei mittlere (s3-s5) und vier späte (s6-s9) Sinks in einem Zeitfenster von 600 ms definiert werden. Eine Veränderung der Stimulusfrequenz eine Oktave über und unter der charakteristischen Frequenz (CF), beziehungsweise des Lautstärkepegels = 24 dB über der minimalen Schwelle, führt zu qualitativen Veränderungen in der CSD-Profilstruktur. Die Sink s7 wird durch Stimuli mit niedrigem Lautstärkepegel weniger verlässlich evoziert, wohingegen die Sink s9 bei Stimuli eine Oktave über der CF verlässlicher evoziert wird. Die Ergebnisse weisen darauf hin, dass im AI die spektralen Informationen eine Oktave über und unter der CF asymmetrisch integriert werden.
Auf Einzelschichtebene konnte bereits gezeigt werden, dass spektrotemporale Eigenschaften von Stimuli durch MUAs schlechter reflektiert wurden als durch LFPs, was vermutlich eine direkte Konsequenz der unterschiedlichen Ursprünge der Signaltypen ist. Daher werden im zweiten Teilprojekt die spezifischen Unterschiede der MUA-, LFP- und CSD-Antworten auf Ebene kortikaler Schichten und kompletter laminarer Profile untersucht, um die Unterschiede und den Informationsgehalt der drei Signaltypen zu charakterisieren. Signifikante Unterschiede, welche durch zwei Reintöne und sieben Kommunikationssignale evoziert werden, können verstärkt im mittleren und späten Latenzbereich und in granulären und infragranulären Schichten vorgefunden werden. Der Grad der Rufspezifizität ist in LFP und CSD-Antworten im Vergleich zu demjenigen in MUA-Antworten größer. Die Segregationsleistung ist im Vergleich zu einzelnen kortikalen Schichten in den von kortikalen Kolumnen abgeleiteten laminaren Profilen um den Faktor 1,8-2,6 erhöht. Die Neuronenpopulationen einzelner kortikaler Kolumnen sind vermutlich wichtig für die Kodierung von Geräuschen, welche sich in ihren spektrotemporalen Eigenschaften unterscheiden.
Viele vorangegangene Studien konnten zeigen, dass die Gehirnhemisphären akustische Signale asymmetrisch verarbeiten. Daher werden im dritten Hauptteil die laminaren Profile der LH und RH quantitativ und statistisch verglichen. Die MUA-, CSD-Profile und im geringeren Maße auch die LFP-Profile zeigen systematische Unterschiede auf signifikantem Niveau in der Dauer, Onset Latenz und vertikalen Ausdehnung bestimmter Aktivitäten. Kommunikationsrufe evozieren in der LH, welche beim Menschen auf Sprachstimuli spezialisiert ist, im Vergleich zur RH komplexere CSD-Profile. Die neuronale MUA-, LFP- und CSD-Aktivitätsstärke ist in der RH für weniger komplexe Stimuli teilweise signifikant erhöht. Die Asymmetrie in der Auftrittsverlässlichkeit der Sink s6 lässt vermuten, dass sich die intrakolumnäre Vernetzung in Schicht VIa zwischen der LH und RH unterscheidet. Die wenigen, signifikanten und nicht systematischen Unterschiede zwischen den Sink-Parametern der LH und RH nach kortikaler Ausschaltung mit dem GABAA-Rezeptor Agonist Muscimol weisen darauf hin, dass die Hemisphärenasymmetrie durch Prozesse des ipsilateralen Kortex maßgeblich beeinflusst wird.
The adult mammalian heart is unable to regenerate lost myocardial tissue after injury. In contrast, some lower vertebrates including zebrafish are able to undergo complete epimorphic regeneration following multiple types of cardiac injury. During the process of regeneration, spared zebrafish cardiomyocytes in the vicinity of the injured area undergo dedifferentiation and proliferation, thereby giving rise to new cardiomyocytes which replace the injured muscle. Insights into the molecular networks controlling these regenerative processes might help to develop novel therapeutic strategies to restore cardiac performance in humans.
While TGF-β signaling has been implicated in zebrafish cardiac regeneration, the role of individual TGF-β ligands remains to be determined. Here, I report the opposing expression response of two TGF-β ligand genes, mstnb and inhbaa, during zebrafish heart regeneration. Using gain- and loss-of-function approaches, I show that these ligands exert opposite effects on cardiac regeneration and specifically on cardiomyocyte proliferation. Notably, I show that overexpression of mstnb and loss of inhbaa negatively regulate cardiomyocyte proliferation and therefore disturb cardiac regeneration. In contrast, loss of mstnb and activation of inhbaa not only promote physiological cardiomyocyte proliferation but also enhance cardiac regeneration. I also identify Inhbaa as a mitogen which promotes cardiomyocyte proliferation independent of the well-established Nrg-ErbB signaling. Mechanistically, I unraveled that Mstnb and Inhbaa function through alternate Activin type 2 receptor complexes to control the activities of the signal transducers, Smad2 and Smad3, thereby regulating cardiomyocyte proliferation.
Altogether, I reveal novel and unidentified opposite functions of two TGF-β ligands during cardiac development and regeneration, resulting in a pro-mitogenic as well as an anti-mitogenic effect on cardiomyocytes. This study should therefore stimulate further research on targeting specific TGF-β family members to generate novel regenerative therapeutic strategies.
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.
Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
Cardiovascular disease is the leading cause of death worldwide. Aging is among the greatest risk factors for cardiovascular disease. Cardiovascular disease comprises several diseases, for example myocardial infarction, elevated blood pressure and stroke. Many processes are known to promote or worsen cardiovascular disease and in the present study, cellular senescence and inflammatory activation were of special interest, as they have a strong association to aging and can be seen as hallmarks of cellular aging.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. In recent years, numerous regulatory functions were shown for these transcripts and lncRNAs were shown to directly interact with DNA, RNA and proteins. The long noncoding RNA H19 was among the first described noncoding RNAs and was initially shown to act as a tumor suppressor. More recently, several studies showed oncogenic roles for H19. In regards to the cardiovascular system, H19 was not analyzed before.
We show that H19 is the most profoundly downregulated lncRNA in endothelial cells of aged mice compared to young littermates. Microarray analysis of human primary endothelial cells upon pharmacological H19 depletion revealed an involvement of H19 in cell cycle regulation. Loss of H19 in human endothelial cells in vitro led to reduced proliferation and to increased senescence. H19 depletion was shown to counteract proliferation before, but none of the described mechanisms applied to endothelial cells. We show that the reduction in proliferative capacity and the pro-senescent function of H19 is most probably mediated by an upregulation of p16ink4A and p21 upon H19 depletion.
When we compared the angiogenic capacity of aortic endothelial cells from young and aged mice in an aortic ring assay, rings from aged mice showed a reduced cumulative sprout length. Interestingly, pharmacological inhibition of H19 in aortic rings of young animals, where H19 is highly expressed, was sufficient to reduce the cumulative sprout length to levels we observed from aged animals. Furthermore, overexpression of human H19 in aortic rings of aged mice, where H19 is poorly expressed, rescued the impaired angiogenic capacity of aged endothelial cells.
We generated inducible endothelial-specific H19 knockout mice (H19iEC-KO) and subjected these animals to hind limb ischemia surgery followed by perfusion analysis in the hind limbs by laser-doppler velocimetry and histological analysis. Perfusion in the operated hind limb was increased in H19iEC-KO compared to Ctrl littermates, which was in contrast to a reduction in capillary density in the operated hind limbs of H19iEC-KO animals compared to Ctrl littermates and to our previous results. Analysis of arteriogenesis revealed an increase in collateral growth upon EC-specific H19 depletion in the ischemic hind limbs, which explains the increase in perfusion despite the reduction in capillary density. Further characterization of the animals revealed an increase in leukocyte infiltration into the tissue in the ischemic hind limbs upon endothelial-specific H19 depletion, indicating a potential role of H19 in inflammatory tissue activation.
Reanalysis of the microarray data from human primary endothelial cells upon H19 depletion revealed an association of H19 with inflammatory signaling and more specifically with IL-6/JAK2/STAT3 signaling. Analysis of cell surface adhesion molecule expression revealed an upregulation of ICAM-1 and VCAM-1 on mRNA level and an increase of the abundance of the two proteins on the cell surface of human primary endothelial cells. Consequently, adhesion of isolated human monocytes to human primary endothelial cells was increased upon H19 depletion in vitro. Interestingly, TNF-α mediated inflammatory activation of primary human endothelial cells repressed H19 expression. H19 did not function via previously described mechanisms. We excluded a competitive endogenous RNA (ceRNA) function for H19 in endothelial cells and showed that miR-675, which is processed from H19, does not play a role in the endothelium. Furthermore, H19 did not regulate previously described genes or pathways.
Analysis of transcription factor activity upon H19 depletion and overexpression revealed a differential activity of STAT3. STAT3 phosphorylation at TYR705 and thus activation was increased upon H19 depletion. Inhibition of STAT3 activation using a small compound inhibitor abolished the effects of H19 depletion on mRNA expression of p21, ICAM-1 and VCAM-1 and on proliferation, indicating that the effects of H19 are at least partially mediated via STAT3. STAT3 was shown to have positive effects on the cardiovascular system before, most likely due to upregulation of VEGF in a STAT3-dependent manner. We were not able to confirm previously described mechanisms for STAT3 in the present study and propose a new mechanism of action for the H19-dependent regulation of STAT3. Taken together, these results identify the long noncoding RNA H19 as a pivotal regulator of endothelial cell function. Figure 38 summarizes the described functions of H19 in endothelial cells.
The cardiovascular system (CVS) consists of heart and blood vessels, forming a close circulatory loop. All tissues depend on the nutrients and molecular oxygen (O2) delivered by the blood. Therefore, it is not surprising that the CVS is one of the first working systems and the heart is the first functional organ in the forming embryo (Baldwin 1996). The building blocks of blood vessels are endothelial cells (ECs), which form the endothelium, a specialized epithelium that defines the luminal surface of the vessels (Pugsley and Tabrizchi 2000). The process of blood vessel development comprises several steps. The first events occurring are the formation of new vessels de novo to constitute the primary vascular loop known as vasculogenesis. During vasculogenesis the vascular precursors, known as angioblasts, migrate and coalesce to form the axial vessels. Subsequently, the main vessels undergo a specification step where they acquire either arterial or venous identity. As the embryo increases in size, the main vascular loop needs to increase in complexity. In order to reach all the different parts of the developing organs, new blood vessels are formed from pre-existing ones, a phenomenon known as angiogenesis (Gore et al. 2012).
Mature blood cells have a short lifespan. Therefore, hematopoietic stem cells (HSCs) are required throughout lifetime to constantly form new blood cells in a process called hematopoiesis. Interestingly, endothelial and immune cells development have been shown to converge at different points during their development, one of which is developmental hematopoiesis. During embryogenesis, definitive hematopoiesis occurs in a tissue called hemogenic endothelium (HE), a specialized subset of ECs at the ventral wall of the dorsal aorta (DA). HE acquires hematopoietic potentials and gives rise to HSCs, through a process known as endothelial-to-hematopoietic transition (EHT). During EHT, these specialized ECs extrude from DA and colonize the so-called aorta-gonadmesonephros (AGM) region, forming the native HSCs (Paik and Zon 2010).
As vascular development requires different steps, the molecular pathways involved are many. The Notch signaling pathway has been demonstrated to be one of the main players in vascular development. Among other functions, Notch signaling has been shown to be important during EHT. In the murine model, Runx1, a master regulator of HSC formation, has been shown to be transcriptionally regulated by NOTCH1 through GATA2 activation. This observation was later corroborated by knockdown studies for notch1a and notch1b in zebrafish (Butko, Pouget, and Traver 2016). Another essential pathway for vascular development is the HIF pathway. Hif-1α, Hif-1β and Hif-2α mouse mutants show severe vascular defects that result in early embryonic lethality (Simon and Keith 2008), which hinders a deep analysis of the phenotypes incurring in the mutant embryos. In addition, deletion of Hif-1α specifically in myeloid cells showed abnormalities in the motility, invasiveness, and adhesion of macrophages (Cramer et al. 2003). Intriguingly, Hif-1α deletion in vascular endothelial cadherin-expressing cells led to a significant but partial reduction of HSC number, suggesting that other players may be involved in this pathway (Imanirad et al. 2014).
Zebrafish embryos have been shown to be tolerant to hypoxia at very early stages of development (Padilla and Roth 2001). Also, zebrafish embryos develop externally and this allows to finely manipulate the environment where they grow (Lieschke and Currie 2007). These features make zebrafish an ideal model to investigate how hypoxia and Hif transcription factors affect vertebrate vascular development. In this study, I will examine the impact of hypoxia on zebrafish vascular development. Specifically, I will dissect the role of hif-1α in macrophage-EC interactions during vascular development and repair. Moreover, I show redundant functions for hif-1α and hif-2α in HSC development upstream of Notch signaling.
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.
Tissue integrity is defined by the composition and connection of cells as a structural and functional unit. It is modulated by a magnitude of processes including differentiation, survival, controlled death and adhesion of cells. Besides, external factors such as physical forces are also involved. A suitable model system to study all modalities of tissue integrity is the mammary gland. Postnatally and within the reproductive phase, the mammary gland undergoes morphological and functional modifications that periodically loosen or strengthen tissue integrity. An important point in the development of the mammary gland is the regression during weaning, also termed involution. The transition from lactation to involution is important for a controlled loss of tissue integrity. In this transition, collective cell death is initiated but not yet prominent enabling the mammary gland to fully recover lactation.
In this thesis, modalities of tissue integrity were investigated using three-dimensional cell cultures (i.e. spheroids) and the mammary gland as model systems. In the context of this thesis, I established (1) an immunofluorescence staining protocol and its detailed evaluation. Furthermore, I studied (2) the role of cell survival during mammary gland development, (3) the effect of physical forces that modulate tissue integrity and (4) the contribution of proteins to cell adhesion and growth.
Since a homogeneous fluorescence stain of the specimen is necessary for quantitative analysis, an immunofluorescence staining protocol was established to stain large spheroids in toto. The evaluation contributes qualitative and quantitative criteria that judge the specificity, intensity and homogeneity of the stain. Based on this approach, it was possible to demonstrate the morphological and functional characteristics that spheroids share with the mammary gland in vivo. These characteristics included the synthesis of extracellular matrix, the development of polarized acinar structures and lactogenic differentiation.
The role of cell survival during mammary gland development was analyzed by means of the expression profile of the pro-survival protein BAG3. The expression of BAG3 differed in the progress of mammary gland development. While the expression was low during pregnancy, it rose in the lactation phase and peaked within the first days of involution, indicating that BAG3 is associated with early involution in the mammary gland. In vitro experiments related the expression of BAG3 to cell survival in mammary epithelial cells.
Physical forces naturally occur during developmental processes influence tissue integrity during the initiation of mammary gland involution. The influence of physical force applied as compression on mammary epithelial spheroids was investigated. A morphological analysis showed that following a lag, the cell nuclei volume changed upon compression. A short-term compression induced the activation of caspases. A prolonged compression reduced the activity of caspases. This suggests the induction of a process that allows cells the adaption to changing environmental conditions. BAG3 is known to be involved in mechanical stress-induced autophagy, also known as chaperone assisted selective autophagy (CASA). Compression of spheroids did not induce CASA. The experimentally applied strain was not comparable to the strain found in the alveolar cells during involution in vivo. Thus, whether or not CASA is activated during mammary gland involution remains elusive. Nevertheless, the methodical approach to apply compression on spheroids in vitro is a model to study the influence of physical forces on cell aggregates.
Apart from cell survival and physical forces, growth and adhesion of cells affect tissue integrity. A spheroid formation assay and subsequent data analysis and computational modeling enabled the investigation of these processes in a non-adhesive environment. The analysis suggested that spheroid formation follows a reaction-controlled process, in which cells do not necessarily form a connection when they collide. The loss of function of either E-cadherin or actin strongly inhibited the formation of a spheroid. The analysis further revealed that neither E-cadherin nor actin influence the chance of the cells to form a connection when they collide. Both molecules are more important in stabilizing established connections. Depolymerization of microtubules still allowed spheroids to form, but the formation was decelerated and growth of the final spheroids was inhibited. The results from computational modeling suggested that microtubules act on cell adhesion through different mechanisms, which also vary among different cell types. The inhibition of FAK phosphorylation at Y397, a downstream target of integrin signaling, and the analysis of FAK protein levels in spheroids showed that integrin-mediated signaling is not prominent in three-dimensional spheroids formed from non-invasive cells. A deletion of BAG3 gene expression increased the number of dead cells in forming spheroids suggesting that BAG3 predominantly affects cell survival.
The results of this thesis identified and characterized adhesion- and survival-associated proteins that are important for tissue integrity. This thesis suggests that a BAG3-dependent cell survival mechanism is prominent at the beginning of mammary gland involution. Future studies will have to identify the related factors and inducers of tissue integrity loss in the mammary gland. This will shed light on the physiology of the organ and could explain the disorders that destroy its integrity. In addition, this thesis contributes to a better understanding of spontaneous cell aggregation, the aggregate organization and implies a role of cell migration in these processes. Future studies that focus on three-dimensional cell migration could explain, how cell migration is promoted and to which extent it supports tissue integrity.
Savannas provide essential ecosystem services for human well-being in West Africa. Thus, ecosystem change not only directly affects biodiversity but also human livelihoods. Human land use considerably shaped these savanna ecosystems for millennia, particularly agriculture, livestock grazing, logging and the collection of non-timber forest products (NTFPs). NTFPs are wild plant products and comprise all organic matter from herbaceous plants, shrubs, and trees (excluding timber). Current increasing land use pressure through fast demographic changes is widely esteemed as a severe threat for savanna biodiversity and the socio-economy of rural communities. In consideration of the pivotal role of NTFP species for biodiversity and livelihoods, it is important to evaluate the effect of increasing land use change on savanna vegetation and on its provisioning service for human well-being. Thus, the major aim of this thesis is to investigate the impacts of land use intensification on vegetation composition, diversity and function and its consequences for provisioning ecosystem services (NTFPs) and human well-being in a West African savanna.
The research for this study was conducted in the North Sudanian vegetation zone of south-eastern Burkina Faso, where population growth exceeds the nationwide trend. Generally, Burkina Faso belongs to the worldwide poorest countries, where nearly one quarter of the population suffers from malnutrition (FAO 2014). The integration of NTFPs and particularly wild food species into rural household economies is, thus, an important measure in the national combat against poverty and food insecurity (FAO 2014). Against this background, I focus on vegetation changes, the economic importance of NTFPs as well as the decrease and substitution of wild food species in this study.
