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Institute
- Biowissenschaften (246) (remove)
Baleen whales (Mysticeti) are a clade of highly adapted carnivorous marine mammals that can reach extremely large body sizes and feature characteristic keratinaceous baleen plates used for obligate filter feeding. From a conservation perspective, nearly all baleen whale species were hunted extensively over a roughly 100 years lasting time period that depleted many of the respective whale stocks with so far unknown consequences for e.g. their molecular viability. From an evolutionary perspective, the lack of fossil records together with conflicting molecular patterns resulted in a still unclear and debated phylogeny of modern baleen whales, particularly in rorquals (Balaenopteridae). In this dissertation, I will demonstrate the application of baleen whale genomes to tackle these open questions by using modern approaches of conservation and evolutionary genomics.
Conservation genomic aspects of baleen whales were addressed in two projects, both using whole genome data of either an Icelandic fin whale (Balaenoptera physalus) population or multiple blue whale (Balaenoptera musculus) populations to evaluate the impact of the industrial whaling era on their molecular viability. The results suggest a substantial drop in effective population size of both species but also a lack of manifestation in genotypes of the fin whale population when compared to the blue whale populations. Especially the rare and short runs of homozygosity (ROH), usually indicative for inbreeding, suggest frequent outcrossing in fin whales while all analyzed blue whale populations featured long and frequent ROH. In addition to these analyses, genome data of blue whale populations was further used to evaluate if northern hemisphere blue whales diverged into different subspecies. Population genetic and gene flow analyses showed clearly separated and well isolated populations in accordance with their assumed geographical distance. In contrast, the genome-wide divergence between all blue whale populations was low compared to other cetacean populations and to the next closely related sei whale species. Because this includes the morphologically different and well recognized pygmy blue whale subspecies, a proposal was made to equally categorize the two northern-hemisphere blue whale populations as subspecies.
Evolutionary aspects were addressed in a third project, by constructing the genome of the pygmy right whale (Caperea marginata) and testing its potential in phylogenetics and cancer research. Phylogenomic analyses using fragments of a whole-genome alignment featuring nearly all extant baleen whales, allowed the revision of the complex evolutionary relationships of rorquals by quantifying and characterizing the amounts of conflicts in early diverging branches. These relationships were further used to identify phylogenetically independent pairs of baleen whales with a maximum of diverging body size differences to compare rates of positive selection between their genomes. The results suggest nearly evenly distributed frequencies of alternative topologies which supports the representation of the early divergence of rorquals as a hard polytomy with high amounts of introgression and incomplete lineage sorting. Within the set of available genomic data, three independent pairs of baleen whales with diverging body sizes were found and comparisons of positive selection rates resulted in many potentially body size and cancer related genes. The lack of conserved selection patterns, however, suggest a more convergent evolution of size and cancer resistance like previously discussed in paleontology.
In conclusion, the application of whole genome data using methods of conservation genetics allowed for a comprehensive estimation about the molecular viability of blue and fin whales as well as an assessment of the taxonomic status of northern-hemisphere blue whale populations. The rather different results between blue and fin whales underlines the importance of genomic monitoring of baleen whales because different species show rather different molecular consequences of their potentially varying depletions. Furthermore, as showcased for the northern-hemisphere blue whale, many important isolated populations of baleen whales may still be unknown to conservation management and genome-wide comparisons will most likely contribute to overcome this under-classification problem. The application of whole genome data in evolutionary research allowed the characterization of the complex patterns of molecular conflicts within baleen whales and especially rorquals that will contribute to the still rather unclear understanding of their evolution. The here found molecular support for the idea of convergent evolution of gigantism in whales will further guide the search for molecular patterns responsible for Peto’s paradox.
The production of ribosomes is a complicated multistep, that is susceptible to changes occurring within the cell and its environment. The process itself requires many proteins, known as ribosome biogenesis factors (RBFs) and many non-coding RNAs like the small nucleolar RNAs (snoRNAs). While RBFs are required for the accurate processing of the pre-rRNA into mature rRNAs, the snoRNAs act to coordinate and guide enzymes for post-transcriptional modifications, chiefly 2´-O-ribose methylation and pseudouridylation. While ribosome biogenesis is mostly described in human and yeast model eucaryotes, similar detailed studies in the model plant Arabidopsis thaliana are far less explored and understood. Furthermore, for many experimentally confirmed modification sites the according snoRNAs and for many pre-rRNA processing steps the responsible RBFs are missing. Therefore, it is expected that a high number of snoRNAs and RBFs are not identified till yet. For this reason, RNA-deep sequencing was performed in order to identify novel snoRNAs and MS analysis data of nucleoli and nuclei of A. thaliana from a former PhD student were used in order to find new proteins involved in pre-rRNA processing.