Vegetation resurveys of different vegetation types since the early 1990s showed that land use change led to more pronounced changes in the herbaceous than in the woody vegetation layer. Most woody vegetation types stayed stable in species composition and richness, even though some highly useful tree species (Vitellaria paradoxa, Parkia biglobosa) declined in some woody vegetation types. In contrast, in most herbaceous vegetation types species richness increased and species composition considerably changed. This change might be explained by a general ruderalisation process through a pronounced increase of wide-ranging herbaceous species. However, in spite of a general species increase in the herbaceous layer, a decrease of preferred herbaceous fodder species was found. Thus, the decline of useful species in both layers is alarming. Herbaceous vegetation types also showed more pronounced changes in plant functional trait characteristics in comparison to woody vegetation types. However, an increase of smaller plant species and species with a high diaspore terminal velocity (VTerm) was found in both vegetation layers. Since these two trait responses are generally related to grazing and browsing, the strong increase of livestock herds is likely to be responsible for the detected vegetation changes.
In addition to the vegetation study, interviews showed that all useful food species were widely considered to decline. The two economically most important tree species, the shea tree (Vitellaria paradoxa) and the locust bean tree (Parkia biglobosa) that contribute with 70% to wild food income, were considered among the most declining species of all cited wild food species. On this matter, local perceptions of species decline and results from field observations are in accordance. However, a wide range of cited substitutes indicated a great knowledge on alternative plant species in the area. Most wild food species are, however, substituted by other highly valued wild food species. Although our results suggest that rural communities are able to cope with the decrease or absence of wild food species, growing decline of one species would concurrently increase the pressure on other native food species. Therefore, the need to counteract the decrease of highly useful wild food species should be of high priority in management measures. In general, I showed that NTFPs are an essential component in rural households, since it contributed with 45 % to total household income. Significant differences in NTFP dependency between the two investigated villages and across the three main ethnic groups were detected, reflecting different traditional uses and harvesting practices. In general, it was shown that poorer households depend more on NTFP income than wealthier households. Against the background of this study, management strategies for agroforestry systems and poverty alleviation should consider local differences, and ethnicity-dependent NTFP-use patterns.
Overall, the combination of field studies on temporal and functional vegetation change with socio-economic and ethno-botanic interviews increases the knowledge on qualitative and quantitative vegetation changes and on the consequences for rural populations. This thesis gives a thorough insight into decreasing trends of economically valued plant species and thus gives evidence on the consequences of vegetation changes for ecosystem services of West African savanna ecosystems. Further, different NTFP-dependencies and use preferences according to socio-economic and cultural variables, such as ethnicity, present a valuable basis for specific decision-making and should be considered in management plans.
Surface water can contain a complex mixture of organic micropollutants (i.e. residues of pharmaceuticals or biocides). Conventional wastewater treatment plants (WWTPs) do not completely remove a broad range of anthropogenic chemicals and therefore represent a leading point source. To upgrade WWTPs, technical solutions based on oxidative and sorptive processes have been developed and successfully implemented. Acknowledging these substantial advances, this thesis focuses on another key topic and aims to investigate whether improved biological treatment processes likewise effectively remove anthropogenic micropollutants from wastewater. The work conducted on this topic was part of two European research projects (ATHENE, ENDETECH).
The ATHENE project aimed to go beyond the state-of-the-art by developing biological wastewater treatment processes that exploit the full potential of biodegradation. With the objective to explore the potential of complementary strictly anaerobic conditions within the biological wastewater treatment, combinations of aerobic and anaerobic treatments on site of a WWTP were implemented. Based on pre-experiments, two promising treatment combinations were selected for a more comprehensive evaluation. An aerobic treatment was paired with an anaerobic pre-treatment under iron-reducing conditions, and an activated sludge treatment was combined with an anaerobic post-treatment under substrate-limiting conditions. For the evaluation of these processes, an effect-based assessment was applied and combined with chemical data of 31 selected target organic micropollutants as well as ten metabolites. To assess the removal of endocrine disrupting chemicals (EDCs), yeast based reporter gene assays covering seven receptor-mediated mechanisms of action including (anti-)estrogenicity, (anti-) androgenicity, retinoid-like, and dioxin-like activity were conducted. Furthermore, the removal of unspecific toxicity (Microtox assay) and oxidative stress response as a marker for reactive toxicity (AREc32 assay) were analyzed to cover micropollutants acting via a non-specific mechanism of action. Moreover, to assess toxicity of the whole effluent in vivo, standardized in vivo bioassays with four aquatic model species (Desmodesmus subspicatus, Daphnia magna, Lumbriculus variegatus, Potamopyrgus antipodarum) were performed.
The combination of aerobic and anaerobic treatments resulted in a low additional removal of the selected target organic micropollutants (by 14-17%). In contrast, the removal of endocrine and dioxin-like activities (by 17-75%) and non-specific in vitro toxicities (by 27-60%) was significantly enhanced. Compared to technical solutions (i.e. ozonation), the combination with an anaerobic pre-treatment under iron-reducing conditions was likewise effective in removing the estrogenic activity as well as the unspecific toxicity, whereas anti-androgenic activity and dioxin-like activity were less effectively removed. Exposure to effluents of the conventional activated sludge treatment did not induce adverse in vivo effects in the investigated aquatic model species. Accordingly, no further improvement in water quality could be observed. In conclusion, the combination of aerobic and anaerobic treatment processes significantly enhanced the removal of specific and non-specific in vitro toxicities. Thus, an optimization of the biological wastewater treatment can lead to a substantially improved detoxification. These capacities of a treatment technology can only be uncovered by complementary effect-based measurements.
The global objective of the ENDETECH project was to develop a biotechnological solution to eliminate recalcitrant pharmaceuticals in wastewater direct from sites, where high loads are expected (i.e. hospitals). For this purpose, laccase, an enzyme mainly found in wood decaying fungi, was immobilized on ceramic membranes for application in bioreactors. In a proof of principle experiment, the performance of immobilized laccase in removing a mixture of 38 antibiotics without and in combination with a natural mediator (syringaldehyde; SYR) was investigated. For the evaluation of the enzymatic membrane bioreactors, chemical data on the elimination of the selected target antibiotics was combined with the outcomes of two in vitro bioassays. Growth inhibition tests with an antibiotic sensitive Bacillus subtilis strain were conducted to assess the residual antibiotic activity of the effluents, and Microtox assays were performed to detect a potential formation of toxic by-products.
The treatment by laccase without SYR did not reduce the load of antibiotics significantly. In contrast, in combination with a SYR concentration of 10 µmol L-1, 26 out of 38 antibiotics were removed by >50% after 24 h treatment. Moreover, increasing the SYR concentration to 1000 µmol L-1 resulted in a further improvement of the antibiotic removal. 32 out of 38 antibiotics were removed by over 50%, whereby 17 were almost completely eliminated (>90%). However, the treatment with laccase in combination with SYR resulted in a time-dependent increase of unspecific toxicity. While SYR alone did not affect B. subtilis, the combination of laccase with SYR led to a strong time-dependent growth inhibition up to 100%. Similar to that, a time-dependent increase of unspecific toxicity in the Microtox assay was observed. In conclusion, the laccase-mediator process successfully degrades a broad spectrum of antibiotics and thus represents a promising technology to treat wastewater from sites, where high loads are expected. However, further research is required to reduce the formation of unspecific toxicity before an implementation of this technology can be considered.
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.
Taxonomy, phylogeny and zoogeography of the hexaploid Torini of the Middle East and North Africa
(2017)
Fishes of the tribe Torini Karaman, 1971 (Teleostei: Cyprinidae) are a diverse group of primary freshwater fishes, distributed in Africa, the Middle East, and Indomalaya. They are an important component of the native freshwater-fish fauna of the Middle East and North Africa, and occur in most large river systems of the Levant, Arabia, Mesopotamia, southern Iran, and Morocco. They belong to the subfamily Cyprininae, are characterised by being tetraploid or hexaploid, having large scales, and a smooth and ossified last unbranched ray in the dorsal fin. As primary freshwater fishes they are not able to tolerate marine conditions and depend on direct freshwater connections for their dispersal. This makes them an ideal model for zoogeographic studies.
Prior to this study, the diversity of the Torini species in the Middle East and North Africa was not well understood. The validity of several genera and species was unclear, and the generic assignment of several species changed frequently.
In this PhD project the taxonomy, phylogeny, and zoogeography of the Torini of the Middle East and North Africa were investigated with morphological, as well as molecular methods. More than 1550 fish specimens were examined morphologically. Some of the specimens, including the types of most nominal species, were already available from museum collections. The remaining specimens were collected during expeditions to Ethiopia, Iran, Jordan, Morocco and Syria. Tissue samples were collected for molecular genetic analyses. The mitochondrial genes for cytochrome b, NADH dehydrogenase subunit 4 and the tRNAs for serine and histidine were sequenced from more than 120 specimens, representing 20 species of Torini and two small, diploid African barbs (Cyprinidae, tribe Smiliogastrini). Molecular data were analysed with Bayesian inference and other methods.
The analyses confirmed that the hexaploid Torini of Africa and the Middle East form a monophyletic group. In the Middle East and North Africa the Torini are represented by the genera Arabibarbus, Carasobarbus, Mesopotamichthys, and Pterocapoeta. These genera are each morphologically diagnosable, monophyletic, and genetically distinct. The species 'Labeobarbus' reinii cannot be assigned to any of these genera, because it is morphologically dissimilar and genetically clearly separated from each of them. A generic name for this species is presently not available and until the description of a new genus it is preliminarily assigned to the genus 'Labeobarbus'.
Out of the 28 species-group taxa described from the Middle East and North Africa until now, 15 are valid: Arabibarbus arabicus, A. grypus, A. hadhrami, Carasobarbus apoensis, C. canis, C. chantrei, C. exulatus, C. fritschii, C. harterti, C. kosswigi, C. luteus, C. sublimus, Mesopotamichthys sharpeyi, Pterocapoeta maroccana, and 'Labeobarbus' reinii.
The phylogenetic relationships between the Middle Eastern and North African Torini are well resolved, based on the analysis of mitochondrial DNA sequences from nearly all relevant species.
The interspecific and intraspecific morphological and genetic diversity is shaped by the zoogeographic history. Conclusions can be drawn about the events that shaped the evolution of this group. The Torini originated in the Indomalayan biogeographical realm and colonised the Middle East and Africa during the Miocene via the Gomphotherium landbridge. The Indomalayan Torini are tetraploid, whereas those of the Middle East and Africa are hexaploid. Molecular phylogenetic analyses showed that the hexaploid Torini cluster within the tetraploid Torini. This makes the tetraploid Torini a paraphyletic group with respect to the hexaploid Torini. Morocco was colonised in two independent waves. The first came from sub-Saharan Africa and is represented by Pterocapoeta maroccana. The second originated in the Middle East and gave rise to C. fritschii, C. harterti, and probably 'L.' reinii. The Tigris-Euphrates system is the largest freshwater system in the Middle East. Its central position between the Orontes River and Jordan River in the West, the Iranian tributaries to the Persian Gulf in the East, and the Arabian Peninsula in the South made it an important crossroad for the colonisation of the Middle East by Torini and other freshwater biota. During the Miocene the predecessors of the Jordan and Orontes rivers were connected to the Tigris-Euphrates system. The Jordan River was separated from the Euphrates before the Orontes. Arabia was colonised in two waves. The first (A. arabicus, A. hadhrami, C. exulatus) dates to the Pliocene, whereas the second (C. apoensis) ended as recently as the late Pleistocene or early Holocene.
The process of urbanization is one of the major causes of the global loss of biodiversity; however, cities nowadays also have the potential to serve as new habitats for wildlife. The European rabbit (Oryctolagus cuniculus, L. 1758) is a typical example of a wildlife species that reaches stable population densities in cities. Due to intense plant and soil damages, German city authorities aim to control high rabbit densities through the application of a yearly hunting regime (e. g., in Munich, Berlin or Frankfurt am Main). In contrast, population densities of O. cuniculus are on decline in German rural areas, i. e., numbers of yearly hunting bags decreased. The aim of my doctoral thesis was to answer the following research questions: Do population densities of the European rabbit correlate with the intensity of urbanization in and around Frankfurt am Main and if so, which factors play a role in varying densities? How are burrow construction behaviors and group sizes, daytime activity patterns and anti-predator behaviors as well as communication behaviors of this mammal affected by urbanization?
In my first study, I focused on population dynamics across 17 different study sites in and around Frankfurt. As one of yet few studies, I invented an approach that quantified the intensity of urbanization (degree of urbanity) of each study site base on four variables: (1) intensity of anthropogenic disturbance per min and ha, (2) number of residents within a radius of 500 m, (3) proportion of artificial ground cover and (4) numbers of anthropogenic objects per ha. Spearman rank correlations confirmed that with increasing degree of urbanity also rabbit and burrow densities increased. The access to dense shrubs, bushes etc. as suitable sites for burrow construction is the most determining factor for rabbit abundances, and therefore I presumed different densities along the rural-to-urban gradient to be driven by shifts in the availability of thick vegetation.
In the second study, I calculated two indices that in both cases classified burrows to be either accumulated, evenly or randomly distributed within study sites. Additionally, in cooperation with local hunters the number of burrow entrances and animals that occupy the same burrow had been determined during the hunting season. With increasing degree of urbanity burrow distribution patterns shifted from accumulated in rural areas towards more evenly distributed within the city center of Frankfurt. This is a clear sign for an increasing access to sites suitable for burrow construction along the rural to-urban gradient. Additional Spearman rank correlations revealed that the external dimensions of burrows decreased (shorter distances between entrances) and that burrows became less complex (fewer entrances) along the rural-to-urban gradient. In accordance, the number of rabbits that commonly shared the same burrow system was highest within rural areas, whereas I found mainly pairs and single individuals within highly urbanized study sites.
In the last study I compared activity patterns, burrow use and percentages of anti-predator behaviors from one hour before sunrise until one hour after sunset of rural, suburban and urban rabbit groups. A linear mixed model (LMM) and Spearman rank correlations confirmed that rabbits located at urban and suburban sites spent more time outside their protective burrows compared to their rural conspecifics. At suburban sites, individuals invested the least amount of time in anti-predator behavior. Results of this third study gave evidence that suburban rabbit populations on one hand benefit from less predation pressure by natural predators in comparison to rural sites, whereas on the other hand are exposed to less intense disturbance by humans compared to urban study sites.
The last study focused on the effects that urbanization had on the latrine-based communication behavior of rabbits. As many other mammals, O. cuniculus exchange information via the deposition of excreta in latrines, and depending on the intended receiver(s), latrines are either formed in central areas for within-group communication or at territorial boundaries, e. g., for between-group communication. The relative importance of within- vs. between-group communication depends on, amongst other factors, population densities and group sizes which I proved both to shift along the considered rural-to-urban gradient. I determined latrine sizes, latrine densities and latrine utilization frequencies relative to their distance to the nearest burrow at 15 different study sites. Latrine densities and utilization frequencies increased with increasing distance from the burrow in suburban and urban populations whereas at rural sites, largest latrines and those containing the most fecal pellets were close to the burrow, suggesting that within-group communication prevailed.
To sum up, for the first time, I was able to relate shifts in the ecology and behavior of the European rabbit as adaptations to a gradual anthropogenic habitat alteration that are typical for “urban exploiters”. Especially the suburban habitat provides high landscape heterogeneity (“edge habitat“) which is essential for high and stable rabbit populations. Moreover, here, comparably low human disturbance and predation pressure are given in contrast to the agriculturally transformed, open landscapes which are nowadays typical for most rural areas in central Europe. I argue that this mainly leads to the observed behavioral changes along the rural-to-urban gradient. Future plans for rural land management actions should aim to increase refuge availability by generating networks of ecotones. This would also benefit species that depend on similar ecosystem structures as the European rabbit and are on decline in Germany.
Die Verarbeitung während des Hörprozesses von Säugetieren verläuft von der Kochlea mit den inneren und äußeren Haarsinneszellen (äHZ) über afferente Nervenbahnen bis zum auditorischen Kortex (AK). Die daran beteiligten Schaltstationen und deren Funktion sind überwiegend aufgeklärt. Die Hörbahn ist zudem in besonderer Weise durch efferente Rückkopplungen gekennzeichnet, die interne Modulationen sowie sekundäre Reaktionen auf den Reiz ermöglichen. Anatomisch betrachtet verlaufen efferente Projektionen vom AK zu sämtlichen am Hörprozess beteiligten Kerngebieten. Vom Olivenkomplex erfolgt über mediale und laterale Fasern eine Innervation der äHZ bzw. des Hörnervs. Trotz der gut beschriebenen Anatomie ist die funktionelle Beziehung zwischen dem AK und der Peripherie weitgehend ungeklärt. In der vorliegenden Arbeit wurde der funktionelle Zusammenhang vom AK zu den äHZ in der mongolischen Wüstenrennmaus untersucht. Dafür wurde entweder eine pharmakologische Blockierung der Kortexaktivität durch den Natriumkanalblocker Lidocain erzeugt oder eine Aktivierung der Kortexaktivität durch die Anwendung elektrischer Reize ausgelöst. Der Einfluss der Manipulationen wurde in der Kochlea mittels Messungen von Distorsionsprodukt-otoakustischen Emissionen (DPOAE) erfasst. Diese entstehen durch die nichtlineare Verstärkung leiser Schallsignale durch die äHZ zur Erzielung hoher Sensitivität und Frequenzauflösung. Die DPOAE treten als kubische (z. B. 2f1-f2) und quadratische (z. B. f2-f1) Verzerrungen auf und geben Aufschluss über unterschiedliche Parameter der äHZ-Verstärkungsfunktion.
Die Lidocainversuche wurden entweder kontra- oder ipsilateral zur DPOAE-Messung durchgeführt. In beiden Konstellationen traten nach der Lidocaininjektion Erhöhungen und Verringerungen der DPOAE-Pegel im Vergleich zur Basismessung oder unveränderte DPOAE-Pegel auf. Im Mittel lagen die Pegeländerungen bei ca. 11 dB, in Einzelfällen betrugen sie bis zu 44,8 dB. In den Gesamtdaten waren die Effekte nach kontralateraler Injektion oft signifikant größer als nach ipsilateraler Injektion. Ebenso waren die Effekte in der 2f1-f2 Emission meist signifikant größer als in der f2-f1 Emission. Zudem wurde beobachtet, dass signifikant größere Effekte bei einer Stimulation mit Pegeln von 60/50 dB SPL im Vergleich zu 40/30 dB SPL erreicht wurden. Grundsätzlich trat in allen Datensätzen eine Reversibilität der DPOAE-Pegel mit zunehmender Versuchsdauer auf. Die Effekte waren direkt nach der Injektion am größten und erreichten je nach Stimuluspegel und Emissionstyp nach 28-100 min die Basispegel. In keinem der Datensätze lag eine Abhängigkeit der im Kortex gereizten charakteristischen Frequenz (CF) zum betroffenen Frequenzbereich in der Kochlea vor. Die Effekte waren über den gesamten gemessenen Frequenzbereich von 1-40 kHz nachweisbar. Allerdings waren die Frequenzbereiche von 1-10 kHz und 30,5-40 kHz besonders stark von der Lidocaininjektion betroffen.