In here, it is shown that with RNA deep-sequencing still new snoRNAs and snRNAs can be identified and that detection of predicted snoRNAs can be fulfilled with a) antisense oligonucleotides tagged with fluorescence dyes and b) with radioactive labeled antisense probes. Furthermore, a secondary structure map of the 60S and 40S subunit highlighting the predicted and moreover verified modification sites in 5.8S, 25S and 18S rRNA was created. Especially, the correlation between the modification sites and the guiding snoRNA is highlighted further shedding light on overview about current pre-rRNA modification sites and corresponding guiding snoRNAs. The next chapter reveals the complex and multi-layered existence of the 5.8S rRNA and its numerous precursors. The mutant prp24 (also known as seap1) encoding AtPRP24, is recognized as factor being important for splicing as it is promoting the recruitment of the U4 and U6 snRNAs to the spliceosome. In here, it was found that AtPRP24 is involved in processing of 5.8S rRNA precursors, recognizable by precursors that are over accumulating in the mutant. Moreover, it could be shown for the first time that the plant-specific precursor 5´-5.8S is exported to the cytoplasm, where final cleavage steps of 5.8S rRNA takes place. In the prp24.2 mutant, this precursor is exported at an increased rate to the cytoplasm, where it can be detected in the actively translating ribosomes (polysomes). A lower sensitivity of the mutant seeds to cycloheximide (CHX) suggests that due to the extension at the 5´-end of 5.8S, the structure of the 60S subunit has altered CHX binding. In conclusion, this work highlights the importance and complexity of 5.8S rRNA and its precursors for ribosome biogenesis and displays new insights into pre-rRNA processing in A. thaliana.
Xylose, an abundant sugar fraction of lignocellulosic biomass, is a five-carbon skeleton molecule. Since decades, utilization of this sugar has gained much attention and has been in particular focus as a substrate for production of biofuels like ethanol by microbial hosts, including Saccharomyces cerevisiae. In this yeast, xylose is naturally not used as a carbon source, but its utilization could be achieved by metabolic engineering either via the oxidoreductive route or through the isomerase pathway. Both pathways share xylulose as a common intermediate that must be phosphorylated before entering the endogenous metabolism via the non-oxidative pentose phosphate pathway (noxPPP). Besides this, in some bacteria a non-phosphorylating oxidative pathway for xylose degradation exists, known as Weimberg pathway, where a molecule of xylose is converted by a series of enzymes - xylose dehydrogenase (XylB), xylonate dehydratase (XylD), 3-keto-2-deoxy-xylonate dehydratase (XylX) and α-ketoglutarate semialdehyde dehydrogenase (KsaD) - to form α-ketoglutarate (AKG). Besides having several useful properties as a product, AKG could also be used for cell growth as an intermediate of the tricarboxylic acid (TCA) cycle. One target of the present study is to establish a functional Weimberg pathway in S. cerevisiae. Previous studies have shown that this task is not trivial, for instance due to the toxicity of xylonate (the first metabolite of the pathway) and the involvement of an iron-sulfur cluster dependent enzyme, the D-xylonate dehydratase. The assembly of iron-sulfur clusters on a heterologous protein in yeast is known to be challenging.
To establish the Weimberg pathway in yeast, the genes xylB, xylD, and xylX were obtained from Caulobacter cresentus and ksaD was from Corynebacterium glutamicum. In a variant, the dehydratase xylD was replaced with orf41 from Arthrobacter nicotinovorans, which is believed to be independent of iron-sulfur clusters. Growth of yeast cells on xylose as a sole carbon source was expected as an indicator of a functional Weimberg pathway. However, the heterologous expression of the codon optimized genes was not sufficient to reach this goal. Due to the complexity of the interactions of the heterologous pathway with the endogenous cellular processes, it was assumed that potential limitations could be overcome by adaptive laboratory evolution, using xylose as a sole source of carbon. Increasing selection pressure was applied on a strain with Weimberg pathway genes integrated into the genome over several generations. As a variant of the evolutionary engineering approach, mutator strains were generated. For this, RAD27 and MSH2 genes were deleted, which are involved in nucleotide excision and mismatch repair mechanisms, respectively. Some of the resulting strains PRY24, PRY25, PRY27 and PRY28 were able grow in xylose as a sole carbon source after evolutionary engineering. As a control, a non-mutator strain PRY19 was also included. Strikingly, only the mutator strains were able to consume xylose as a sole carbon source, which shows the feasibility of the approach.
In addition to the mutator strain strategy, a further approach employed in the present study was the simultaneous expression of the Weimberg pathway in the cytosol and mitochondria. This was based on the reasoning that the iron-sulfur cluster biogenesis on XylD may be improved in the organelle and that the AKG is an intermediate of the TCA cycle. In the strain AHY02, all enzymes of the pathway were tagged with mitochondrial targeting signals in addition to a full cytosolically localized pathway. The localization of the mitochondrial variants was confirmed by fluorescence microscopy. Together with AHY02, CEN.PK2-1C wild type strain was also included as a control for evolution. When a selection pressure on xylose was applied, both strains - AHY02 and CEN.PK2-1C - were able to grow in the course of evolution. Deletion of the xylulokinase (XKS1) gene was found to be detrimental for both evolved strains in xylose-containing media. This suggests that the evolution of the endogenous oxidoreductive and noxPPP genes is responsible for growth of the evolved cells. For the evolved strain AHY02, it could also be possible that the Weimberg pathway genes supported to growth in addition to the oxidoreductive route. To elucidate the underlying molecular mechanisms, genome sequencing and reverse engineering approaches would be necessary in future.
In addition to screening for growth on xylose as a sole carbon source, a less stringent screening system was created to examine even a minor flux of xylose towards AKG. For this, all genes necessary for conversion of isocitrate to AKG where deleted, yielding a glutamate auxotrophic strain. In this system, the cells can grow on other carbon sources, whereas xylose is only provided as a source of AKG for the synthesis of glutamate...