Auch nach der elektrischen Reizung wurden die drei oben beschriebenen Effekttypen definiert. Mit 54,6 % war der Prozentsatz unveränderter DPOAE-Pegel allerdings sehr hoch. In den anderen beiden Kategorien konnten zusätzlich Differenzierungen im zeitlichen Verhalten der DPOAE-Pegel vorgenommen werden. In 21,6 % bzw. 16,5 % der Datensätze waren die Verringerungen bzw. Erhöhungen bis zum letzten gemessenen Zeitpunkt nach der elektrischen Reizung irreversibel und nur in jeweils 2,8 % der Datensätze war eine Reversibilität zu verzeichnen. In diesen Fällen war die Effektdauer mit im Mittel 31 bzw. 25 min kürzer als in den Lidocainversuchen. Auch die Effektstärken waren mit maximal 23,9 dB und je nach Effekttyp im Mittel 5,1-13,7 dB geringer als nach der Lidocaininjektion. Die größten Effekte traten in einem mittleren Stimuluspegelbereich von 45-55 dB SPL auf. Wiederum konnte keine Abhängigkeit des betroffenen Frequenzbereichs von der kortikal gereizten CF nachgewiesen werden. In Einzelfällen waren auf DPOAE-Ebene nur die Frequenzen ober- und unterhalb der kortikalen CF beeinflusst, wohingegen bei der CF selbst keine Effekte auftraten.
Durch Kontrollexperimente (Salineinjektion bzw. Einführen der Elektrode ohne elektrische Reizung) konnte nachgewiesen werden, dass die Effekte durch die Manipulation der Kortexaktivität hervorgerufen wurden. Somit liegt eine funktionelle Beziehung zwischen dem AK und der Peripherie vor, die langanhaltende massive Ausmaße annehmen kann. Die Effektrichtung ist vermutlich durch die exzitatorisch oder inhibitorisch wirkenden Neurone vom Colliculus inferior zum Olivenkomplex bedingt. Die größeren Effekte in der kontralateralen Konfiguration lassen sich durch die Diskrepanz in der Anzahl der gekreuzten (2/3) und ungekreuzten (1/3) medialen Efferenzen erklären. Die kubischen Komponenten der äHZ-Verstärkungsfunktion scheinen stärker beeinflusst zu sein als die quadratischen Komponenten, was in größeren Pegeländerungen in der 2f1-f2 Emission resultiert. Die teils großen Effektstärken sowie die nicht vorhandene Frequenzabhängigkeit zwischen AK und Kochlea sind vermutlich auf den großen Kortexbereich zurückzuführen, der von den gewählten Injektionsvolumina bzw. elektrischen Reizstärken betroffen war. Die großen Effekte im mittleren Stimuluspegelbereich lassen sich sowohl mit einer möglichen Schutzfunktion der Efferenzen vor zu lauten Schallereignissen als auch mit einer Verbesserung des Signal-Rausch-Verhältnisses zur erleichterten Detektion akustischer Signale in Einklang bringen. Insgesamt deuten die Ergebnisse darauf hin, dass die Aktivität des AK einen starken Einfluss auf periphere auditorische Mechanismen hat, wodurch die kochleäre Verarbeitung akustischer Signale je nach kortikalem Verarbeitungsstatus massiv modifiziert werden kann.
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.
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.
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 mitochondriale Innenmembran (IM) besteht aus zwei Subkompartimenten. Der Cristae Membran (CM) und der inneren Grenzmembran (IBM), welche durch die runden und schlitzartige Strukturen der Christa Junctions (CJs) verbunden werden Der MICOS-Komplex ist an den CJs lokalisiert und besteht aus mindestens 6 Komponenten, Mic60, Mic27, Mic26, Mic19, Mic12 und Mic10. Es ist bekannt, dass der MICOS-Komplex essentiell für die Stabilität der CJs ist. Die in dieser Arbeit gezeigten Ergebnisse, geben Aufschluss darüber, wie sich einzelne MICOS-Komponenten auf die Stabilität von Cristae und CJs im Modellsystem Hefe (S cerevisiae) auswirken. Zu Beginn dieser Arbeit war zum einen bekannt, dass die MICOS-Komponente Mic60 essentiell für die Bildung von CJs ist. Zum Anderen wurden im Vorfeld dieser Arbeit Interaktionen von Mic60 mit Proteinen in der mitochondrialen Außenmembran, vor allem Proteinkomplexe mit β-barrel-Proteinen identifiziert. Diese Interaktionen werden über den evolutionär, konservierten C-Terminus von Mic60 vermittelt.
β-barrel Proteine besitzen eine charakteristische Peptidsequenz, die β-Sequenz. Diese dient nach dem Import der β-barrel Proteine in die Mitochondrien als Signalpeptid für den SAM-/TOB-Komplex, welcher daraufhin die Proteine in die Außenmembran insertiert. In dieser Arbeit wurde ebenfalls eine β-Sequenz im C-Terminus von Mic60 identifiziert, diese zeigte einen Einfluss auf die Cristae-Stabilität. Zellen die eine Mic60-Variante mit einer Deletion oder Punktmutation der β- Domäne exprimieren, zeigten eine reduzierte Anzahl an CJs. Auch das Verkürzen des C-Terminus von Mic60 hatte diesen Effekt auf die mitochondriale Ultrastruktur. So konnte gezeigt werden, dass die β-Domäne und die Integrität des C-Terminus essentiell für die Stabilität von CJs sind.
Der Fokus dieser Arbeit lag in der Charakterisierung der MICOS-Komponenten Mic26 und Mic27. Es konnte bewiesen werden, dass beide Proteine genetisch mit der MICOS-Kernkomponente Mic60 interagieren. Die Untersuchung der mitochondrialen Ultrastruktur von Δmic26- und Δmic27-Zellen zeigte, dass eine Deletion vom Mic26 keinen Einfluss auf die Organisation der mitochondrialen Innenmembran hat. Im Gegensatz dazu, ist im Vergleich zum Wildtyp die Anzahl an CJs in Δmic27-Zellen um zwei Drittel reduziert. Auch die Innenmembranoberfläche ist in diesen Zellen stark vergrößert. Die Untersuchung der Morphologie der mitochondrialen Innenmembran in Zellen ohne Mic27 durch Kryo-Elektronentomographie isolierter Mitochondrien, veranschaulichte die Struktur der CJs in diesen Zellen genauer. Es zeigten sich hier breitere CJs, und der Übergang von der Cristaemembran in den Bereich der inneren Grenzmembran ist sehr flach und undefiniert. In Wildtyp-Mitochondrien waren die CJs schmal und schlitzartig und haben einen scharfkantigen Übergang von der Cristaemembran zur inneren Grenzmembran. Des Weiteren wies die Cristaemembran in Δmic27-Zellen unregelmäßige zackige Strukturelemente auf, was auf eine Anhäufung an Dimeren der F1FO-ATP Synthase hinweist.
Diese Beobachtungen in den Kryo-Tomogrammen, wurde durch Analysen des Oligomerisierungszustands der F1FO-ATP Synthase in Δmic27-Zellen, bestätigt. Hier fanden sich deutlich weniger höhere Oligomere und vermehrt Dimere. So kann aus diesen Befunden geschlossen werden, dass Mic27 die Oligomere der F1FO-ATP Synthase stabilisiert.
Um zu untersuchen, wie der MICOS-Komplex mit der F1FO-ATP Synthase in Verbindung steht, wurde mittels 2D-BNE-Analysen und einem Complexome Profiling die Komplexierung der nativen Komplexe in Wildtyp- und Δmic27-Mitochondrien analysiert. Zum einen konnte durch diese Untersuchungen gezeigt werden, dass Mic27 neben der F1FO-ATP Synthase auch stabilisierend auf den MICOS-Komplex wirkt. Die Komplexe im hochmolekularen Bereich der MICOS-Komponenten zerfielen in Δmic27-Zellen, was darauf hinweist, dass die anderen MICOS-Komponenten hier nicht mehr assemblieren können. Mic10 war die einzige MICOS-Komponente die in Δmic27-Zellen noch stabile Komplexe im hohen Massenbereich ausbildete. Mic10 findet sich zudem nicht nur in Klustern mit anderen MICOS-Komponenten sondern auch mit der F1FO-ATP Synthase.
Die Interaktion von Mic10 und der F1FO-ATP Synthase wurde auch biochemisch, mittels chemischer Quervernetzern und Ko-Immunpräzipitationsexperimenten bestätigt. Dies legt nahe, dass Mic10 die CJs mit hoher Wahrscheinlichkeit, durch die Verbindung mit der F1FO-ATP Synthase, mit der Cristaemembran verbindet und so stabilisiert.
Aufgrund der Erkenntnisse dieser Arbeit konnte ein neuartiges Modell postuliert werden. Die MICOS-Komponente Mic60 stabilisiert die CJs durch eine Interaktion seines C-Terminus mit Proteinen in der Außenmembran. Mic27 vermittelt über Mic10 die Interaktion zur F1FO-ATP Synthase. Somit ist diese neu identifizierte Interaktion des MICOS-Komplex zur F1FO-ATP Synthase essentiell für die Stabilität von CJs ist, indem es den MICOS-Komplex mit den Oligomeren der F1FO-ATP Synthase verbindet.
Der Gyrus dentatus ist eine anatomische Region im Hippocampus und besitzt die einzigartige Fähigkeit auch im adulten Gehirn lebenslang neue Nervenzellen zu generieren. Dieser Prozess wird als adulte Neurogenese bezeichnet, stellt eine besondere Form struktureller Plastizität dar und es wurde gezeigt, dass adult neugebildete Körnerzellen im Gyrus dentatus essentiell am Prozess des hippocampalen Lernens und der Gedächtnisausbildung beteiligt sind. Es wird vermutet, dass neue Körnerzellen aufgrund ihrer charakteristischen Eigenschaften verstärkt auf neue Informationsmuster reagieren können und darauf spezialisiert sind Muster, die eine hohe Ähnlichkeit zueinander haben zu separieren und diese Unterschiede zu kodieren. Obwohl bereits eine Vielzahl von wissenschaftlichen Studien zum Verständnis der Entwicklung und Funktion adult neugebildeter Körnerzellen beitragen konnte, bestehen immer noch Unklarheiten darin, wie sich diese neuen Nervenzellen strukturell entwickeln, wann es zu einer funktionellen Integration kommt und wie diese beiden Prozesse miteinander zusammenhängen. In den vorliegenden Arbeiten wurde die strukturelle Entwicklung und synaptische Integration adult neugebildeter Körnerzellen in das bestehende hippocampale Netzwerk der Ratte und Maus unter in vivo Bedingungen untersucht. Zur Beantwortung dieser Fragen wurden Methoden aus der Anatomie, Histologie und in vivo Elektrophysiologie kombiniert. Der Nachweis neuer Körnerzellen erfolgte entweder durch immunhistologische Färbungen gegen spezifische Marker für unreife und reife Körnerzellen, Markierungen mit Bromdesoxyuridin oder retro- bzw. adenovirale intrazerebrale Injektionen und Expression von GFP. Es wurde eine in vivo Stimulation des Tractus perforans in der anästhesierten Ratte zur Langzeitpotenzierung der Körnerzellsynapsen und anschließend eine immunhistologische Analyse der Expression von synaptischen Aktivitäts- und Plastizitätsmarkern in neugebildeten und reifen Körnerzellen nach der Stimulation durchgeführt. Zusätzlich wurden detaillierte drei-dimensionale Rekonstruktion dendritischer Bäume erstellt und dendritische Dornenfortsätze an retroviral markierten Zellen analysiert.
Die vorliegenden Daten belegen den generellen Verlauf der Entwicklung neugeborener Körnerzellen in zwei unterschiedliche Phasen: eine frühe dendritische Reifung und eine späte funktionelle und synaptische Integration. Neugeborene Körnerzellen zeigten ein rasches dendritisches Auswachsen, dass innerhalb der ersten drei bis vier Wochen abgeschlossen war. Während dieses Wachstumsprozesses passieren Dendriten nacheinander die Körnerzellschicht und anschließend die innere, mittlere und äußere Molekularschicht. Dadurch sind sie innerhalb ihrer morphologischen Entwicklungsphasen anatomisch auf spezifische präsynaptische Partner limitiert. In der wissenschaftlichen Literatur wird eine transiente kritische Phase beschrieben, in der neugeborene Körnerzellen eine starke Plastizität und sensitivere synaptische Erregbarkeit aufweisen. Obwohl die vorliegenden Resultate keine direkten Hinweise auf eine stärkere bzw. sensitivere Plastizität neugeborener Körnerzellen liefern, konnte eine Phase zwischen vier und fünf Wochen identifiziert werden, in der neue Körnerzellen einen sprunghaften Anstieg in ihrer Fähigkeit zur Expression synaptischer Aktivitätsmarker (z.B. Arc und c-fos) und Ausbildung struktureller Plastizität (Dendriten und Dornenfortsätze) zeigten. Die präsentierten Resultate machen deutlich, dass Dornenfortsätze neuer Körnerzellen nach elf Wochen eine vergleichbare Dichte, Größenverteilung und Plastizität aufzeigen, die vergleichbar mit denen vorhandener Körnerzellen sind. Die Fähigkeit zur dendritischen Plastizität nach synaptischer Aktivierung zeigten jedoch nur neugeborene Körnerzellen zwischen der vierten und fünften Woche. Diese Ergebnisse implizieren, dass die Integration neugebildeter Körnerzellen kontinuierlich verläuft und obwohl die vorliegenden Daten die Existenz einer dendritischen Plastizität und einen sprunghaften Anstieg synaptischer Plastizität in der vierten und fünften Woche belegen, wurden keine weiteren Hinweise auf eine transiente kritische Phase gefunden. Des Weiteren zeigten dendritische Bäume von gereiften adult neugeborenen und reifen Körnerzellen Unterschiede, die daraufhin deuten, dass neue Körnerzellen eine eigene Subpopulation darstellen.
Multicellular organisms require that cells adhere to each other. This cell-cell adhesion is indispensable for the formation and the integrity of epithelial structures, tissues and organs. Mammals have developed four different cell-cell adhesion structures, the adhering junctions, which ensure the tight contact between cells but are also important platforms for communication and exchange in tissues. Two of these adhering junctions are cadherin based, the belt-like adherens junctions and the spot-like desmosomes. Both structures have in common that they are composed of single membrane spanning proteins, the cadherins, which accomplish adhesion in a calcium-dependent manner. The intracellular parts of classical as well as desmosomal cadherins bind to different adaptor proteins of the armadillo-protein family and others which build a protein plaque underneath the membrane and link the cadherins to the actin or intermediate filament cytoskeleton.
Desmosomes are of special importance for tissues that have to withstand mechanical stress. Although they are essential to stabilize tissues they have to be highly flexible and dynamic structures, as processes like wound healing or tissue remodeling require that adhesive interactions can be modulated. The molecular dynamics within desmosomes are not jet understood in detail, but it is assumed that two different membrane associated pools of desmosomal cadherins exist in cells. Cadherins that are incorporated in mature desmosomes are part of the junctional pool, whereas cadherins that are not associated with firm desmosomes and the intermediate filament cytoskeleton belong to the non-junctional pool. Lateral movements between the two pools results in a dynamic equilibrium and allows for example the exchange of old cadherins. Little is known about the breakdown of desmosomal cadherins. Several studies found that desmosome assembly or endocytosis are cholesterol dependent processes and claimed that membrane microdomains play a role in the regulation of desmosome dynamics. Moreover, membrane rafts may be involved in the pathomechanism of the desmosome associated disease pemphigus, were autoantibodies bind to the cadherin desmoglein-3 and trigger its internalization which results in a loss of adhesion in skin cells.
Membrane rafts are cholesterol dependent nanoscale structures of cellular membranes that are able to regulate the distribution of proteins within the plasma membrane and thus form platforms for cell signaling and membrane trafficking. Flotillins are proteins that are associated with membrane rafts and are reported to be involved in processes like endocytosis, endosomal sorting and a multitude of different signaling events. We could recently show that the membrane raft associated proteins flotillin-1 and flotillin-2 bind directly to the armadillo protein y-catenin which can be part of both, the adherens junction and the desmosome. The aim of this study was to eluciadate a possible role of flotillins in the regulation of desmosomes.
HaCaT keratinocytes were chosen as the main cell system for this study and at first the association of desmosomal components with flotillins was analyzed in detail. It was found that flotillins are clearly associated with desmosomal proteins. They colocalize with desmoglein-3 at cell borders and precipitate the other desmogleins. Further binding assays revealed that both flotillins bind to all desmogleins and the long isoforms of the second class of desmosomal cadherins, the desmocollins. The interaction is a direct one and was mapped to the ICS sequence within the cadherins. This close association rendered the question whether flotillins are functionally implicated in desmosome regulation. To address this issue, stable flotillin knockdown HaCaT cells were analyzed in detail. The molecular morphology of desmoglein-3, desmoglein-1 and two plaque proteins was clearly altered in the absence of flotillins. The membrane staining of all tested desmosomal proteins was derailed and disordered. Furthermoore, the loss of flotillins had an impact on the adhesive capacity of HaCaT keratinocytes. The cell-cell adhesion was weakened in the absence of flotillins, which was monitored by an increased fragmentation of knockdown cells in a cell dissociation assay.
In order to find out the mechanism by which flotillins influence the membrane morphology and the adhesiveness in keratinocytes, the association of desmosomal proteins with membrane microdomains was examined, at first. A predominant part of desmoglein-3 is associated with membrane rafts in HaCaT keratinocytes, whereas only a minor part of desmoglein-1 is found there. However, the raft-association of none of the examined proteins was altered in the absence of flotillins. Furthermore, flotillin depletion did not change the distribution of desmogleins with the two different cadherin pools. Less desmoglein-3 is found in the junctional pool of the flotillin depleted cells compared to the control cells, but this is due to an overall diminished desmoglein-3 protein level in these cells.
Flotillins are involved in endocytic processes but their exact role there is under debate. The endocytic uptake of desmosomal cadherins requires intact membrane rafts, but the precise mechanism is still unknown. A possible involvement of flotillins on the endocytosis of desmoglein-3 was addressed next. It is known that the internalization of desmoglein-2 is dependent on the GTPase dynamin, arguing for an involvement of dynamin in the endocytosis of desmoglein-3 as well. When dynamin and thus desmoglein-3 endocytosis was inhibited using chemical compounds, the mislocalization of desmoglein-3 that was observed in flotillin knockdown cells was restored. This suggest that inhibition of desmoglein-3 endocytosis enhances the amount and/or availability of desmoglein-3 at the plasma membrane, which then normalizes the morphological alterations caused by a knockdown of flotillins. Furthermore the morphological alterations in the flotillin knockdown HaCaT cells were found to be similar to the localization of desmoglein-3 that was observed upon treatment of keratinocytes with PV IgG These structures have been described before as linear arrays and are assumed to be sites of endocytic uptake. This strengthens the idea that enhanced desmoglein-3 internalization takes place in the absence of flotillins, which then results in a weakened adhesion.
Altogether this study revealed flotillins as novel players in desmosome mediated cell-cell adhesion processes. By binding to desmosomal cadherins and desmosomal plaque proteins, flotillins stabilize desmosomes at the plasma membrane and are required for a proper cell-cell adhesion.
In the dentate gyrus (DG) of the mammalian hippocampus, neurogenesis continues to take place throughout an organism’s life. Adult neurogenesis includes proliferation and differentiation of neural stem cells into dentate granule cells (GCs) that mature and integrate into the existing cellular network. This thesis work presents a novel approach that enables longitudinal examination of living postnatally generated GCs in their endogenous niche by using retroviral (RV) labeling in organotypic entorhino-hippocampal slice cultures (OTCs). Older GCs were fluorescence-labeled with an adeno-associated virus controlled by the synapsin 1 promoter (AAV-Syn). The combination of time-lapse imaging and 3-D reconstruction of newborn developing GCs and older, more mature GCs enabled comparative analyses of dendritic growth and cellular dynamics as well as investigations of spine formation and the establishment of synaptic contacts.
Postnatal neurogenesis was studied in the mouse and rat DG in vivo by analysis of the distribution of chemical neuronal maturation markers doublecortin (DCX) and calbindin in combination with the GC marker Prox1 between P7 and P42. The marker expression patterns at different time points indicated that the number of mature GCs increased gradually over time and that young, immature GCs were added to the inner layers of the granule cell layer (GCL), as is the case in the adult brain. The most substantial shift in GC maturation took place between P7 and P14, though GCs in the rat DG matured faster (i.e. by ~5 days) than GCs in the mouse. Immunocytochemical in vitro analysis in OTCs at DIV 7, 14, and 28 exhibited a distribution of marker expression over time that was comparable to in vivo, though the number of DCX-expressing GCs was low at DIV 28, indicating a considerable decrease in neurogenesis rate over time in the OTC. Nevertheless, RV-labeling of newborn GCs at DIV 0 yielded successful visualization and enabled time-lapse imaging of complete developing GCs up to 4 weeks after mitosis. During the second week of development, newborn GCs exhibited a high level of structural dynamics, including extension and retraction of dendritic segments. In the third week, newborn GCs displayed high dendritic complexity which was followed by pronounced dendritic pruning. Finally, a phase of structural stabilization and local refinement could be observed during the fourth week. Older AAV-Syn-labeled GCs did not exhibit such dynamic structural remodeling. Anterograde tracing of entorhinal projection fibers using the biotinylated dextran amine Mini Ruby showed innervation of the outer molecular layer (OML) by entorhinal axons at early time points, i.e. DIV 8 when newborn GCs started to extend dendrites into the ML, as well as at DIV 20 when RV-labeled GCs exhibited elaborate dendritic trees with processes in the OML intermingling with entorhinal fibers. This shows that newborn GCs in the OTC grow into an area of existing entorhinal axon terminals, which is highly similar to the situation in the adult brain. Hence, the results show that postnatal neurogenesis can be studied effectively in the OTC system as a model of adult neurogenesis. The first appearance of spine-like protrusions in newborn GCs was observed two weeks post RV injection. Ultrastructural electron-microscopic images revealed that spines established synaptic contacts with axonal boutons. These findings suggest that newborn GCs are successfully integrated into the existing cellular circuitry in the OTC system. The high level of structural flexibility found in this study might be a necessary requisite of new neurons for successful dendritic maturation and functional integration into a neuronal network. Thus, live imaging of postnatally born GCs in the OTC appears as a useful novel approach to elucidate the mechanisms that affect cellular dynamics of neurogenesis.
Heat stress transcription factors (Hsfs) play essential role in heat stress response and thermotolerance by controlling the transcriptional activation of heat stress response (HSR) genes including molecular chaperones. Plant Hsf families show a striking multiplicity, with more than 20 members in the many plant species. Among Hsfs, HsfA1s act as the master regulators of heat stress (HS) response and HsfA2 becomes one of the most abundant Hsfs during HS. Using transgenic plans with suppressed expression of HsfA2 we have shown that this Hsf is involved in acquired thermotolerance of S. lycopersicum cv Moneymaker as HsfA2 is required for high expression and maintenance of increased levels of Hsps during repeated cycles of HS treatment.
Interestingly, HsfA2 undergoes temperature-dependent alternative splicing (AS) which results in the generation of seven transcript variants. Three of these transcripts (HsfA2-Iα-γ), generated due to alternative splicing of a second, newly identified intron encode for the full length protein involved in acquired thermotolerance. Another 3 transcripts (HsfA2-IIIα-γ) are generated due to alternative splicing in intron 1, leading in all cases to a premature termination codon and targeting of these transcripts for degradation via the non-sense mRNA decay mechanism (NMD).
Interestingly, excision of intron 2, results into the generation of a second previously unreported protein isoform, annotated as HsfA2-II. HsfA2-II shows similar transcriptional activity to the full-length protein HsfA2-I in the presence of HsfA1a but lacks the nuclear export signal (NES) required for nucleocytoplasmic shuttling which allows efficient nuclear retention and stimulation of transcription of HS-induced genes. Furthermore, stability assays showed that HsfA2-II exhibits lower protein stability compared to HsfA2-I.
The presence of a second intron and the generation of a second protein isoform we identified in other Solanaceae species as well. Remarkably, we observed major differences in the splicing efficiency of HsfA2 intron 2 among different tomato species. Several wild tomato accessions exhibit higher splicing efficiency that favors the generation of HsfA2-II, while in these species the splice variant HsfA2-Iγ is absent. This natural variation in splicing efficiency specifically occurring at temperatures around 37.5oC is associated with the presence of 3 intronic polymorphisms. In the case of wild species these polymorphisms seemingly restrict the binding of RS2Z36, identified as a putative splicing silencer for HsfA2 intron 2.
Tomato accessions with the polymorphic “wild” HsfA2 show enhanced thermotolerance against a direct severe heat stress incident due to the stronger increase of Hsps and other stress induced genes. Introgression of the “wild” S. pennellii HsfA2 locus into the cultivar M82, resulted in enhanced seedling thermotolerance highlighting the potential use of the polymorphic HsfA2 for breeding.
We conclude that alterations in the splicing efficiency of HsfA2 have contributed to the adaption of tomato species to different environments and these differences might be directly related to natural variation in their thermotolerance.
The transition from the marine to the terrestrial realm is one of the most fascinating issues in evolutionary biology for it required the appearance, in different organisms, of several novel adaptations to deal with the demands of the new realm. Adaptations include, for instance, modifications in different metabolic pathways, development of body structures to facilitate movement and respiration, or tolerance to new conditions of stress. The transition to the land also gives an extraordinary opportunity to study whether evolution used similar changes at the genomic level to produce parallel adaptations in different taxa. Mollusks are among taxa that were successful in the conquest of the land. For instance, several lineages of the molluscan clade Panpulmonata (Gastropoda, Heterobranchia) invaded the intertidal, freshwater and land zones from the marine realm. In my dissertation, using tools from bioinformatics, phylogenetics, and molecular evolution, I used panpulmonates as a suitable model group to study the independent invasions into the terrestrial realm and the adaptive signatures in genes that may have favored the realm transitions. My work includes two peer-reviewed published papers and one manuscript under review. In Publication 1 (Romero et al., 2016a), I used mitochondrial and nuclear molecular markers to resolve the phylogeny of the Ellobiidae, a family that possesses intertidal and terrestrial species. The phylogeny provided an improved resolution of the relationships within inner clades and a framework to study the tempo and mode of the land transitions. I showed that the terrestrialization events occurred independently, in different lineages (Carychiinae, Pythiinae) and in different geological periods (Mesozoic, Cenozoic). In addition, the diversification in this group may not have been affected by past geological or climate changes as the Cretaceous-Paleogene (K-Pg) event or the sea-level decrease during the Oligocene. In Publication 2 (Romero et al., 2016b), I generated new mitochondrial genomes from terrestrial species and compared them with other panpulmonates. I used the branch-site test of positive selection and detected significant nonsynonymous changes in the terrestrial lineages from Ellobioidea and Stylommatophora. Two genes appeared under positive selection: cob (Cytochrome b) and nad5 (NADH dehydrogenase 5). Surprisingly, I found that the same amino acid positions in the proteins encoded by these genes were also under positive selection in several vertebrate lineages that transitioned between different habitats (whales, bats and subterranean rodents). This result suggested an adaptation pattern that required parallel genetic modifications to cope with novel metabolic demands in the new realms. In Manuscript 1 (Romero et al., under review), I de novo assembled transcriptomes from several panpulmonate specimens resulting in thousands of genes that were clustered in 702 orthologous groups. Again, I applied the branch-site test of positive selection in the terrestrial lineages from Ellobioidea and Stylommatophora and in the freshwater lineages from Hygrophila and Acochlidia. Different sets of genes appeared under positive selection in land and freshwater snails, supporting independent adaptation events. I identified adaptive signatures in genes involved in gas-exchange surface development and energy metabolism in land snails, and genes involved in the response to abiotic stress factors (radiation, desiccation, xenobiotics) in freshwater snails. My work provided evidence that supported multiple land invasions within Panpulmonata and provided new insights towards understanding the genomic basis of the adaptation during sea-to-land transitions. The results of my work are the first reports on the adaptive signatures at the codon level in genes that may have facilitated metabolic and developmental changes during the terrestrialization in the phylum Mollusca. Moreover, they contribute to the current debate on the conquest of land from the marine habitat, a discussion that has been only based in vertebrate taxa. Future comparative genome-wide analyses would increase the number of genes that may have played a key role during the realm transitions.
Ziel der vorliegenden Arbeit war es, vor- und nachbereitenden Unterricht zu Biodiversitätsführungen an den vier außerschulischen Lernorten Palmengarten, Senckenbergmuseum, Stadtwaldhaus und Zoo Frankfurt zu evaluieren. Durch den Unterricht mithilfe neu entwickelter Arbeitsmaterialien sollte die aktuelle Motivation der Schüler und weitere pädagogisch-psychologische Lernvariablen gefördert werden. Es stellte sich die Frage, ob so eine erhöhte Auseinandersetzung mit dem Themenkomplex Biodiversität erreicht werden kann und welche Einflussfaktoren dabei eine Rolle spielen.
Theoretische Grundlage war dabei das Risikowahlmodell der Leistungsmotivation nach Atkinson, das von Rheinberg zum handlungstheoretischen Modell der Motivation erweitert wurde (Rheinberg & Vollmeyer, 2012). Auf dieses bezieht sich der von Rheinberg et al. (2001) entwickelte und hier eingesetzte Fragebogen zur aktuellen Motivation (FAM).
Die Stichprobe setzte sich aus insgesamt 523 Schülern der Klassen 5 bis 9 zusammen. Davon nahm jeweils die Hälfte mit (Versuchsgruppe) und die andere ohne (Kontrollgruppe) vor- und nachbereitendem Unterricht an den Biodiversitätsführungen teil. Die Erhebung der aktuellen Motivation, des erworbenen Fachwissens und weiterer Variablen erfolgte in einem Pre/Post/Follow-Up-Design mit Fragebögen, deren Auswertung analytisch statistisch durgeführt wurde.
Es zeigte sich, dass in der Gesamtstichprobe die Teilnahme an der Biodiversitätsführung die aktuelle Motivation der Schüler erhöhte. Dauerhafte Lernparameter wie die Biologieeinstellung und die Interessenshandlung wurden jedoch nicht signifikant verändert. Ein eindeutiger Effekt der unterrichtlichen Vorbereitung konnte jedoch nicht ermittelt werden. Einzig beim gemessen Fachwissen zu den Führungsinhalten schnitt die Versuchsgruppe signifikant besser ab. Insgesamt wird angenommen, dass der Effekt des Besuchs des außerschulischen Lernortes an sich den Effekt der Vor- und Nachbereitung überdeckt oder vom Einfluss anderer Parameter beeinflusst wird. Hier stach besonders das Alter der Jugendlichen hervor, das vor allem in der hier evaluierten Schülergruppe bedingt durch die Pubertät eine große Rolle spielt. Weitere Einflussfaktoren waren die Biologieeinstellung und die Unterrichtsvariablen der Führung. In den Stichproben der einzelnen außerschulischen Lernorte zeigten sich leichte Abweichungen von der Gesamtstichprobe. Diese waren meist auf die leicht unterschiedliche Zusammensetzung der Stichproben zurückzuführen. Aber auch Besonderheiten der Lernorte hatten dabei ein bedeutendes Gewicht.
Bezüglich der Lernbedingungen für die Lernorte ließen sich aus den Ergebnissen vor allem zwei Komponenten ermitteln: Zum einen die Architektur/räumliche Struktur der Lernorte. Hier können Faktoren wie drinnen/ draußen, Größe und die räumliche Orientierung unterschieden werden. All dies hat Auswirkungen auf das physische Wohlbefinden der Schüler, was wiederum eine Voraussetzung für eine hohe Lernmotivation ist. Die andere Hauptkomponente ist das am Lernort behandelte Thema. Hier kann grob zwischen Pflanzen und Tieren unterschieden werden. Pflanzen wurden dabei in mehreren Studien von den Schülern als weniger attraktiv eingeschätzt. Trotzdem sollten aber die Möglichkeiten, auch botanische Themen außerhalb der Schule zu behandeln, von den Lehrkräften zur Vermittlung biologischer Vielfalt genutzt werden.
Als Konsequenz der Ergebnisse kann der Besuch eines außerschulischen Lernrotes im Biologieunterricht bezüglich der Förderung der Lernmotivation unbedingt empfohlen werden. Da kein klarer Effekt des vor- und nachbereitenden Unterrichts der Biodiversitätsführungen erkennbar war, wären hier weitere Untersuchungen vonnöten, um genauere Aussagen machen zu können. Hier böten sich Studien mit Schülern anderer Altersgruppen und der Vergleich nur zweier außerschulischer Lernorte an.
Saccharomyces cerevisiae is a natural producer of isobutanol, which has more advantages as biofuel than ethanol, i.e. superior combustion energy, weaker corrosive action and reduced aqueous miscibility. Isobutanol is produced by the combination of the valine biosynthesis and the Ehrlich pathway. In this work, an industrial strain was employed for isobutanol production, in which the valine pathway was relocated into the cytosol. The valine pathway in yeast has a cofactor imbalance, since the glycolysis produces NADH, while Ilv5 employs NADPH for the reaction. Therefore, the cofactor specificity of the pathway was rebalanced with exchange of Ilv5 by an NADH-consuming mutant, IlvC6E6. Furthermore, Ilv6, which regulates the feed-back inhibition of the valine biosynthesis, was tested to boost isobutanol production; however, none of these Ilv6 alternatives could greatly enhance isobutanol production. Therefore, due to a still low production yield, the bottlenecks of the isobutanol pathway were deeper studied.
The major observed bottleneck concerned the conversion of DIV into KIV, since high concentrations of acetoin, 2,3-butandiol and, specially, DIV were observed in the fermentation supernatant, while neither KIV nor isobutyraldehyde were detected. This step is performed by the dihydroxy-acid dehydratase, Ilv3, which needs iron-sulfur clusters for its activity. Therefore, the first approach to circumvent this limitation was to increase the FeS assembly and its transference into the cytoplasm; however, Ilv3Δ19 activity was not improvement. Afterwards, Ilv3 alternatives were screened for substitution of Ilv3Δ19. Heterologous ILV3 orthologous with possible advantages were investigated, but Ilv3Δ19 was still the most promising alternative. Furthermore, sugar-acid enolases were tested as Ilv3Δ19 substitutes. These enolases also catalyze the dehydration of the substrate in the same way as Ilv3, but uses Mg2+ as cofactor. One of the employed enolases could complement valine auxotrophy; however, it allowed just a very slow growth of the Δilv3 strain and its activity could not be enhanced by mutagenesis studies.
Interestingly, we observed that once DIV is secreted out of the cell, it cannot be re-uptaken from the medium and this possibly further aggravates the pathway flux and Ilv3Δ19 activity. In order to suppress DIV waste, two strategies were formulated: the deletion of the possible DIV transporter, and the substrate channeling of DIV from IlvC6E6 to Ilv3Δ19. In order to find possible DIV export proteins, a transcriptome analysis of a strain producing high amounts of DIV against a strain producing no detected DIV were compared. Several transporters were found upregulated in the DIV producing strain, but, alone, none of these were responsible for the DIV efflux. For the substrate channeling, an artificial enzymatic net was constructed by the fusion of IlvC6E6 and Ilv319 with synthetic zippers, which have high affinity to each other, and as both enzymes are alone organized as oligomers. The use of this enzymatic net enhanced not only the isobutanol production in about 17%, but also 3-methyl-butanol production yield was 25% increased.
Nevertheless, together with bottlenecks arising from Ilv3 activity, the isobutanol production is limited by the ethanol production, which is the main product of S. cerevisiae. Therefore, in order to abolish ethanol production, PDC1 and PDC5 were deleted. Moreover, BDH1 and BDH2 were also deleted to create an NADH-driving force towards isobutanol production. However, the isobutanol yield of this mutant was even lower than that of the strain without the mentioned deletions. As a high production of isobutyric acid was observed, and it could be produced directly from KIV, different KIV decarboxylases and isobutanol dehydrogenases were investigated; but without improvement. Then, alternative pathways were abolished in other to favor isobutanol production, e.g. valine, leucine, isoleucine and panthotenate biosyntheses. Nevertheless, isobutanol yields were still low and the main byproducts were glycerol, acetoin, DIV and isobutyric acid. Despite the outcomes were not enough to enhance isobutanol production up to commercially required yields, these results help in the comprehension of the bottlenecks surrounding the isobutanol production pathway and serve as basis for further studies within the branched-chain amino acids biosynthesis and Ehrlich pathway.