Identification of new natural products from nematode-associated bacteria using mass spectrometry
(2023)
This work aims to find unknown natural products produced by bacteria, that live in close association with nematodes and to elucidate their structure by using mass spectrometry.
The first chapter of this work is dedicated to the detection of hitherto unknown natural products by using a metabolomics approach and subsequent structure elucidation of said compounds. This chapter includes metabolomics analysis of Xenorhabdus szentirmaii wild type and knockout mutants, overproduction of the target compound, identification of derivatives from other strains and MS based structure elucidation.
The second and third chapters are about natural products that protect C. elegans from B. thuringiensis infections.
The second chapter deals with natural products that protect the nematode host without killing the pathogen. I deployed molecular biology methods to generate deletion and overproduction strains of a target compound, identified it via LC-MS/MS analysis and used LC-MS/MS and lipidomics to analyse the chemical properties of the active compound.
The third chapter aims at finding natural products, which are produced by Pseudomonas strains MYb11 and MYb12, respectively. These natural products display the ability to protect C. elegans by killing B. thuringiensis. I identified said compounds via fractionation and subsequent bioactivity testing. After identification, I generated production strains of the target compounds and elucidated the structure of the bioactive derivative.
The last chapter deals with the structure elucidation of peptides produced by an unusual GameXPeptide synthetase in Xenorhabdus miraniensis. I analysed producer strains of GameXPeptides using LC-MS and elucidated the structural differences between the known GameXPeptides, produced by P. luminescens TT01, and the unusual ones produced by X. miraniensis.
Adhesion to host cells is the first and most crucial step in infections with pathogenic Gram negative bacteria and is often mediated by trimeric autotransporter adhesins (TAAs). TAA-producing bacteria are the causative agent of many human diseases and TAA targeted anti-adhesive compounds might counteract such bacterial infections. The modularly structured Bartonella adhesin A (BadA) is one of the best characterised TAAs and serves as an attractive adhesin to study the domain-function relationship of TAAs during infection. BadA is a major virulence factor of B. henselae and is essential for the initial attachment to host cells via adhesion to extracellular matrix proteins. B. henselae is the causative agent of cat scratch disease and adheres to fibronectin using its long BadA fibres. The life cycle of this pathogen, with alternating host conditions, drives evolutionary and host-specific adaptations.
Human, feline, and laboratory adapted B. henselae isolates display genomic and phenotypic differences. By analysing the genomes of eight B. henselae strains using long-read sequencing, a variable genomic badA island with a diversified and highly repetitive badA gene flanked by badA pseudogenes was identified. Moreover, numerous conserved flanking genes were characterised, however, their influence on the regulation of badA expression and modification remains to be explored. It seems that B. henselae G 5436 is the evolutionary ancestor of the other B. henselae strains analysed in this work. The diversity of the badA island among the B. henselae strains indicates that the downstream badA-like domain region might be used as a ‘toolbox’ for rearrangements in the badA gene. Overall, it is suggested that badA-domain duplications, insertions, and/or deletions are the result of active phase variation via site-specific recombination and contribute to rapid host adaptation in the scope of pathogenicity, immune evasion, and/or enhanced long-term colonisation.
The model strain B. henselae Marseille expresses a badA gene that includes 30 repetitive neck/stalk domains, each consisting of several predicted structural motifs. To further elucidate the motif sequences that mediate fibronectin binding, various modified badA constructs were generated. Their ability to bind fibronectin was assessed via whole-cell ELISA and fluorescence microscopy. In conclusion, it is suggested that BadA adheres to fibronectin in a cumulative fashion with quick saturation via unpaired β-strands appearing in structural motifs present in BadA neck/stalk domains 19, 27, and other homologous domains. Furthermore, antibodies targeting a 15-mer amino acid sequence in the DALL motif of BadA neck/stalk domain 27 were able to reduce fibronectin binding of the B. henselae mutant strain S27. Moreover, this DALL motif sequence is conserved in the genome of all analysed B. henselae strains. The identification of common binding motifs between BadA and fibronectin supports the development of new anti-adhesive compounds that might inhibit the initial adherence of B. henselae and other TAA-producing pathogens during infection.