In the adult mammalian brain stem cells within defined neurogenic niches retain the capacity for lifelong de novo generation of neurons. The subventricular zone (SVZ) of the lateral ventricles and the subgranular layer (SGL) of the hippocampal dentate gyrus (DG) have been identified as the two major sites of adult neurogenesis. Moreover, the third ventricle in the hypothalamus is emerging as a new neurogenic niche in the adult brain. Extracellular purine and pyrimidine nucleotides are involved in the control of both embryonic and adult neuro-genesis. These nucleotides act via ionotropic P2X or metabotropic P2Y receptors and studies of the adult SVZ and the DG provide strong evidence that ATP promotes progenitor cell proliferation in this stem cell rich regions. Previous studies have shown that the extracellular nucleotide-hydrolyzing enzyme NTPDase2 is highly expressed by adult neural stem and progenitor cells of the SVZ and the rostral migratory stream (RMS), the hippocampal SGL, and the third ventricle. NTPDase2 preferentially hydrolyzes extracellular nucleoside triphosphates (NTPs) and, to a lower extent, diphosphates, thus modulating their effect on nearby nucleotide receptors. Deletion of the enzyme increases extracellular NTP concentrations, and might indicate roles of purinergic signaling in adult neurogenesis. As shown by enzyme histochemistry, genetic deletion of NTPDase2 essentially eliminates ATPase activity in neurogenic niches but does not affect protein expression levels and activity of other ectonucleotidases. Lack of NTPDase2 leads to expansion of the hippocampal stem cell pool as well as of the inter-mediate progenitor type-2 cells. Cell expansion is lost at around type-3 stage, paralleled by increased labeling for caspase-3, indicating increased apoptosis, and decreased levels in CREB phosphorylation in doublecortin-expressing cells, diminishing survival in this cell population. In line with increased cell death, P2Y12 receptor-expressing microglia is enriched at the hilus orientated side of the granule cell layer. These data strongly suggest that NTPDase2 functions as central homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion in the adult brain by balancing extracellular nucleotide concentrations and activation of purinergic receptors.
In order to further characterize the role of purinergic signaling in adult neurogenesis, the ADP-sensitive P2Y13 receptor was identified as a potential candidate whose activation might inhibit neurogenesis in the hippocampal dentate gyrus and the newly identified neurogenic niche at the third ventricle. Deletion of P2ry13 increased progenitor cell proliferation and long-term progenitor survival as well as new neuron formation in the hippocampal neurogenic niche. This was further paralleled by increased thickening of the granule cell layer, CREB phosphorylation, and expression of the neuronal activity marker c-Fos. Increased progenitor cell proliferation and progenitor survival persist in aged P2ry13 knockout animals. However, in the ventral dentate gyrus proliferation and expansion levels of progenitor cells did not differ significantly from the wild type. This study strongly supports the notion that extracellular nucleotides significantly contribute to the control of adult neurogenesis in the dentate gyrus in situ. Data in this work suggest that activation of the P2Y13 receptor dampens progenitor cell proliferation, new neuron formation, and neuronal activity. In contrast to several in vitro studies and studies in the SVZ in situ, a contribution of the ATP/ADP-sensitive P2Y1 receptor could not be confirmed in the dentate gyrus in vivo.
To unravel implications of purinergic signaling and P2Y13 receptor action in the control of adult hypothalamic neurogenesis a pilot study was performed. Mice null for P2ry13 revealed increased progenitor cell proliferation at the third ventricle as well as long-term progeny survival and new neuron formation in the hypothalamus. In contrast to results obtained in the dentate gyrus expression of the neuronal activity marker c-Fos was significantly decreased in hypothalamic nuclei, indicating increased inhibition of appetite-regulating neuronal circuits by surplus neurons in knockout animals. These data provide first evidence that extracellular nucleotide signaling contributes to the control of adult hypothalamic neurogenesis in situ. Activation of the P2Y13 receptor inhibits progenitor cell proliferation, long-term survival and neuron formation and therefore controls inhibition of appetite-regulating circuits in the adult rodent hypothalamus.
The baker’s yeast Saccharomyces cerevisiae is a valuable and increasingly important microorganism for industrial applications (Hong and Nielsen, 2012). Its robustness concerning process conditions like low pH, osmotic and mechanical stress as well as toxic compounds is an advantage. Moreover, S. cerevisiae is ‘generally regarded as safe’ (GRAS). The model organism has been studied intensively. The collected data, including genomic, proteomic and metabolic information, can be used to genetically modify and improve its metabolism. Fatty acids and fatty acid derivatives have wide applications as biofuels, biomaterials, and other biochemicals. Several studies have been dealing with the overproduction of fatty acids and derivatives thereof in S. cerevisiae. The fatty acid biosynthesis starting with acetyl-CoA requires two enzymes, the acetyl-CoA carboxylase (Acc1p) and the fatty acid synthase complex (FAS), to produce acyl-CoA esters with predominantly 16 to 18 carbon atoms chain length (Lynen et al., 1980). For the synthesis of monounsaturated fatty acids in S. cerevisiae the ER bound acyl-CoA desaturase, Ole1p is essential (Tamura et al., 1976; Certik and Shimizu, 1999).
Using S. cerevisiae, the first section of this work dealt with the heterologous characterization of potential ω1-desaturases. Due to the fact that unsaturated fatty compounds can be modified further by hydrosilylations, hydrovinylations, oxidations to epoxides, acids, aldehydes, ketones or metathesis reactions, the interest in ω1-fatty acids is tremendous (Behr and Gomes, 2010). With the intention to find enzymes in fungi, that have a terminal desaturase activity a search in different genome databases was performed. The sequences of Pex-Desat3 and Obr-TerDes were used as reference sequences. The analysed proteins from Schizophyllum commune (EFI94599.1), Schizosaccharomyces octosporus (EPX72095.1), Wallemia mellicola (EIM20316.1), Wallemia ichthyophaga (EOR00207.1) and Agaricus bisporus var. bisporus (EKV44635.1), however, finally turned out to be Δ9 desaturases. A fungal desaturase with ω1-activity could not be found. The Δ9 desaturase SCD1 from Mus musculus was crystallized by Bai et al. (2015) and the information for specific amino acids responsible for the substrate specificity or enzyme activity were allocated. In combination with sequence and enzyme activity data form ChDes1 from Calanus hyperboreus, Desat2 from Drosophila melanogaster, Pex-Desat3 from Planotortrix excessana and Obr-TerDes from Operophtera brumata single amino acid exchanges were performed in the Δ9 desaturase Ole1p from S. cerevisiae. For all mutants, only fatty acids (C16 - C18) with a double bond between carbon C9 and C10 could be found. This indicates, that all inserted amino acid exchanges do not affect the substrate specificity or the position of the introduced double bond.
In the second section the focus was in the development of a production system for fatty acids in S. cerevisiae with regard to the previously established procedures by metabolic engineering. The combination of cytosolic malate dehydrogenase (MDH3), cytosolic malate enzyme (MAE1) and a citrate- α-ketoglutarate- carrier (YHM2) should improve the availability of acetyl-CoA in the cytosol, which is an important precursor for the fatty acid biosynthesis. If the major pathway (acetyl-CoA carboxylase and fatty acid synthase) was already optimized by high expression levels than no positive effect on increased fatty acid synthesis was detectable. Only non-optimized strains, with the additional overexpression of ATP-citrate lyase and cytosolic malate dehydrogenase, lead to a 41 % (20 mg/g dcw) improvement of fatty acid synthesis. In order to increase the fatty acid content further, the additional overexpression of DGA1 and TGL3 was performed. Hence, the highest amount of fatty acids could be observed with the strain S. cerevisiae WRY1ΔFAA1ΔFAA4 (2.5 g/L ± 0.8 g/L). The additional elimination of acyl-CoA synthetase Fat1p did not improve the yield.
It was recently reported, that chain length control of the fatty acid synthesis of bacterial FAS can be changed by rational engineering (Gajewski et al., 2017a). The knowledge about bacterial FAS was transferred in this work to S. cerevisiae FAS. Mutating up to five amino acids in the FAS complex enabled S. cerevisiae to produce medium chain fatty acids (C6 - C12). Further improvement was done by metabolic pathway engineering (promoter of alcohol dehydrogenase II from S. cerevisiae (pADH2), deletion of acyl-CoA synthetase FAA2) and optimization of fermentation conditions (YEPD-bacto medium buffered with potassium phosphate). The production of medium chain fatty acids resulted in the highest yield of 464 mg/L (C6 to C12 fatty acids). Furthermore, strains were created specifically overproducing hexanoic acid (158 mg/L) and octanoic acid (301 mg/L). The characterization of transferases, which could be responsible for the de-esterification of CoA-bound fatty acids, was analysed in an additional approach. It could be shown, that the genes EHT1, EEB1 and MGL2 have an influence on the MCFA yield in the supernatant. Generally speaking, the data from the single and double deletion strains suggest that Eeb1p has a selective hydrolytic activity for hexanoic acid-CoA ester, while Eht1p shows selective hydrolytic activity for octanoic acid-CoA ester, which is in line with Saerens et al. (2006).
In der vorliegenden Arbeit werden funktionale Details der Okklusion während der Mastikation bei ausgewählten fossilen und rezenten Primaten quantitativ vergleichend untersucht. Dazu wurden die Okklusionsflächen von antagonistischen Molarenpaaren mit modernen virtuellen Verfahren eingescannt und anhand von 3D Kronenmodellen kartiert und funktional ausgewertet. Die in der Forschergruppe DFG FOR 771 entwickelte Software „Occlusal Fingerprint Analyser“ (OFA) kam erstmals bei einer großen Stichprobe von Primaten zum Einsatz.
Aus dem ursprünglichen tribosphenischen Molarentyp der frühen eozänen Primaten haben einige Nahrungsspezialisten im Laufe der Evolution Modifikationen entwickelt um ihre Nahrung mechanisch besser aufzubereiten. So sind neue Funktionselemente auf den Molaren entstanden, wie z.B. ein distolingualer Höcker (Hypoconus) auf den Oberkiefermolaren.
Die Auswertung der Parametermessungen, wie die Facettenlage und -größe, der Okklusale Kompass, der Mastikationskompass und die Messungen der Okklusionsreliefs ergaben, dass die basalen Primatenvertreter aus dem Eozän einen flachen Hypoconus als vergrößerte Fläche zum Quetschen der Nahrung genutzt haben. Das weist auf eine frugivore Nahrungspräferenz hin. Der distolinguale Höcker ist unter den rezenten Spezies mit insektivorer Nahrungspräferenz besonders häufig ausgebildet. Es konnte gezeigt werden, dass eine zweite Kauphase, die nach der maximalen Verzahnung mit der Öffnung des Kiefers eintritt, unter den rezenten Strepsirrhini mit Hypoconus nur sehr schwach ausgeprägt ist.
Eine weitere evolutionäre Modifikation sind buccolingual ausgerichtete komplementäre Kantenpaare auf den Molaren, die sogenannte Bilophodontie, die sich in der Familie der Cercopithecidae entwickelt hat. Die Unterfamilie der Colobinae zeigt eine besonders stark reliefgeführte Okklusion und hat deshalb während der Mastikation weniger Bewegungsspielraum als die der Cercopithecinae. Die zweite Kauphase der Cercopithecinae ist gegenüber den folivoren Colobinae zum Teil auffällig verlängert. Da die Colobinae Vormagenverdauer und die Cercopithecinae Monogastrier sind, kann vermutet werden, dass die Zahnmorphologie eng mit der entsprechenden chemischen Verdauungsweise verknüpft ist. Das dryopithecine Höckermuster der Hominoidea hat eine wesentlich flachere Höckermorphologie als die bilophodonten Molaren. Daher war ein höherer Bewegungsspielraum während der Mastikation beobachtbar. Es konnte gezeigt werden, dass steilere Facetten bei den folivoren Nahrungsspezialisten zu finden sind, wie den Colobinae bei den bilophodonten, oder den Gorillas unter dem dryopithecinen Molarentyp. Mit einem flacheren Molarenrelief kann auf ein breiteres Nahrungsspektrum zugegriffen werden.
Mit den OFA-Analysen und den Ergebnissen der Quantifizierung des Kronenreliefs von rezenten und fossilen Primatenzähnen konnte in der vorliegenden Untersuchung eine relevante Vergleichsbasis für ein funktionelles Verständnis der Evolution der vielfältigen Kronenformen bei Primaten erarbeitet werden. Für zukünftige Studien sollte die innerartliche Stichprobe erweitert werden um die Variabilität näher zu untersuchen.
The fungal genus Pestalotiopsis s.l. contains approximately 300 described species and is globally distributed. The monotypic genus Pestalotia is considered the closest relative of Pestalotiopsis s.l. This study aims to investigate the diversity and systematics within Pestalotiopsis s.l. and its relation to Pestalotia. Therefore, an integrative approach is used considering molecular phylogeny methods as well as examination of morphological characters.
Recently, Pestalotiopsis s.l. was split into three genera with the addition of the newly erected Neopestalotiopsis and Pseudopestalotiopsis. The species of these genera are usually saprotrophic, phytoparasitic, or endophytic, and have been isolated from soil, air, and many kinds of anorganic material. The asexual fruiting bodies appear on infected plant material as black acervuli that release conidia. The conidia are important to examine for morphological taxon recognition. The number of conidial cells is the feature that distinguishes Pestalotiopsis s.l. spp. with five celled conidia, from Pestalotia pezizoides with six celled conidia. However, the significance of morphological characters is controversially discussed among mycologists. In recent years, 55 new species were described based on minor genetic distances and marginal or no morphological differences. Thus, the value of certain morphological characters and genetic markers need to be reconsidered.
In this study, 102 herbarium specimens of 26 described species, with an emphasis on plant pathogenic species from North America, have been morphologically examined and documented through drawings and photographs. Morphological examination was complemented with a comprehensive molecular dataset obtained from 191 cultures representing the genera Neopestalotiopsis, Pestalotia, Pestalotiopsis, Pseudopestalotiopsis, and Truncatella. One novelty of this work is that, besides the well-established markers ITS, TEF1, and ß-tubulin, the protein-coding genes MCM7 and TSR1 were successfully sequenced and included in the analyses. Phylogenies using Maximum Likelihood and Bayesian inference methods of single loci and the combined dataset were calculated. By comparison of these phylogenies, MCM7 was identified as the most powerful one in terms of phylogenetic resolution and statistical support of nodes and is proposed as an additional barcoding marker in Pestalotiopsis s.l.
In Pestalotiopsis, species delimitation was tested using the Baysian Phylogenetics and Phylogeography (BP&P) program that tests an existing species scenario against Bayesian inference methods under a multispecies coalescent model. The program supported only ten species out of the predetermined 19 species scenario. Measurements of conidia for species detected by BP&P were explored using a TukeyHSD-Test in the program R to find means that are significantly different from each other. This test revealed that combinations of morphological characters are required to distinguish between the ten species found by BP&P.
Another purpose of this work was to clarify the status of Pestalotia with regard to Pestalotiopsis s.l. Therefore, fresh epitypic material of Pestalotia pezizoides, was collected, isolated, and cultivated. The molecular analysis of a combined dataset of the gene regions ITS and LSU for species of Amphisphaeriales nested P. pezizoides in the genus Seiridium. Thus, synonymy of Pestalotia with Seiridium is proposed here. This is supported by morphology of the conidia. Further, an epitype is proposed for the type species of Pestalotiopsis, P. maculans. On the other hand, the recently proposed epitype of P. adusta is rejected here as it conflicts with the taxonomic hypothesis obtained in this study and its introduction is inconsistent with the formal requirements for epitypification. A new topotypic specimen is proposed instead. Additionally, several nomenclatural changes become necessary in many species examined. These include three new combinations and six synonyms of species of Pestalotiopsis s.l.
The conclusion of this work is that morphological data have potential as a valuable, inexpensive and easy way to recognize species. However, it is not the best method for species discovery and delimitation bearing in mind that in microfungi and many other organisms, individual plasticity and analogous structures are inadequately investigated. By phylogenetic analyses of molecular sequence data, it is possible to compare a great amount of equivalent characters and to delimit species that are morphologically cryptic. This is especially important since species of Pestalotiopsis s.l. mostly lack sexual structures that are helpful for morphological species delimitation in other groups of fungi. Thus, the Genealogical Concordance Species Concept (GCSC) finds its application in many fungal taxa. Conflicts in the genealogy between phylogenetic trees of different markers are interpreted as recombination of the genetic material within a linage. Accordingly, the change from conflict to congruence in a set of different phylogenetic trees can be seen as the species limit. It can be expected that increased application of the GCSC will lead to further approximation of described species numbers to the real number of species, especially in complicated groups like asexual microfungi.
Embryonale Stammzellen (ESCs) sind ein wichtiges Werkzeug zur Untersuchung der frühen embryonalen Entwicklung. ESCs können mit Hilfe neuer Technologien zur Modifikation von Genen (z.B. mit dem CRISPR/Cas9 System) genetisch manipuliert werden. Daraus resultierende „knockout“ ES Zelllinien können helfen, die physiologische Rolle von Proteinen während der Differenzierung zu verstehen.
Transkriptionsfaktoren, die schnell und spezifisch Signalwege regulieren, spielen während der Embryonalentwicklung und während der Differenzierung von ESCs in vielen verschiedenen Zelltypen eine essentielle Rolle. Der Transkriptionsregulator „Far Upstream Binding Protein 1“ (FUBP1) ist ein Protein, welches eine ganz bestimmte einzelsträngige DNA Sequenz, das „Far Upstream Sequenz Element“, erkennt, bindet, und dadurch Gene wie z.B c-myc oder p21 reguliert. Mit der Entwicklung zweier Fubp1 Genfallen Mausstämme (Fubp1 GT) sollte die Frage nach der physiologischen Funktion von FUBP1 beantwortet werden. Die homozygoten FUBP1-defizienten GT Embryonen sterben im Mutterleib ungefähr am Tag E15.5 der Embryonalentwicklung. Sie sind kleiner als Wildtypembryonen und zeigen ein anämisches Aussehen. Daher wurden diese Mausmodelle hinsichtlich der Hämatopoese untersucht, die zu diesem Zeitpunkt vor allem in der Leber stattfindet. Es konnte eine signifikante Reduktion der hämatopoetischen Stammzellen (HSCs) festgestellt werden und zusätzlich war die langfristige Repopulation der FUBP1-/--Stammzellen im Knochenmark in Transplantationsexperimenten reduziert.
In der vorliegenden Arbeit wurde die Rolle von FUBP1 in einem weiteren Stammzellsystem analysiert und gleichzeitig seine Bedeutung in anderen Zelltypen der frühen Embryonalentwicklung untersucht.
Die Quantifizierung der FUBP1 Expression in den ESCs und während der Differenzierung zu sogenannten `embryoid bodies` (EBs) zeigten eine starke Expression auf mRNA- und auf Proteinebene. Nach der erfolgreichen Optimierung der Differenzierung von murinen ESCs wurden Fubp1 „knockout“ (KO) ESC Klone mit Hilfe der CRISPR/Cas9 Technologie etabliert. Die molekularbiologische Analyse der ESCs zeigte eine signifikante Erhöhung der Oct4 mRNA-Expression, während Nanog und die Differenzierungsmarker Brachyury, Nestin und Sox17 unverändert und in vergleichbarer Menge zu den Kontrollen vorhanden waren. Während der Differenzierung der Fubp1 KO Klone zu EBs zeigte sich eine signifikante Reduktion mesodermaler Marker wie Flk-1, SnaiI, Snai2, Bmp4 und FgfR2. Mit Hilfe durchflusszytometrischer Analysen bestätigte sich die verzögerte Bildung mesodermaler Zellen (Brachyury- und Flk-1-exprimierender Zellen) in den Fubp1 KO Klonen der EBs an den Tagen 3, 4 und 5 nach Beginn der Differenzierung.
Die Anwendung einer Ko-Kultivierung auf OP9 Zellen zur Differenzierung der ESCs in hämatopoetische Linien sollte zeigen, ob der Fubp1 KO ESCs ein Defekt in der frühen Entwicklung hämatopoetischer Stammzellen zu beobachten ist. Erneut konnte am Tag 5 der ESC-Differenzierung in der OP9 Ko-Kultur eine signifikante Reduktion der mesodermalen (Flk-1+) Zellen festgestellt werden. Die weitere Differenzierung zu hämatopoetischen CD45+ Zellen zeigte jedoch keinen Unterschied im prozentualen Anteil CD45+ Zellen am Tag 12 der Differenzierung. Auch die gezielte Differenzierung zu erythroiden Zellen durch Zugabe des Zytokins EPO zum Medium zeigte keinen signifikanten Unterschied im Differenzierungsgrad der erythroiden Zellen zwischen Kontroll- und Fubp1 KO Klonen.
In weiteren Experimenten habe ich in dieser Arbeit die Expression von FUBP1 in WT Embryos an den Tagen E9.5 und E13.5 der Embryonalentwicklung untersucht. Hierbei zeigte sich in beiden Entwicklungsstadien eine immunhistochemische Anfärbung von FUBP1 in den meisten Zellen des Embryos. Die Annahme, dass die Abwesenheit von FUBP1 in der Embryonalentwicklung zu verstärkten apoptotischen Vorgängen führen könnte und gleichzeitig die massive Expansion von Zellen gestört sein könnte wurde mit Hilfe immunhistochemischer Färbung von „cleaved Caspase 3“ (Apoptosemarker) und „Ki-67“ (Proliferationsmarker) in den homozygoten Fubp1 GT Embryos an den Tagen E9.5 und E13.5 nicht bestätigt.
Die Ergebnisse dieser Arbeit lassen darauf schließen, dass die Regulation von Apoptose und Proliferation durch FUBP1 während der Embryonalentwicklung nicht die Hauptrolle von FUBP1 darstellt. Es zeigte sich jedoch, dass FUBP1 als Transkriptionsregulator wichtig für die mesodermale Differenzierung von ESCs ist. Zu beobachten war, dass es in den FUBP1-defizienten ESCs zu einer Verzögerung der mesodermalen Differenzierung kommt. Es konnte bereits gezeigt werden, dass FUBP1 essenziell für die Selbsterneuerung von HSCs ist. Dies macht deutlich, dass FUBP1 neben der Proliferation und Apoptose ein breiteres Spektrum an Signalwegen reguliert, die für Stammzellen und deren Differenzierung von Bedeutung sind.
Bei Cryptochromen handelt es sich um Blaulichtrezeptoren der Cryptochrom-Photolyase-Proteinfamilie (CPF). Mitglieder dieser Proteinfamilie sind in allen Domänen des Lebens zu finden und haben eine essentielle Rolle in der Reparatur der DNA sowie der lichtgesteuerten Regulation der Expression. Cryptochrome sind in der Regel keine DNA-reparierenden Proteine. Sie sind regulativ an der Steuerung der inneren Uhr und des Zellzyklus der Organismen beteiligt. In der Kieselalge Phaeodactylum tricornutum konnten bisher sechs phylogenetisch unterschiedliche Mitglieder der CPF identifiziert werden. Bei CryP handelt es sich um das einzige pflanzenähnliche Cryptochrom der photoautotrophen Diatomee. Für das Protein CryP konnte bereits ein blaulichtinduzierter Photozyklus durch die Absorption der Chromophore 5-Methenlytetrahydrofolat (MTHF) und Flavinadenindinukleotid (FAD) gezeigt werden. Außerdem ist eine regulative Wirkung des Proteins auf die Lichtsammelkomplexe (Lhc) der Diatomee bekannt. Für eine weitere Charakterisierung des CryPs wurde in dieser Arbeit zunächst das Absorptionsverhalten unter verschiedenen Wellenlängen beobachtet, um so einen Einblick in eine mögliche Aktivierung und Deaktivierung des Proteins durch Licht unterschiedlicher Wellenlängen zu erlangen. Es zeigte sich hierbei eine mit pflanzlichen Cryptochromen vergleichbare Anreicherung verschiedener Redoxzustände des FADs in Abhängigkeit von der Wellenlänge.
Für eine Aufklärung der Wirkungsweise des CryP-Proteins wurden verschiedene Hypothesen untersucht: Die phylogenetische Nähe und ein ähnliches Absorptionsverhalten des CryPs zu Cryptochromen mit Reparaturfähigkeit für einzelsträngige DNA (Cry-DASH) führte zu einer Untersuchung des Proteins als möglicher Transkriptionsfaktor. Hierfür konnte eine Kernlokalisation des Proteins nachgewiesen werden, was Rückschlüsse auf eine potentielle Regulation der Expression mittels DNA-Bindung zulässt. Außerdem wurde gezeigt, dass CryP DNA-Bindefähigkeit besitzt. Die bisher nachgewiesenen Bindungen waren jedoch unspezifischer Art. Dies konnte auch für die Promotersequenz eines der durch CryP regulierten Gene lhcf1 festgestellt werden. Auf Grund der unspezifischen DNA-Bindung wurde eine zweite Hypothese für CryP untersucht: CryP wirkt regulativ auf die Expression verschiedener Gene durch Protein-Protein-Interaktionen und ist Teil einer Reaktionskaskade zur Signalweiterleitung in P. tricornutum.
Durch die Untersuchung der zweiten Hypothese konnten drei Interaktionspartner für CryP identifiziert und eine Interaktion verifiziert werden. Hierbei handelt es sich um das Protein AAA mit einer bisher unbekannten Funktion und das Protein BolA, welches Teil der zuerst in Escherichia coli identifizierten BolA-like-Proteinfamilie ist. Außerdem konnte eine Interaktion mit dem Cold-Shock-Domänen-Protein CSDP gezeigt werden. Bei den Proteinen BolA und CSDP handelt es sich um potentiell regulierende Faktoren der Transkription und Translation, was Teil einer Reaktionskaskade sein kann. Die aus anderen Organismen bekannten Funktionen des BolA-Proteins überschneiden sich mit den in CryP-Knockdown-Mutanten beobachteten Effekten. Sie zeigen eine erhöhte Sensitivität für Stresssituationen wie abweichende Nährstoffkonzentration, Osmolaritäten und Temperaturen. Diese Beobachtungen stellen einen Zusammenhang der durch einen CryP-Knockdown beobachteten Effekte und der CryP-BolA-Interaktion her. Durch Homologien zu Cold-Shock-Proteinen aus Chlamydomonas reinhardtii gibt die CryP-Interaktion mit dem Protein CSDP Hinweise auf einen potentiellen Mechanismus zur Regulation der Lhc-Proteine, für welche zuvor ein CryP-abhängiger Effekt beschrieben war.
Über die Protein-Protein-Interaktionen hinaus wurde die Phosphorylierung des CryPs als Möglichkeit der Signalweiterleitung untersucht. Es konnte eine reversible Phosphorylierung des heterolog aus E. coli isolierten CryPs gezeigt werden. Diese zeigt Ähnlichkeiten zu bekannten Phosphorylierungen pflanzlicher Cryptochrome und gibt Hinweise auf einen Mechanismus der Signalweiterleitung.
Durch die Untersuchung der CryP-regulierten Transkription mit P. tricornutum CryP-Knockdown-Mutanten durch Next-Generation-Sequencing (NGS) konnte die Hypothese der regulativen Proteinkaskade und der Signalweiterleitung weiter bestätigt werden. Die Auswirkungen des CryPs auf die Transkription erwiesen sich als nicht auf einen Teilbereich des Metabolismus begrenzt, sondern sind in einem großen Teil der funktionellen Gengruppen in P. tricornutum zu sehen. Außerdem konnten drei Klassen CryP-regulierter Gene festgestellt werden. Kategorie 1: die ausschließlich unter Blaulicht regulierten Gene; Kategorie 2: die sowohl unter Blaulicht als auch im Dunkeln regulierten Gene und Kategorie 3: die ausschließlich im Dunkeln regulierten Gene. Ein im Dunkeln und unter Blaulicht jeweils unterschiedlicher regulativer Effekt deutet auf eine Doppelfunktion des CryPs hin. Möglicherweise hat das Cryptochrom unterschiedliche lichtabhängige und lichtunabhängige Funktionen.
Durch die Analyse der CryP-regulierten Genexpression konnte außerdem ein Zusammenhang zwischen CryP und weiteren Photorezeptoren gezeigt werden. Der CryP-Proteingehalt in der Zelle hat einen regulativen Einfluss auf das CPF1-Protein, eine Photolyase mit dualer Funktion aus der gleichen Proteinfamilie. Zusätzlich konnte auch ein Einfluss auf die Lichtsensitivität der Genexpression des Rotlichtrezeptors Phytochrom (DPH) durch CryP gezeigt werden. Vergleichbar mit höheren Pflanzen scheint ein regulatives Netzwerk der Photorezeptoren auch in der Diatomee P. tricornutum vorhanden zu sein.
Es wird davon ausgegangen, dass das ehemalige Larven-Mikrohabitat der Asiatische Tigermücke Aedes albopictus (synonym: Stegomyia albopicta) die Phytotelmata in den Waldgebieten von Südostasien darstellte. In den letzten vier Jahrzehnten adaptierte sich die Art jedoch an urbanere Regionen und ihre Antrotelmata. Dank ihrer Eigenschaft, Eier mit einer gewissen Trocken- und Kältetoleranz zu produzieren, verbreitete sich die Art zusammen mit den international gehandelten Waren weltweit. Zudem ist Ae. albopictus ein theoretischer Vektor für mindestens 27 Viren sowie Parasiten und spielt eine Hauptrolle bei der Übertragung von Dengue-Viren und Chikungunya-Viren und Zika-Vieren. Daher wird die Art als große Gefahr für die öffentliche Gesundheit betrachtet.
Die vorliegende Arbeit thematisiert drei Untersuchungen zum Anpassungs- und Etablierungs-potential der invasiven Asiatischen Tigermücke.
In einem ersten Ansatz wurde das Problem behandelt, dass es lediglich zwei standardisierte toxikologische Testverfahren für Culicidae gab. Daher wurde ein Dosis-Wirkungs-Testsystem entwickelt, das den Weg für weitere biologische Endpunkte und ihre integrativen Parameter freimachte und dadurch ein besseres Verständnis für die Wirkweisen von Insektiziden ermöglicht. Hierdurch konnte nun der Frage nachgegangen werden, ob es Unterschiede in der ökotoxikologischen Reaktion zwischen der invasiven tropisch-subtropischen Asiatischen Tigermücke und der einheimischen nördlichen Hausstechmücke Culex pipiens auf das Insektizid λ-Cyhalothrin gibt. Weiter wurde der Einfluss von Temperatur und die Verfügbarkeit von Nahrung auf die Insektizidsensitivitäten der Arten getestet. Schließlich konnte in einer Risikobewertung festgestellt werden, dass bei falsch angewendeten Bekämpfungsmaßnahmen höhere Temperaturen sowie der Ausfall von aquatischen Top-Prädatoren zu Fitnessvorteilen für die Art führen können.
In einer zweiten Untersuchung wurde der Mechanismus der Kältetoleranz der Eier (Kälteakklimatisierung und Diapause) näher untersucht, da dieser für die erfolgreiche Invasion in gemäßigten Breitengraden verantwortlich gemacht wird. Nachdem eine lang vorherrschende Hypothese verworfen wurde, dass die Einlagerung von Polyolen die Frosttoleranz bewirken würde, war der aktuelle Stand der Wissenschaft, dass eine Verdickung der Wachsschicht des Chorions dafür verantwortlich sei. Jedoch lag keine detaillierte Evaluierung von Stechmücken-Eihüllen vor. Mittels einer transmissionselektronenmikroskopischen Studie konnte gezeigt werden, dass nicht nur die Wachsschicht nicht in der Serosa-Cuticula zu verorten ist, sondern im Endochorion und sie zudem im Zuge der Diapause in der Mächtigkeit schrumpft. Daher wird auf Basis der gewonnenen Erkenntnisse auf eine Kompaktierung der Schicht geschlossen.
Die dritte Untersuchung schließlich hatte das hohe Adaptationspotential in gemäßigten Breiten zum Gegenstand. Eine Adaptation auf genetischen Level gilt als unwahrscheinlich, da Gründerpopulationen in den neu besiedelten Gebieten eine niedrige genetische Diversität aufwiesen und ein regelmäßiger Neueintrag von Allelen unwahrscheinlich ist. Jedoch bietet das Konzept der epigenetischen Temperatur-Adaptation einen Erklärungsansatz für dieses Phänomen. Daher wurde die Frage gestellt, ob es möglich ist, eine vererbbare Diversifizierung dieses kältetoleranten Phänotyps nach einer randomisierten epigenetischen Behandlung der DNA zu detektieren. Es wurde eine transgenerationale Untersuchung der Effekte von zwei epigenetischen Agenzien (und einem Lösemittel) auf die Kältetoleranz der Eier durchgeführt. Die Ergebnisse zeigten ein Korrelationsmuster, das den durch die Agenzien veränderten Methylierungsgrad der DNA mit der Forsttoleranz verband, was die gestellte Hypothese unterstützte.
In Folge dieser drei Untersuchungen wurde festgestellt, dass Ae. albopictus ein hohes Potential hat, in weiteren Ländern – vor allem in gemäßigten Breiten – ein Gesundheitsrisiko darzustellen. Da die Art einerseits Fitnessvorteile durch falsche Bekämpfungsmaßnahmen und andererseits möglicherweise eine hohes epigenetisches Adaptationspotential besitzt, kann zusammenfassend empfohlen werden, dass der Fokus für weitere Forschung maßgeblich auf der Entwicklung von Impfstoffen für die übertragenen Viren und Pathogene liegen sollte. Dadurch kann die Bevölkerung geschützt werden, ohne Ökosysteme und ihre Dienstleistungen zu gefährden, und dies wäre zudem ökonomisch gesehen die effektivere Lösung.
In der vorliegenden Arbeit wurde erstmals die Interaktion von A. baumannii mit humanem Plasminogen untersucht. Mit dem Translations-Elongationsfaktor TufAb, dem äußeren Membranprotein OmpW sowie dem Lipoprotein p41 konnten insgesamt drei Plasminogen-bindende Proteine von A. baumannii identifiziert werden. Außerdem wurde ein grundlegender Beitrag zur funktionellen Charakterisierung von TufAb sowie p41 von A. baumannii erbracht.
Es konnte nachgewiesen werden, dass gereinigtes TufAb humanes Plasminogen bindet und diese Interaktion teilweise durch Lysin-Reste vermittelt und von der Ionenstärke beeinflusst ist. An TufAb-gebundenes Plasminogen war für den Plasminogen-Aktivator u-PA zugänglich und konnte zu Plasmin aktiviert werden, welches das chromogene Substrat S-2251, das physiologische Substrat Fibrinogen und die zentrale Komplementkomponente C3b proteolytisch spaltete. Schließlich konnte TufAb als „Moonlighting“-Protein auf der Zelloberfläche von A. baumannii identifiziert werden.
Für das Lipoprotein p41 konnte ebenfalls gezeigt werden, dass dieses an Plasminogen bindet. Die Bindung von Plasminogen an p41 erfolgte ebenfalls über Lysin-Reste, zeigte sich allerdings von der Ionenstärke unbeeinflusst. Im Fall von p41 konnte mit Hilfe von C-terminal verkürzten p41-Konstrukten gezeigt werden, dass C-terminale Lysin-Reste an der Bindung von Plasminogen beteiligt sind. Weitere Versuche mit p41-Proteinen, bei welchen vier C-terminale Lysin-Reste durch Alanin-Reste substituiert wurden, ergaben, dass die beiden Lysin-Reste K368 und K369 essentiell für die Bindung von Plasminogen an p41 sind. Zudem konnte gezeigt werden, dass sowohl Kringle-Domäne 1 als auch Kringle-Domäne 4 von Plasminogen bei der Interaktion mit p41 involviert sind. An p41 gebundenes Plasminogen ließ sich durch u-PA zu Plasmin aktivieren, welches Fibrinogen sowie die zentrale Komplementkomponente C3b degradierte. p41 ist außerdem in der Lage, die Komplementkomponenten C3, C3b und C5 zu binden und den alternativen Weg zu inhibieren. Zudem ergaben Untersuchungen im Rahmen dieser Arbeit erste Hinweise darauf, dass zumindest die Plasminogen-bindende Region auf der Zelloberfläche von A. baumannii lokalisiert ist.
Die Inaktivierung des p41-kodierenden Gens führte zu einer signifikanten Abnahme im Überleben von A. baumannii-Zellen in der Gegenwart von NHS. Zudem zeigte die Mutante Δp41 einen Defekt in der Plasmin-abhängigen Transmigration durch einen Endothelzell-Monolayer. Beide Versuche untermauern die physiologische Relevanz für die Interaktion von A. baumannii mit Plasminogen.