Auf der Oberfläche von Erythrozyten, Thrombozyten und Neutrophilen befinden sich mehrere hundert verschiedene polymorphe, ungekoppelt vererbte Blutgruppenantigene. Dementsprechend birgt jede Bluttransfusion das Risiko einer Immunisierung gegen fremde Blutgruppenmerkmale. Auch während der Schwangerschaft können aufgrund väterlich vererbter Antigene Alloantikörper induziert werden. Deshalb muss das Blut vor jeder Transfusion oder während einer Schwangerschaft auf das Vorhandensein irregulärer erythrozytärer Antikörper untersucht werden. Dabei greifen die aktuellen diagnostischen Verfahren auf primäre, stabilisierte Testerythrozyten von Blutspendern zurück, deren relevante Blutgruppenantigene bekannt sind. Antikörperspezifitäten können anhand von Agglutinationsreaktionen der Testzellen mit dem zu untersuchenden Patientenplasma auf ein oder mehrere Antigene zurückgeführt werden. Ist jedoch ein Antikörper gegen ein häufiges, ein hochfrequentes oder ein nicht-polymorphes, ubiquitäres Antigen gerichtet, kann in Ermangelung Antigen-negativer Testzellen keine adäquate Diagnostik gewährleistet, die Verträglichkeit der Transfusion also nicht definitiv sichergestellt werden. Auch der medizinische Einsatz therapeutischer Antikörper, welche Antigene adressieren, die auch auf Erythrozyten exprimiert werden, führt zunehmend zu Problemen. Tests auf granulozytäre Antikörper sind mangelhaft bezüglich ihrer Robustheit, besitzen eine unzureichende Auflösung und sind zudem meist zeitaufwändig und daher teuer. Antikörper gegen humane Plättchenantigene spielen insbesondere in der Schwangerschaft eine Rolle; sie vermögen bei Neugeborenen thrombozytopenische Blutungen bis hin zu massiven Hirnblutungen zu verursachen, die zu schweren Entwicklungsstörungen führen können. Bisher erfolgt jedoch mangels geeigneter Reagenzien keine standardisierte pränatale Untersuchung auf thrombozytäre Antikörper. In dieser Arbeit wurde ein neuartiges Verfahren für die Identifikation und Differenzierung irregulärer Blutgruppenantikörper etabliert, welches auf gentechnisch hergestellten, xenogenen Testzellen basiert, die einzelne definierte humane Blutgruppenantigene auf ihrer Oberfläche präsentieren. Die nicht humanen Zellen co exprimieren Fluorochrome, anhand derer Antikörper-markierte Testzellen durchflusszytometrisch voneinander unterscheidbar sind. Weiterhin können die generierten Testzellen zur Depletion von Antikörpern aus polyagglutinierenden Plasmen unter Erhalt der anderen Antikörperspezifitäten verwendet werden. Diese Technologie könnte die konventionelle Diagnostik erheblich erleichtern und bietet zudem die Möglichkeit, therapeutische Antikörper (wie z. B. anti-CD38, anti CD47, etc.), die häufig zu Interferenzen mit der Routinediagnostik führen, spezifisch prädiagnostisch aus Patientenproben zu entfernen.
Die akute myeloische Leukämie (AML) ist eine aggressive Erkrankung des Knochenmarks, welche die Hämatopoese beeinträchtigt und zu Knochenmarksversagen führt. Trotz des Fortschritts in der AML-Therapie bleibt die Prognose für die meisten Patienten schlecht, sodass neue Therapieansätze für die Behandlung dringend benötigt werden. Autophagie, ein kataboler Abbauprozess von zellulären Komponenten, ist nachweislich an der Entstehung von AML beteiligt. Als zentraler Regulator von Zellüberleben, Homöostase und Stoffwechsel, dient die Autophagie als Nährstoffquelle durch die Wiederverwertung von Makromolekülen während begrenzter Energieversorgung. AML-Zellen benötigen ein konstantes Nährstoff- und Energieniveau, um ihre Vermehrung aufrechtzuerhalten. Dies wird durch eine Umstellung von Stoffwechselwegen, insbesondere des mitochondrialen Stoffwechsels einschließlich der oxidativen Phosphorylierung (OXPHOS) und des Tricarbonsäurezyklus (TCA), erreicht.
Mehrere Studien haben die Hemmung der Autophagie für die Behandlung von Krebs als vielversprechenden Ansatz vorgestellt. Doch eine Monotherapie mit Autophagie-Inhibitoren erzielte nur eine geringfügige Wirksamkeit. Eine mögliche Erklärung hierfür ist die Entstehung von Kompensationsmechanismen, die zum Ausgleich der Autophagie-Hemmung in Krebszellen entstehen. Bis heute sind diese Kompensationsmechanismen kaum untersucht. Ziel dieser Arbeit ist es, ein geeignetes Autophagie-Gen zu identifizieren, mit dem sich die Rolle der Autophagie-Hemmung für das Überleben von AML-Zellen untersuchen lässt. Zusätzlich sollen die kompensatorischen Mechanismen, die durch die Autophagie-Hemmung in AML-Zellen entstehen können, untersucht werden, um neue metabolische Angriffspunkte zu identifizieren, die für Kombinationstherapien genutzt werden können.
Zu Beginn der Arbeit wurde ein gezielter CRISPR/Cas9 Screen in zwei humanen AML-Zelllinien durchgeführt, um Autophagie-Gene zu identifizieren, deren Verlust eine Proliferationsstörung in AML-Zellen verursacht, welche überwunden werden kann. Validierungsexperimente zeigten, dass der Verlust von ATG3 das Zellwachstum signifikant verminderte. Außerdem zeigte die Messung des Autophagie-Fluxes, dass der Verlust von ATG3 die Autophagie stark beeinträchtigte. Dies wurde durch eine Western-Blot-Analyse, die eine beeinträchtigte LC3-Lipidierung zeigte, und durch eine Immunfluoreszenzanalyse der Autophagosomen-Bildung mittels konfokaler Mikroskopie, die eine geringere Anzahl von Autophagosomen in ATG3-defizienten Zellen ergab, bestätigt. Deshalb wurde der Knockdown von ATG3 in AML Zellen verwendet, um die Mechanismen, die zum Ausgleichen der Autophagie-Hemmung entstehen, zu untersuchen. Zuerst wurde die Zellproliferation in fünf verschiedenen AML Zelllinien über sieben Tage betrachtet. In allen Zellenlinien führte der Verlust von ATG3 mittels small hairpin RNA zu verminderter Zellproliferation. Diese Ergebnisse zeigen die wichtige Rolle von ATG3 in der Autophagie und dass Autophagie-Hemmung durch ATG3-Verlust das Wachstum von AML-Zellen beeinträchtigt.
Da der Verlust von ATG3 die Proliferation von AML-Zellen beeinträchtigte, wurde eine Zellzyklusanalyse durchgeführt. Eine reduzierte S-Phase bestätigte die verminderte Proliferation in ATG3-depletierten AML-Zellen, doch der Zellzyklus war grundsätzlich nicht gestoppt. Darüber hinaus ergab die Analyse der Apoptose, dass diese unter dem Verlust von ATG3 erhöht war, aber etwa 50% der Zellen blieben vital. Diese Beobachtungen deuten darauf hin, dass AML-Zellen trotz des Verlusts der ATG3-abhängigen Autophagie weiter proliferieren können.
Um die Mechanismen zur Kompensation der Autophagie-Hemmung zu untersuchen, wurden die Auswirkungen des ATG3-Verlusts auf die mitochondriale Homöostase untersucht. Die Mitophagie sowie das mitochondriale Membranpotenzial und die Masse unterschieden sich zwischen Kontroll- und ATG3-depletierten AML-Zellen nicht, was darauf hindeutet, dass die mitochondriale Homöostase durch den Verlust von ATG3 nicht beeinträchtigt ist. Als nächstes wurde die mitochondriale Funktion durch Messung des ATP-Spiegels und der OXPHOS untersucht. Die ATP-Level und die OXPHOS waren nach dem Verlust von ATG3 in AML-Zellen erhöht, was auf eine gesteigerte mitochondriale Aktivität bei Autophagie-Defizienz hinweist.
Due to their sessile nature, plants are constantly exposed to an everchanging environment. When these changes exceed certain limits, they can significantly impact plant growth and development, which, in case of crop plants, has consequences on food security. Exposure to high temperatures causes heat stress (HS), one of the most devastating stresses that plants can face. The survival and recovery from HS are dependent on the activation of the HS response (HSR), a collection of molecular mechanisms conferring HS tolerance by maintaining the cellular homeostasis. Stress responses follow a strictly orchestrated network of signal perception and -transduction, ultimately resulting in an adaptive cellular output. Thereby, the massive reshaping of the transcriptome plays a major part, in which heat stress transcription factors (HSFs) play the key role by inducing the expression of HS-responsive genes, including heat shock proteins and other transcription factors. Additionally, alternative splicing (AS), the selective usage of splice sites, contributes to the rapid adjustment of the transcriptome landscape by producing different mRNA variants from a single gene. Consequently, this results in the reduction of translatable transcripts by nonsense-mediated mRNA-decay or nuclear retention, but also enhances the proteome diversity by allowing the synthesis of protein isoforms with distinct functions. AS thereby modulates the activity of important regulatory factors like HSFA2 in Solanum lycopersicum (tomato). HSFA2 is the key factor of acquired thermotolerance (ATT), which enables the ability to survive a potentially lethal HS through pre-exposure to a preceding mild HS. Temperature-dependent AS leads to the synthesis of two HSFA2 protein variants, whereby inhibition of splicing ensures the synthesis of the stable isoform HSFA2-I that is required for ATT.
Transcriptome analysis of several plant species exposed to HS has highlighted the strong impact of high temperatures on the regulation of pre-mRNA splicing. Despite its importance, little is known about the molecular basis of the AS regulation in plants. Particularly for an economically important crop like tomato, understanding the regulation of HS-sensitive AS will contribute to the description of such an important regulatory mechanism but also might offer new insights for increasing HS resilience. Serine/arginine-rich proteins (SR proteins) are central regulators of constitutive and AS by modulating the splice site selection by the spliceosome. This study describes two members of the RS2Z subfamily of SR proteins in tomato, namely RS2Z35 and RS2Z36, which act as core regulators of AS under HS and consequently as central factors for thermotolerance. This study investigates the interaction of the two RS2Z proteins with the HSFA2 pre-mRNA and provides evidence for their function as splicing repressors in this particular AS event. Thereby, RS2Z proteins play an important role in the HSR by modulating the AS of the key factor of the ATT. Furthermore, based on global transcriptome analysis of knockout mutants of single or both RS2Z genes, it is demonstrated that RS2Z proteins are involved in the splicing of pre-mRNAs of almost 2000 genes. Moreover, RS2Z proteins act as splicing regulators and take part in a large portion of HS-induced AS events, thus playing a broader role in AS regulation. Furthermore, the HS-induced RS2Z36 is involved in basal thermotolerance (BTT), highlighting its importance for the basic HS resilience capacity of tomato. In addition, RNA sequencing demonstrates that RS2Z proteins–especially RS2Z36–regulate the expression of proteins involved in plant immunity. The study thereby provides experimental evidence for the important and essential role of SR proteins for plant thermotolerance and suggests the existence of RS2Z-mediated crossroads of different stress responses.
This work characterizes the post-PKS modifications of AQ-256. Additionally, the second part describes the establishment of an AQ production platform for electrolyte generation that can be utilized in redox-flow-batteries. Lastly, a silent BGC that encodes the genes for terpenoid biosynthesis was described and characterized with regards to product formation and putative ecological function.
Using walls to navigate the room: egocentric representations of borders for spatial navigation
(2021)
Spatial navigation forms one of the core components of an animal’s behavioural repertoire. Good navigational skills boost survival by allowing one to avoid predators, to search successfully for food in an unpredictable world, and to be able to find a mating partner. As a consequence, the brain has dedicated many of its resources to the processing of spatial information. Decades of seminal work has revealed how the brain is able to form detailed representations of one’s current position, and use an internal cognitive map of the environment to traverse the local space. However, what is much less understood is how neural computations of position depend on distance information of salient external locations such as landmarks, and how these distal places are encoded in the brain.