Phenology is the study of periodic life cycle events of living organisms and how these are influenced by environmental factors. Late phenological phases such as the timing of seed release and subsequent seed dispersal considerably affect ecology and evolution in plants. Since plants are mostly sessile organisms, seed dispersal is a crucial life cycle event for the ecology and evolution of plants. In fact, long-distance seed dispersal (LDD) is a very complex process in plant biology and significantly shapes the spatial and temporal dynamics of plant populations. For example, wind dispersal in plants is influenced by a variety of factors such as plant traits, habitat type and environmental conditions (e.g. wind speed). Considering the variability of wind conditions throughout the year, the timing of seed release and dispersal is known to have considerable effects on LDD. Even though late phenologies such as ripening duration and timing of seed release and subsequent dispersal are vital in estimating ecologically highly relevant LDD, these phenologies are not appropriately addressed in ecological research. The aim of this thesis is to gain insights into the factors that shape late plant phenologies. In particular, we address the following questions: which ecologically or evolutionary parameters drive the ripening process of plant species? How does the seasonal variability of wind affect the seed release phenology of plant species? How do these factors interact for plant species in different habitat types?
In order to address these questions, we applied different methodological approaches, ranging from fieldwork and monitoring phenology to computational simulation studies and statistical modeling. To study the ripening process of species, we monitored the flowering, ripening and seed release phenology of more than 100 Central European plant species. We conducted computational simulation studies for estimating LDD by wind to study the phenology of seed release and the parameters determining LDD by wind. In conjunction with phenological data from literature, we used the obtained simulation results to investigate evidence for the existence of phenological adaptations towards LDD in 165 plant species. Further, we used the results from simulation studies of LDD by wind to disentangle the effects of species, habitat types and meteorological conditions and their interactions on the spatial spread of plant species.
The results of the relationship between plant traits, phylogeny, the ripening process and climatic factors provide insights into the basic understanding of the ripening process of plants. We identified ecological factors that shape species’ ripening phenology and seed release timing. In particular, we suggest that the species’ seed weight, life form and phylogeny shapes ripening and seed release phenology. With the statistical models on species’ temperature demands for reproduction, we introduce data that that are well suitable for parametrization and further development of plant dispersal models. The results from the simulation study based on a seasonal perspective showed that heavier seeded tree species with medium wind dispersal potential (including genera Abies, Acer, Fraxinus and Larix) have a clear synchronisation of seed abscission with periods favouring LDD. These species, which are both ecologically and economically important, showed significant synchronisation of the highest rate of seed release with high wind-speed that promoted LDD by wind in wintertime. For the tree species mentioned, we suggest strong seasonal synchronisation as evidence for phenological adaptations in order to match favourable conditions during seed release. With a closer look at the wind conditions that promote LDD by wind, our results showed considerable differences in how specific wind conditions affect LDD in different species and habitat types. We suggest that LDD by wind in species from open habitats with high wind dispersal potential is likely to be driven by thermal updrafts that are mainly driven by the sun providing energy to the ground. By contrast, LDD of heavier-seeded species from open and forested habitats is more likely to be driven by storms that produce shear-driven turbulence. The results from this thesis contribute to an increased understanding of the complete dispersal process of plants and to making more realistic projections of (future) plant distribution.
The results obtained on factors driving ripening and release phenology provide valuable insights into their ecological and phylogenetic factor constraints. The implementation of more realistic assumptions in assessing species’ dispersal potential throughout the year could help considerably in improving landscape management (e.g. timing of mowing) and in the conservation of plant populations. The evidence found for phenological adaptations towards LDD in plants is an important step in understanding the evolutionary basis of LDD in these species.
The neural crest gives rise to the neurons and glial cells of the peripheral nervous system (PNS) (Bronner-Fraser and Fraser, 1989; Frank and Sanes, 1991). Self-renewing neural crest-derived stem cells (NCSCs) are present in migratory neural crest and various postmigratory locations, including peripheral ganglia (Duff et al., 1992; Morrison et al., 1999; Kruger er al., 2002). It is demonstrated that NCSCs from embryonic mouse dorsal root ganglia (DRG) are reprogrammed in neurosphere (NS) cultures in the presence of EGF and FGF. Reprogrammed NCSCs (rNCSCs) generate exclusively central nervous system (CNS) progeny, both in vitro and upon transplantation into the mouse brain (Binder et al., 2011). In this study the timing and mechanisms underlying the reprogramming were addressed. Most of the cells acquire CNS characteristics at passage 2, reaching a stable proportion of >90% of Olig2-positive cells at passage 3, which is maintained at least up to passage 10. The PNS marker p75 is completely lacking from passage 3 onwards. Furthermore, it was shown that the reprogramming does not involve a transient pluripotency state. This suggests a direct reprogramming of NCSCs to cells with CNS identity. The reprogramming leads to a stable CNS identity as shown by delayed BMP4 application. This result is in agreement with the previous observation that rNCSCs only generate CNS progeny, in particular mature myelinating oligodendrocytes, upon transplantation into embryonic, postnatal and lesioned adult mouse brains (Binder et al., 2011). Genome wide gene expression profiles of rNCSC NS demonstrates already in culture a complete switch to a (spinal cord stem cell) SCSC CNS identity. These results demonstrate a complete reprogramming of PNS progenitors to CNS identity without genetic modification and imply PNS cells as a source for stem cell-based CNS therapy.
The reprogramming of NCSCs is completely blocked in the presence of BMP4 in NS cultures, as shown by the expression of neural crest markers p75 and Sox10. In addition, BMP4 NCSCs generate PNS neurons (Tuj1/Phox2b- and Peripherin/Tuj1-coexpressing cells) and Schwann cells (O4/p75-coexpressing cells). Genome wide gene expression profiles of BMP NCSCs demonstrate that BMP NCSCs express genes at high levels which are characteristic for neural crest/neural crest derivatives, mesenchymal derivatives of neural crest and perivascular pericytes/MSCs. On the other hand CNS marker genes are restricted to rNCSCs and are only expressed at background or undetectable levels in BMP NCSCs. These findings imply that the CNS versus PNS identity is controlled by antagonistic functions of FGF and BMP4.
The use of rNCSCs for cell therapies requires an accessible source of these cells in the adult organism. Since the DRG is not an easily approachable tissue source, the adult mouse palate, containing NCSCs, was chosen. These results suggest that pNCSCs arise from Sox10-positive neural crest-derived stem cells, that downregulate PNS marker gene expression, such as Sox10 and p75, in NS culture. Contrary to rNCSCs, CNS marker upregulation was not observed. Notably, genome wide gene expression profiles of pNCSCs demonstrate an enrichment of genes expressed by mesenchymal derivatives and perivascular pericytes/mesenchymal stem cells. Since the cranial crest gives rise, besides PNS neural progeny and melanocytes, to mesenchymal derivatives, the results demonstrate that pNCSCs have a restricted developmental potential in comparison to rNCSCs and acquire mostly normal fates of the cranial neural crest.
Taken together, the results demonstrate that rNCSCs acquire a SCSC identity in the presence of EGF and FGF and that the reprogramming can be efficiently blocked by BMP4. On the other hand, NCSCs derived from adult palate rather acquire mesenchymal fates and do not acquire a CNS identity under the conditions used.
Tulasnella species (Tulasnellaceae, Cantharellales, Basidiomycota) form inconspicuous basidiomata on rotten branches or trunks of trees, difficult to find and recognize in nature. However, according to ultrastrucural and molecular data, species of Tulasnellaceae are the most frequent mycorrhriza forming fungi (mycobionts) of green, photosynthetic orchids worldwide. Species of Tulasnellaceae were also found as prominent mycobionts of the extraordinary diverse orchids in tropical montane rainforest of Southern Ecuador. Orchids obligately depend on mycobionts during the juvenile stage when the fungi have to deliver carbon to the non-photosynthetic protocorm and thus the fungi substantially influence the establishment of orchids in the wild. Species of Tulasnellaceae can acquire carbon from decaying bark or wood by specific saprotrophic capabilities as was recently proven through comparative genomics that included data on decay enzymes from Tulasnella cf. calospora isolated from orchid mycorrhizae (Anacamptis laxiflora, Italy). Thus, species of Tulasnellaceae can be saprotrophs and symbionts simultaneously.
It is currently under discussion, whether specific species of Tulasnella are required for seed germination and establishment of distinct terrestrial and epiphytic orchids in nature or if species of Tulasnella are generalists concerning their association with orchids. The inconsistences in species concepts and taxonomy of Tulasnella spp., however, strongly impede progress in this field of research. The aim of the present study was, therefore, to revise the species concepts by combining, for the first time, morphological and molecular data from basidiomata.
Specimens were collected in tropical Andean forest in Southern Ecuador and in temperate forests in Germany. Additional specimens were loaned from fungaria. In total, 205 specimens, corresponding to 16 own samples and 189 specimens from fungaria were analyzed. The mycobiont relationships of Tulasnella spp. with orchids from the sampling area in Ecuador were studied in populations of Epidendrum rhopalostele. The basis for molecular-phylogenetic analysis was completed by data obtained from own previous investigations on mycobionts from the investigation area and Tulasnella isolates from Australia.
30 morphospecies are illustrated and delimited by a morphological key based on traditional species concepts. Tulasnella andina from Ecuador and Tulasnella kirschneri from China are presented as species new to science. Tulasnella cruciata is described from herbarium material for the first time. Tulasnella aff. eichleriana and T. violea are reported for the first time from Ecuador. Molecular sequences of two Tulasnella spp. isolated from mycobionts of Epidendrum rhopalostele cannot be related to any morphological species concept. Statistical analyses suggest that conventional diagnostic using morphological characteristics is ambiguous for delimiting morphologically similar species.
For the first time sequences of the ITS-5.8S rDNA region were obtained after cloning from fresh basidiomata. Extraction of DNA from herbarium specimens was, however, unsuccessful. Sequences from 16 fresh basidiomata, six pure cultures, and sequences of orchids mycorrhizae (e.g. from Epidendrum rhopalostele) available in the database GenBank were analyzed. Proportional
variability of ITS-5.8S rDNA sequences within and among cultures and within and among specimens were used to designate morphospecies. Results suggest an intragenomic variation of less than 2 %, an intraspecific variation of up to 4 % and an interspecific divergence of more than 9 % for Tulasnella spp.
Four percent of intraspecific divergence was defined as a minimum threshold for delimiting phylogenetic species. This threshold corroborates the so far used 3 % to 5 % divergence in delimitation of operational taxonomic units of Tulasnella mycobionts.
Quite a number of sequences of Tulasnella are available in GenBank, mostly obtained from direct PCR amplification from orchid mycorrhizae. By including closely related sequences in the phylogenetic analysis, several morphological cryptic species of Tulasnella, mostly from Ecuador, were found. Arguments are given for molecular support of the new species Tulasnella andina and the established species Tulasnella albida, T. asymmetrica, T. eichleriana, T. tomaculum, and T. violea. Thus, by combining molecular and morphological data species concepts in Tulasnella are improved. The definitions of Tulasnella calospora and T. deliquescens, however, remain phylogenetically inconsistent.
The present investigation is a first step to expand our knowledge on the intraand interspecific morphological and molecular variability of Tulasnella spp. and to delimit species relevant for studies on ecology and communities of orchids and Tulasnellaceae.
Heutzutage unterliegen insbesondere aquatische Ökosysteme durch den permanenten Anstieg nicht-heimischer Arten einem folgenreichen Wandel. Dabei stellt der Biodiversitätsverlust nur den Endpunkt einer biologischen Invasion dar. Dazwischen wirken sich Faktoren wie Konkurrenz-, Prädationsdruck oder die Übertragung von Krankheitserregern wie z.B. Parasiten auf den Rückgang der Arten aus. Als integraler Bestandteil eines jeden Ökosystems spielen Parasiten bei Invasionsprozessen eine entscheidende Rolle und können durch ihren Einfluss auf heimische und nicht-heimischen Arten Invasionen begünstigen. Fische übernehmen aufgrund ihrer vielseitigen Bedeutung im Nahrungsnetz eine Schlüsselfunktion als Wirte diverser Parasitenarten und sind deshalb ausgezeichnete Untersuchungsobjekte, um durch nicht-heimische Arten verursachte Veränderungen in Nahrungsnetzstrukturen oder Parasitenfaunen aquatischer Ökosysteme aufzudecken.
Vor diesem Hintergrund wurde die vorliegende kumulative Dissertation angefertigt, welche auf drei (ISI-) Einzelpublikationen basiert. Die Arbeit beschäftigte sich unter Verwendung morphologischer und molekularbiologischer Methoden schwerpunktmäßig mit der Parasitendiversität und Nahrungsökologie zweier eingeschleppter Fischarten in stark anthropogen beeinflussten deutschen Fließgewässern. Dabei handelte es sich um die hauptsächlich durch das Ballastwasser von Schiffen verbreitete invasive Schwarzmundgrundel Neogobius melanostomus aus den Flüssen Rhein und Main sowie den von Hobby-Aquarianern in den durch Industrieabwässer thermisch belasteten Gillbach ausgesetzten Zebrabuntbarsch Amatitlania nigrofasciata. Unter Berücksichtigung nahrungsökologischer und räumlich-zeitlicher Aspekte sollten die parasitologischen Risiken und Konsequenzen, welche durch das Einschleppen nicht-heimischer Fischarten auftreten, aufgezeigt werden, um übergeordnet die Folgenabschätzung auf dem Gebiet der Invasionsbiologie zu verbessern. Das Nahrungsspektrum beider Fischarten wies sowohl räumliche (zwischen den Flüssen und Standorten), als auch zeitliche (monatlich) Variationen auf, was die Anpassungsfähigkeit bzw. die optimale Nutzung zur Verfügung stehender Ressourcen von invasiven Arten verdeutlicht. Ebenso wurden Unterschiede in der Parasitierung nachgewiesen, was die Notwendigkeit räumlicher und zeitlicher Analysen zur Erfassung der vollständigen Parasitenfauna einer invasiven Art, inklusive aller Variationen der Befallszahlen, unterstreicht. Beide Fischarten bilden zudem Zwischenwirte für heimische, als auch nicht-heimische Parasitenarten, wobei die direkte Einschleppung von nicht-heimischen Parasiten aus dem ursprünglichen Verbreitungsgebiet der Fische auszuschießen ist und somit die Enemy-Release-Hypothese (Verlust ursprünglicher Gegenspieler wie Prädatoren oder Parasiten) unterstützt. Dies könnte zusammen mit der opportunistischen Ernährungsweise ein Grund für die starke Ausbreitung sowie die anhaltend hohen Individuenzahlen dieser Arten im jeweils betrachteten Verbreitungsgebiet (Rhein, Main sowie Gillbach) sein.
Besonders hervorzuheben ist der Nachweis der nicht-heimischen Nematoden Anguillicoloides crassus und Camallanus cotti. Durch die Aufdeckung einer besonderen Form von Hyperparasitismus konnten erstmalig hohe Befallszahlen von A. crassus in N. melanostomus dokumentiert werden. Die hoch abundante Grundelart gilt somit potentiell als entscheidender Überträger des invasiven Parasiten auf den Europäischen Aal. Der durch seine hohe Virulenz in der Aquaristik bekannte C. cotti gelangte durch das Aussetzen von Zierfischen in den Gillbach und trat mit hohen Befallszahlen in A. nigrofasciata auf und wird bereits auf die heimische Fischfauna übertragen (z.B. auf den Gründling Gobio gobio und den Döbel Squalius cephalus). Ebenso ist der starke Befall der heimischen Parasiten Pomphorhynchus sp. (Acanthocephala) und Raphidascaris acus (Nematoda) bei N. melanostomus sowie A. anguillae (Acanthocephala) bei A. nigrofasciata zu nennen. Durch die zusätzliche Wirtfunktion der Neozoen und die hohen Befallsintensitäten ist mit einem verstärkten Spillback-Effekt (Rückinfizierung) auf heimische Fischarten zu rechnen.
Die stetig steigende Anzahl biologischer Invasionen führt zu immer weitreichenderen Veränderungen heimischer Ökosysteme. Für den Erhalt der Biodiversität sowie einhergehender Ökosystemfunktionen rückt auch die Forschung über Neobiota immer mehr in den Mittelpunkt. In diesem Zusammenhang gewinnen auch Pathogene wie Parasiten immer mehr an Bedeutung, welche durch gebietsfremde Arten eingeschleppt werden und die bestehende Biodiversität gefährden können. Die Ergebnisse der durchgeführten Studien haben gezeigt, dass die Verknüpfung von parasitologischen und nahrungsökologischen Untersuchungen Einblicke in die hoch dynamischen Prozesse der Invasionsbiologie geben können, was ein Vorantreiben der Forschung und Folgenabschätzungen auf diesem Gebiet ermöglicht.
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.
The development of the atrioventricular (AV) canal and the cardiac valves is tightly linked and a critically regulated process. Anomalies in components of the involved pathways can lead to congenital valve malformations, a leading cause of morbidity and mortality in neonates. Myocardial Bmp as well as endocardial Notch and Wnt signaling have been identified as critical factors for the induction of EMT during the formation of the endocardial cushions and cardiac valves. Of these, canonical Wnt signaling positively regulates endocardial proliferation and EMT but negatively regulates endocardial differentiation. Further, elevated Wnt signaling leads to the ectopic expression of myocardial Bmp ligands suggesting a high level of integration of the involved pathways and crosstalk amongst the different cardiac tissues.
Here we have identified a novel role for Id4 as a mediator between Bmp and Wnt signaling. Id4 belongs to the Id family of proteins and is known to be involved in bone and nervous system development. We found that in zebrafish, id4 is expressed in the endocardium of the AV canal at embryonic stages and throughout the atrial chamber in addition to AV canal, in adults. Using transcription activator-like effector nucleases (TALENs) we established an id4 mutant allele. Our analysis shows that id4 mutant larvae are susceptible to retrograde blood flow, and show aberrant expression of developmental valvular markers. These include expanded expression domains of markers like bmp4, cspg2a and Alcam. In contrast, valve maturation as assessed by the expression of spp1 is considerably reduced in id4 mutants. Using conditional transgenic systems, along with elegant in vivo imaging of transgenic reporter lines, we further found that id4 is a transcriptional target of Bmp signaling, and it is capable of dose dependently restricting Wnt signaling in the endocardium of the Atrioventricular Canal.