The work in this thesis explores the role of one brain region in particular, the retrosplenial cortex (RSC), as a key area to implement distance computations in relation to distal landmarks. Previous research has shown that damage to the RSC results in losses of spatial memory and navigation ability, but its exact role in spatial cognition remains unclear. Initial electrophysiological recordings of single cells in the RSC during free exploration behaviour of the animal resulted in the discovery of a new population of neurons that robustly encode distance information towards nearby walls throughout the environment. Activity of these border cells was characterized by high firing rates near all boundaries of the arena that were available to the animal, and sensory manipulation experiments revealed that this activity persisted in the absence of direct visual or somatosensory detection of the wall.
It quickly became apparent that border cell activity was not only modulated by the distance to walls, but was contingent on the direction the animal was facing relative to the boundary. Approximately 40% of neurons displayed significant selectivity to the direction of walls, mostly in the hemifield contra-lateral to the recorded hemisphere, such that a neuron in left RSC is active whenever a wall occupies proximal space on the right side of the animal. Using a cue-rotation paradigm, experiments initially showed that this egocentric direction information was invariant to the physical rotation of the arena. Yet this rotation elicited a corresponding shift in the preferred direction of local head-direction cells, as well as a rotation in the firing fields of spatially-tuned cells in RSC. As a consequence, position and direction encoding in RSC must be bound together, rotating in unison during the environmental manipulations, as information about allocentric boundary locations is integrated with head-direction signals to form egocentric border representations.
It is known that the RSC forms many anatomical connections with other parts of the brain that encode spatial information, like the hippocampus and para-hippocampal areas. The next step was to establish the circuit mechanisms in place for RSC neurons to generate their activity in respect to the distance and direction of walls. A series of inactivation experiments revealed how RSC activity is inter-dependent with one of its communication partners, the medial entorhinal cortex (MEC). Together they form a wider functional network that encodes precise spatial information of borders, with information flowing from the MEC to RSC but not vice versa. While the conjunction between distance and heading direction relative to the outer walls was the main driver of neural activity in RSC, border cells displayed further behavioural correlates related to movement trajectories. Spiking activity in either hemisphere tended to precede turning behaviour on a short time-scale in a way that border cells in the right RSC anticipated right-way turns ~300 ms into the future.
The interpretation of these results is that the RSC’s primary role in spatial cognition is not necessarily on the early sensory processing stage as suggested by previous studies. Instead, it is involved in computations related to the generation of motion plans, using spatial information that is processed in other brain areas to plan and execute future actions. One potential function of the RSC’s role in this process could be to act correctly in relation to the nearby perimeter, such that border cells in one hemisphere are involved in the encoding of walls in the contralateral hemifield, after which the animal makes an ipsilateral turn to avoid collision. Together this supports the idea that the MEC→RSC pathway links the encoding of space and position in the hippocampal system with the brain’s motor action systems, allowing animals to use walls as prominent landmarks to navigate the room.
The main aim of this thesis work was to elucidate the catalytic mechanism of several enzyme complexes on the basis of their three-dimensional structure. All investigated enzyme complexes occur in the anaerobic energy metabolism and have an essential function by the challenging degradation of aromatic compounds and the flavin-based electron bifurcation (FBEB)/confurcation, an energy-coupling mechanism. More specifically, I studied the phthaloyl-CoA decarboxylase of Thauera chlorobenzoica (Pcd) involved in phthalate ester decomposition, the FBEB protein complexes lactate dehydrogenase/electron-transfer flavoprotein (Ldh/EtfAB) of Acetobacterium woodii, the heterodisulfide-related subunit HdrA of the sulfur- oxidizing bacteria Hyphomicrobium denitrificans (sHdrA). In addition, I contributed to the structure determination of the caffeyl-CoA reductase- EtfAB complex of A. woodii and the naphthoyl-CoA reductase of the sulfate-respiring enrichment culture N47 (mentioned in the Appendix E and F).
This work addresses the investigation of the biosynthesis mechanisms of type II polyketide synthase (PKS) and fatty acid synthase (FAS) derived specialized metabolites (SMs) from Photorhabdus laumondii.
The elucidation of the biosynthetic pathway of the bacterial 3,5-dihydroxy-4-isopropyl-trans-stilbene (IPS) was one of the major topics of this thesis. IPS exhibits several bioactive characteristics as it inhibits the phenoloxidase of insects, acts antibacterial, but also influences the soluble epoxide hydrolase which is involved in inflammatory reactions. It was recently approved as a treatment against psoriasis by the FDA and is the first Photorhabdus derived drug.