Taken together, our data identifies Id4 as a novel player in Atrioventricular Canal and valve development. We show that Id4 function is important in valve development acting downstream of Bmp signaling by restricting endocardial Wnt to allow valve maturation
Die vorliegende Studie vermittelt einen epidemiologischen Überblick über das mit Haut- und Nagelläsionen assoziierte Pilzspektrum im Westen Panamas. Hierzu wurden Proben von vermutlich durch Pilzinfektionen verursachten Haut- sowie Nagelläsionen gesammelt und zum Anlegen von Kulturen verwendet. Die isolierten Pilze wurden basierend auf dem D-H-S System (Rieth), anhand morphologischer Merkmale, rDNA Sequenzdaten sowie phylogenetischen Analysen klassifiziert und mit Hilfe von Literaturdaten sowie physiologischen Eigenschaften als saprotrophe, opportunistische oder pathogene Organismen beurteilt. In Panama wurden 52 Proben von 51 Personen gesammelt, wobei das Material von 42 Haut- und Nagelläsionen der Füße, vier Läsionen der Fingernägel, zwei Chromomykosen, einer Tinea nigra und drei sonstigen Hautläsionen stammt. Bei 75 Prozent (n = 39) der Proben konnten Pilze kultiviert und insgesamt 201 Pilzstämme isoliert und subkultiviert werden. Hiervon wurden 50 Isolate (24,9 %) als Dermatophyten, 24 Stämme (11,9 %) als Hefen und 127 Isolate (63,2 %) als Schimmelpilze klassifiziert. Bei 19 Probanden (48,7 %) konnten Dermatophyten isoliert werden, wobei aus dem Probenmaterial von 12 Personen (63,2 %) ebenfalls andere Pilzarten nachgewiesen wurden. Von zwei Läsionen (5,1 %) wurden nur Hefen isoliert, wobei einmal eine Schwarze Hefe kultiviert wurde. In dem Material acht weiterer Proben (20,5 %) wurden Schimmelpilze und Hefestämme nachgewiesen und bei zehn Probanden (25,6 %) konnten aus dem Probenmaterial nur Schimmelpilze kultiviert werden. 172 Isolate wurden taxonomisch klassifiziert und 44 Arten aus 25 Gattungen, 17 Familien, 15 Ordnungen, sechs Klassen sowie den Abteilungen Ascomycota oder Basidiomycota zugeordnet. Die Ascomyceten stellen mit 164 Stämmen 40 verschiedener Arten aus 23 Gattungen, 15 Familien, 11 Ordnungen und vier Klassen die am häufigsten isolierte und vielfältigste Gruppe dar, während die Basidiomycota nur mit acht Isolaten vier verschiedener Arten zwei unterschiedlicher Gattungen, Familien, Ordnungen und Klassen nachgewiesen wurden. Im Rahmen dieser Arbeit wurden in Panama die anthropophilen Dermatophyten Trichophyton rubrum und T. interdigitale dokumentiert, wobei T. rubrum die am häufigsten isolierte Art darstellt. Kultivierte Hefen waren Candida albicans, C. duobushaemulonii, C. tropicalis, Hortaea werneckii, Sporobolomyces sp., Trichosporon asahii, T. japonicum und T. montevideense. Die Schimmelpilze stellen die größte und ökologisch diverseste Organismengruppe der kultivierten Pilze dar. So wurden von den untersuchten Läsionen sowohl humanpathogene Erreger, als auch opportunistische Arten und rein saprotrophe Pilze sowie mehrere Vertreter wahrscheinlich bisher nicht wissenschaftlich beschriebener Arten bzw. Gattungen nachgewiesen. Aus dem Probenmaterial wurden die Pilze Acremonium collariferum, Aspergillus awamori, A. clavatus, A. flavus, A. giganteus, A. heteromorphus, A. niger, A. ochraceus, A. sclerotiorum, A. versicolor, Chaetomium globosum, Chrysosporium tuberculatum, Cladosporium sphaerospermum, C. tenuissimum, Curvularia geniculata, C. lunata, Fonsecaea pedrosoi, Fusarium oxysporum, F. solani, Lophotrichus bartlettii, Microascus cinereus, Neoscytalidium dimidiatum, Penicillium commune, Scolecobasidium sp., Scopulariopsis carbonaria, S. croci, Verticillium cf. epiphytum und Wardomycopsis litoralis isoliert. Zudem wurden vier Isolate von zwei vermutlich neuen Arten der Gattung Acremonium (Bionectriaceae, Hypocreales), zwei Stämme mit einer genetischen Affinität zu der Gattung Cryptendoxyla (Cephalothecaceae, Sordariales) und jeweils ein mit den Gattungen Fusicladium (Venturiaceae, Venturiales), Knufia (Trichomeriaceae, Chaetothyriales) bzw. Rhexothecium (Eremomycetaceae, Dothideomycetidae) assoziierter Stamm kultiviert. Im Rahmen dieser Studie wurden A. giganteus, C. tenuissimum, L. bartlettii, S. carbonaria, S. croci, V. epiphytum und W. litoralis erstmalig von Mykosen des Menschen dokumentiert und die in der Literatur als Verursacher sowie Besiedler von Haut- und Nagelläsionen beschriebenen Organismen A. clavatus, A. flavus, A. niger, A. ochraceus, C. tropicalis, C. globosum, C. sphaerospermum, C. lunata, F. oxysporum, M. cinereus, P. commune, T. asahii, T. japonicum und T. montevideense wurden das erste Mal in klinischem Probenmaterial aus Panama nachgewiesen. Die Arten A. awamori, A. heteromorphus, C. globosum, C. tenuissimum, L. bartlettii, M. cinereus, P. commune, S. croci, T. asahii, T. japonicum, T. montevideense, V. epiphytum, W. litoralis und die Gattung Scolecobasidium wurden zudem erstmalig für Panama dokumentiert. Die Isolation von W. litoralis ist ebenfalls der erste Nachweis dieses Pilzes außerhalb von Spanien und auf dem amerikanischen Kontinent. Die große Anzahl im Rahmen dieser Arbeit beschriebener, bisher für die Wissenschaft unbekannter bzw. nicht in Panama dokumentierter Pilzarten lässt auf eine große mykologische Biodiversität in Panama schließen und zeigt den Bedarf weiterer Forschung.
Panama, a small country between the major continents of North and South America, is one of the lesser studied regions in Central America, but is recognized for its mega-biodiversity. This is particularly true for Eastern Panama, which I am considering as the easternmost portion of the country, covering the area from the Chepo, which is also the beginning of the San Blas mountain range, towards east, up to the Darien Mountain range on the border with its neighboring country Colombia. In the lowland region I visited two physiographic areas: the Isthmian-Atlantic Moist Forests (IAMF) and the Chocó-Darién Moist Forests (CDMF). In the IAMF I worked at the localities of Río Mono, Wacuco, La Moneda, Arretí, Metetí, Filo del Tallo, and Laguna de Matusagaratí. In the CDMF I visited the localities of Cruce de Mono, Cana, Garachiné, Sambú, and Pavarandó. And I have worked in the highlands of Darién (DM), Majé (MM), Jingurudó-Sapo (JSM), Pirre (PM) and San Blas (SSM) in the highlands.
Before my research, 138 reptile and 104 amphibian species had been reported for EP. From 2008 to 2013, I collected specimens to evaluate the diversity of amphibians and reptiles for this region. I applied an integrative approach to evaluate the taxonomy, diversity, biogeography, and conservation of the herpetofauna of EP. I included analyses of morphometrics, molecular genetics (e.g. barcoding), biogeography, bioacoustics (in anurans), hemipenial morphology (in squamates), and ecology. This is the first regional evaluation of the biodiversity in EP applying integrative taxonomy. Aside from morphological and bioacoustic data, my work is based on the barcoding of 608 specimens, from which I obtained 16S mtDNA for 486 specimens and COI mtDNA for 455. In total I have got sequences for 69.2 %of the amphibian and 48.6 % of the reptile species present in EP. For the morphological analyses, I compared 1597 specimens, including my samples complemented by specimens obtained from various museums. The bioacoustic data were obtained from the analysis of 1504 calls of 27 species of frogs. Based on specimens collected in EP and according to external morphology, I could identify 65 species of amphibians and 72 reptiles, but after applying an integrative approach these numbers increased to 79 amphibians and 88 reptiles described species within my collected specimens. Additionally, I uncovered 33 taxonomic units that could not be assigned to any described species until now, 22 of them represent confirmed candidate species (CCS), and 11 were classified as Unconfirmed candidate species (UCS). Thus, increasing the known species of amphibian by 19.4 % and of reptiles by 4.8 %. Currently, there are 145 reptiles and 129 amphibians known to occur in EP. Based on my results, I have initiated several projects to solve taxonomic uncertanties, including the species of the genera Bolitoglossa, Diasporus, Dactyloa, Ecnomiohyla, Lepidoblepharis, and the taxonomic status of the species Pristimantis caryophyllaceus and Norops tropidogaster.
Out of the 22 CCS I found, I described nine species new to science with type locality in EP, six amphibians and four reptiles. Among these is a new species of Bolitoglossa described from Cerro Chucantí, Cordillera de Majé, Provincia de Darién, Panama. Additionally, I include comments on the other species of congeneric salamanders known to occur in the region. Among the tink frogs, only Diasporus quidditus was known to occur in EP. During my field work I collected six additional species of this genus, four of which are new to science, plus two species new for this region.
I also described one new species of Dactyloa (giant anole lizards) related to the former D. chocorum. I synonymized D. chocorum with D. purpurescens, and included information about the other species of the group from EP. The new species of Dactyloa resembles D. ibanezi, D. limon, and D. purpurescens in external morphology but differs from these species in dewlap coloration, dorsal color pattern, morphometrics, and scalation. I discovered one species of the genus Ecnomiohyla, which exhibits significant genetic distances (16S mtDNA gene) and morphological differences to all known Ecnomiohyla species. Along with the description of the new Ecnomiohyla species, I provide detailed comparisons of morphological and molecular characters of almost all members of the genus in Lower Central America, as well as an identification key for the entire genus. Two new species of the genus Lepidoblepharis from EP were described. In the corresponding work, I include an analysis of Lepidoblepharis spp. in the region, including phylogeography and taxonomy. One of the new species, Lepidoblepharis emberawoundule, can be differentiated from most species in the genus by its small size and its low number of lamellae under the fourth toe and finger. The other species described from EP, Lepidoblepharis rufigularis, can be differentiated from all species in the genus by its small size and the reddish throat in males.
Xenorhabdus and Photorhabdus bacteria are gaining more and more attention as a subject of research because of their unique yet similar life cycle with nematodes and insects. This work focused on the secondary metabolites that are produced by Xenorhabdus and Photorhabdus. With the help of modern HPLC-MS methodologies and increasingly available bacterial genome sequences, the structures of unknown secondary metabolites could be elucidated and thus their biosynthesis pathways could be proposed, too.
The first paper reported 17 depsipeptides termed xentrivalpeptides produced by the bacterium Xenorhabdus sp. 85816. Xentrivalpeptide A could be isolated from the bacterial culture as the main component. The structure of xentrivalpeptide A was elucidated by NMR and the Marfey´s method. The remaining xentrivalpeptides were exclusively identified by feeding experiments and MS fragmentation patterns.
The second paper described the discovery and isolation of xenoamicin A from Xenorhabdus mauleonii DSM17908. Additionally, other xenoamicin derivatives from Xenorhabdus doucetiae DSM17909 were analyzed by means of feeding experiments and MS fragmentation patterns. The xenoamicin biosynthesis gene cluster was identified in Xenorhabdus doucetiae DSM17909.
The manuscript for publication focused on the biosynthesis of anthraquinones in Photorhabdus luminescens. The Type II polyketide synthase for the biosynthesis of anthraquinone derivatives was discovered in P. luminescens in a previous publication by the Bode group,1 in which a partial reaction mechanism for the biosynthesis has been proposed. The manuscript reported in this thesis however elucidated the biosynthetic mechanisms in a greater detail as compared to the previous publication. Particularly, the biosynthetic mechanism was deciphered through heterologous expression of anthraquinone biosynthesis (ant) genes in E. coli. Additionally, deactivation of the genes antG encoding a putative CoA ligase and antI encoding a putative hydrolase, was performed in P. luminescens. Selected ant genes were over-expressed in E. coli as well as the corresponding proteins purified for in vitro assays. Model compounds were chemically synthesized as possible substrates of AntI and were used for in vitro assays. Here, it was revealed that the CoA ligase AntG played an essential role in the activation of the ACP AntF. Furthermore, a chain shortening mechanism by the hydrolase AntI was identified and was further confirmed by in vitro assays using model compounds. Additionally, this chain shortening mechanism was supported by homology based structural modeling of AntI.
The lung comprises more than 40 different cell types, from epithelial cells to resident mesenchymal cells. These cells arise from the foregut endoderm and differentiate into specialized cell types that form the respiratory and conducting airways, and the trachea. However, the molecular pathways underlying these differentiation processes are poorly understood, and may be relevant to pathological conditions. According to the World Health Organization (WHO), while the respiratory disease rate is increasing, limited treatment and therapies are available. Thus, there is a growing need for new treatment strategies and alternative therapies. Various in vivo and in vitro studies in the model organism mus musculus have already provided valuable information on lung cell lineages and their differentiation and/ or dedifferentiation during development and pathological conditions. However, there remain many questions regarding the key regulators and molecular machinery driving lung cell differentiation and underlying lung progenitor/stem cell biology.
Aiming to develop new animal models for lung diseases, we used a forward genetic careening approach, which provides an unbiased method for identifying genes with important roles in lung cell differentiation, and thus probable contributors to pathological conditions. We conducted an N-ethyl-N-nitrosourea (ENU) mutagenesis screen in mice and used several histological and immunohistochemical approaches to identify and isolate mutants, focusing on mutations associated with cell differentiation rather than those affecting early development and patterning of the respiratory system. Thus, we screened for phenotypes in the respiratory system of pups from the F2 generation at postnatal day 7 and 0 (P7; P0). I specifically screened 114 families. Each F1 male animal is the founder of 5 to 6 F2 female daughters. For each family, at least 4 F2 females per male founder were analyzed. In total, I screened 630 litters at P7 and P0 with 7 pups on average for each litter. As a result of this extensive screening, 11 different phenotypes in 42 different F2s were discovered at primary screen and later just 2 phenotypes recovered in F3 generation of identified carriers. To identify the causative genes for each of these phenotypes, whole exome sequencing will be conducted in the future to identify recurring SNPs; these can subsequently be linked causatively to the resultant phenotype(s) via complementation studies. In turn, these linkages would enable the creation of mutant mice using CRISPR/Cas9 genomic engineering, which would be invaluable to the further study of respiratory development and disease.
Juvenile neuronal ceroid-lipofuscinosis (JNCL) is a rare lysosomal storage disease in children with lethal outcome and no therapy. The origin of JNCL has been traced to autosomal recessive mutations in the CLN3 gene, and ~85% of the JNCL patients harbor a 1.02 kb deletion that removes the exons 7 and 8 and the surrounding intronic DNA (CLN3Δex7/8). So far, structure, function and localization of the CLN3 protein remain elusive. However, there is strong evidence that CLN3 modulates a process or condition that is essential in many cellular pathways. Lipid metabolism and antero-/retrograde transport, two mechanisms CLN3 was previously implicated in, fulfill these requirements. Notably, also a bioactive group of glycosphingolipids referred to as gangliosides is tightly interrelated with these functions. Furthermore, a-series gangliosides have been shown to be involved in the development and sustenance of the brain, where they are essential for neurite outgrowth and cell survival. Defects in ganglioside metabolism were shown to play a crucial role in many lysosomal storage disorders. However, the contribution of gangliosides to NCL pathology is largely unknown.
The present study analyzed central enzymes and metabolites of the a-series ganglioside pathway in a JNCL cell model. The core finding was, thereby, the reduced amount of the neuroprotective ganglioside GM1 in homozygous CbCln3Δex7/8 cells. This was caused by the enhanced action of the GM1-degrading multimeric enzyme complex and in particular, by the upregulation of protein levels and increased enzyme activity of β-galactosidase (Glb1).
Improved binding of Glb1 to substrate-carrying membranes was provided by an increase in LBPA levels. In combination with other smaller alterations in the ganglioside pattern, a shift towards less complex gangliosides became present. The resulting loss of neuroprotection may be the reason for the multifocal pathology in homozygous CbCln3Δex7/8 cells.
The second part of the present study investigated the cellular mechanisms behind the altered ganglioside profile with regard to the potential role of CLN3. Here, the anterograde transport of GM1 to the plasma membrane presented a positive correlation with the amount of full-length CLN3. In case of the truncated protein this correlation was missing, resulting in reduced PM staining with CTxB-FITC. However, transfection of full-length CLN3 in these cells restored the CTxB-FITC intensity. Based on the neuroprotective role of GM1, the corresponding increase in GM1 levels may be the cause for the restoration effects observed in previous studies using full-length CLN3. Hence, administration of GM1 was expected to improve cell viability of homozygous CbCln3Δex7/8 cells and beyond that to rescue potentially some disease phenotypes. However, no effect could be observed. The reason for this may be reduced caveolar uptake and the mislocalization of ganglioside GM1 to the trans-Golgi network (TGN) and redirection towards degradative compartments.
Both are in line with the idea of an impaired endocytic flux in CLN3 deficiency. The observed localization of CLN3 in the TGN suggests a potential role for CLN3 in the lipid sorting machinery, subsequently altering membrane composition and its regulatory functions. The resulting imbalance may affect many of the cellular processes impaired in JNCL.
Primäre Tumore werden nach ihrem Entstehungsort benannt. Selbst Metastasen zeigen eine gewisse Ähnlichkeit mit ihrem ursprünglichen Gewebe. Somit gehören alle Geschwülste, die ursprünglich der Harnblase entstammen, zu den Harnblasenkarzinomen.
Das Harnblasenkarzinom geht meist (90-95%) von der Schleimhaut der ableitenden Harnwege aus und wird als Urothel bezeichnet. Dementsprechend haben die meisten Patienten mit der Diagnose Blasenkarzinom ein Urothel-Blasenkarzinom. Die restlichen Blasenkarzinome entfallen auf Adenokarzinome, Plattenepithelkarzinome, kleinzellige Karzinome, Sarkome, Paragangliome, Melanome oder Lymphome (Humphrey A. et al. 2016, Moch H. et al 2016).
Die Urothel-Blasenkarzinome können sowohl flach, als auch warzenförmig wachsen. Je nach Diagnostik „oberflächlich“ oder „muskelinvasiv“ lassen sich die Urothel-Blasenkarzinome in zwei Hauptgruppen unterteilen;
Etwa 70% der Erkrankten haben dabei ein oberflächliches Urothel-Blasenkarzinom, das auf die Blasenschleimhaut begrenzt ist und durch eine Basistherapie, sog. Transurethrale Resektion (TUR-B) behandelt wird. Dabei werden die in der Schleimhaut gewachsenen Tumore getrennt reseziert. Therapieergänzend und/oder prophylaktisch wird die Blase danach mit einem Chemotherapeutikum gespült (intravesikale Instillation). Diese Zytostatika-Behandlung soll das Wiederauftreten eines Rezidivs verhindern bzw. eventuell verlangsamen (Iida K. et al. 2016, Celik O. et al. 2016).