The stilbene generation in Photorhabdus requires the formation of the two acyl-carrier-protein (ACP) bound 5-phenyl-2,4-pentadienoyl- and isovaleryl-β-ketoacyl-moieties. The ketosynthase (KS)/cyclase StlD catalyzes a ring formation via a Michael-addition between the two intermediates which is then further processed by an aromatase. The formation of 5-phenyl-2,4-pentadienoyl-ACP was shown via in vitro assays with purified proteins by proving the influence of the KS FabH, ketoreductase FabG and dehydratase FabA or FabZ of the fatty acid metabolism. While E. coli was able to complement most of these enzymes in attempts to produce IPS in the heterologous host, the Photorhabdus derived FabH was not replaceable despite 73 % sequence identity with the E. coli based isoenzyme, acting as a gatekeeper enzyme for cinnamic acid (CA) moieties. Furthermore, the ability to incorporate meta-substituted halogenated CA-derivatives was shown in order to produce 3-chloro- and 3-bromo-IPS. While studying the stilbene biosynthesis, the ability of Photorhabdus and Xenorhabdus to produce hydrazines was also discovered.
The second investigated biosynthesis was the formation of benzylideneacetone (BZA). BZA is produced by Photorhabdus and Xenorhabdus strains acting as a suppressor for the immune cascade of insects and has also antibiotic activities towards Gram-negative bacteria. Due to its structural similarity towards CA and the intermediates during the stilbene formation, a shared mechanism for Photorhabdus and Xenorhabdus budapestensis was proposed due to their ability to produce CA. The production of BZA was also dependent on the stilbene related CoA-ligase, the ACP and FabH. It was verified in vitro and in vivo in E. coli yielding a 150-fold increase of the BZA production compared to the Photorhabdus and Xenorhabdus wildtype (WT) strains.
The second part of this work deals with the optimization of P. laumondii strains regarding the production titers of IPS. Therefore, several deletions of other SM related genes as well as promoter exchanges in front of stilbene related genes were carried out. These approaches were combined with the upregulation of the phenylalanine by heterologous plasmid expression, since it is the precursor of CA. Another approach applied in parallel was the optimization of the cultivation conditions with different media and supplementation with XAD-resins. It was proved that media rich on fatty acids or peptides led to higher optical densities of the cultures and thus to higher titers of stilbenes. Since IPS is inhibiting the phenoloxidase, an enzyme important for the insect immunity, it was hypothesized that cultivation in media containing insects might enhance the output of this SM. Starting from 23 mg/l of IPS in the P. laumondii WT strain, it was possible to increase the production levels to more than 860 mg/l by utilizing the mentioned approaches.
The last topic of this thesis focuses on the production of epoxidated IPS (EPS) and its derivatives. Under laboratory conditions, only a low titer of EPS was observed for the wildtype strain. However, the optimized IPS strains and IPS-production conditions could also be applied for EPS which led to higher productions and also to the detection of many new derivatives. Most of the EPS derivatives were amino acid or peptide derived acting as nucleophiles to open the epoxide ring and yielding β-amino-alcohols. However, purification and chemical synthesis attempts to obtain EPS failed due to its poor stability. Epoxides were utilized in in vitro assays with amino acids, peptides and proteins to get insights whether epoxidations might act as posttranslational modification in Photorhabdus. The reactions were performed with styrene oxide and stilbene oxide replacing EPS based on their structural similarity. The modifications were executed successfully although proteomics approaches with in vivo data are required to confirm these findings. During the purification attempts of EPS, further derivatives were detected. The structures of dimerized stilbenes, a cis-isomer of IPS and another derivative that might incorporate an amino-group in the resveratrol ring were proposed on the basis of the HPLC-MS data.
Heart development is a dynamic process modulated by various extracellular and intracellular cues. Cardiac progenitors in vertebrates such as the zebrafish, migrate over to the midline after differentiation from the epiblast (Bakkers, 2011; Rosenthal & Harvey, 2010; Stainier et al., 1996; Trinh & Stainier, 2004). These progenitors form a cardiac disc at the midline which elongates into the linear heart tube. The differentiation and migration of cardiac precursors is modulated by signaling interactions between cardiac precursor cells and their extracellular environment known as the Extracellular Matrix (ECM). Studies have shown that Cell-ECM interactions play a crucial role in sculpting the heart during early morphogenic events (Davis CL, 1924; Männer & Yelbuz, 2019; Rosenthal & Harvey, 2010). One key factor to these processes is the presence of a specialized ECM known as the Basement Membrane (BM). Extracellular basement membrane proteins such as Fibronectin have been shown to modulate these very early migration processes of the cardiomyocyte progenitors (Trinh & Stainier, 2004). As the heart develops further, the linear heart tube is composed of myocardial cells with an inner endothelial cell lining separated by a layer of thick jelly like substance called the cardiac jelly (Barry A, 1948; Davis CL, 1924; Little et al., 1989). The cardiac jelly also called the cardiac basement membrane, has been shown to regulate distinct developmental events during cardiogenesis. This early CJ contains components of the basal lamina such as laminins, fibronectin, hyaluronan as well as non-fibrillar collagens such as Collagen IV (Little et al., 1989). In this study, I aimed to identify ECM molecules of the Basement Membrane in the heart and identify their role in the modulation of cardiac development and regeneration using the zebrafish as my model organism.
I identified genes belonging to the Zebrafish Matrisome expressed during cardiac developmental and regeneration and performed CRISPR/Cas9 sgRNA mediated mutagenesis. I also developed overexpression tools for these genes.
Agrinp168 mutants exhibited no obvious gross morphology defects during cardiac development and were adult viable. Adult mutants exhibited reduced cardiomyocyte proliferation, but no significant difference in cardiomyocyte dedifferentiation post cardiac cryoinjury.
Decorin overexpression through mRNA injections led to increased myocardial wall thickness and DN dcn overexpression through mRNA injections led to loss of cardiac looping during early development.
Mutants for Small Leucine Rich Proteoglycan (SLRP) prelp generated using CRISPR/Cas9 mutagenesis exhibited cardiovascular defects. Close observation of prelp mutant hearts revealed a reduced heart rate and impaired fractional shortening of the ventricle. prelp mutants exhibited an enlarged atrium at 48 hpf and 72 hpf as well as a reduced ventricle size at 72 hpf. Chamber size in the mutant hearts were enlarged irrespective of contractility of the heart. Mutants showed an increased number of Atrial cardiomyocytes, but no change in cell size. On the molecular level, extracellular Laminin localization was disrupted in prelp mutants along with an increase in thickness and volume of the cardiac HA in the CJ suggesting a potential compensatory role, or retention of immaturity of the cardiac jelly in the prelp mutants. Transcriptomics analysis on the prelp mutant hearts revealed downregulation of ECM organization and ECM-Receptor interaction processes in the mutants. Gene Ontology analysis on prelp mutants hearts transcriptome revealed increased MAPK signaling. Interestingly, genes related to degradation of cardiac HA and maturation of cardiac jelly were downregulated, and genes related to epithelial identity of cardiomyocytes were upregulated. Analysis of the mutant hearts at single cell resolution revealed increased number of mutants exhibiting rounded up cardiomyocytes and loss of apical Podocalyxin. Truncated forms of prelp were generated to identify domain specific roles for Prelp, and reintroduction of N-terminal truncated Prelp into the mutants rescued the basal lamina localization and cardiac jelly volume phenotypes. Myocardium specific re-establishment of prelp expression revealed a marked rescue of the mutant cardiovascular phenotype suggesting that tissue specific expression of prelp is not required so long as Prelp is secreted into the CJ. With these data, I’ve elucidated the role of ECM SLRPs in modulation of cardiac chamber morphogenesis process and regeneration of the heart.
Untersuchungen zur Bedeutung selektiver Autophagie für Alterungsprozesse von Podospora anserina
(2022)
Das Ziel der vorliegenden Arbeit war, die Funktion und die Rolle von Autophagie-assoziierten Proteinen im Alternsmodell Podospora anserina zu untersuchen und einen Einblick in die nicht-selektive Autophagie, die Mitophagie und die Bildung und den Abbau von Autophagosomen im Zusammenhang zur Alterung von P. anserina zu analysieren. Dabei wurden folgende Erkenntnisse erhalten:
1. Die Untersuchungen zu ΔPaAtg8 bestätigen, dass die PaATG8-abhängige Autophagosomenbildung zur Aufrechterhaltung der Lebensspanne benötigt wird. In ΔPaAtg8 kommt es zu einem Verlust der nicht-selektiven Autophagie. Die Mitophagie hingegen ist auch ohne PaATG8 partiell möglich und es liegt ein PaATG8-unabhängiger Abbau von mitochondrialen Proteinen in P. anserina vor.
2. In P. anserina ist PaATG11 an der nicht-selektiven Autophagie beteiligt und auch die Mitophagie erfolgt in Abhängigkeit dieses Gerüstproteins. Während der PaAtg11-Deletionsstamm unter Normalbedingungen keinen zum Wildtyp veränderten Phänotyp zeigt, führt eine Kultivierung auf M2-Medium mit Glycerin als einziger Kohlenstoffquelle zu einer starken Verkürzung der Lebensspanne. Eine mikroskopische Untersuchung der Mitochondrien zeigte, dass im juvenilen Altersstadium von ΔPaAtg11 stark fragmentierte Mitochondrien vorliegen. Während der Alterung normalisiert sich die Mitochondrienmorphologie wieder. Der mitochondriale Funktionsverlust wird möglicherweise von den fragmentierten Mitochondrien ausgelöst, denn eine Kultivierung von älteren ΔPaAtg11-Stämmen auf M2-Medium mit Glycerin führt zu einer Normalisierung der Lebensspanne.
3. Die initialen Untersuchungen zur ΔPaAtg11/ΔPaAtg24-Doppelmutante zeigen, dass es bei der Kultivierung unter Normalbedingungen zu einem additiven Effekt der beiden Genverluste kommt. Bei der Anzucht auf M2-Medium mit Glycerin hingegen kann eine im Vergleich zum ΔPaAtg11-Stamm längere Lebensspanne festgestellt werden. Die Mikroskopie der Mitochondrien in ΔPaAtg11/ΔPaAtg24 zeigt, dass im juvenilen Alter zum Wildtyp vergleichbare filamentöse Mitochondrien vorhanden sind.
4. In P. anserina ist PaATG24 kein Mitophagierezeptorprotein, da im PaAtg24-Deletionsstamm eine Beeinträchtigung der nicht-selektiven Autophagie vorliegt. Auch die Mitophagie ist in diesem Stamm geschädigt. Die mikroskopische Betrachtung der Mitochondrien zeigt keinen Unterschied zum Wildtyp. Bei der Untersuchung zur Mitochondrienfunktion durch M2-Medium mit Glycerin ist wie unter Normalbedingungen eine verkürzte Lebensspanne feststellbar.
5. Der Abbau von GFP::PaATG8 ist in der PaAtg24-Deletionsmutante signifikant verringert und es kommt zu einer Akkumulation von Autophagosomen, somit liegt in diesem Stamm eine Beeinträchtigung des autophagosomalen Flusses vor. Bei der mikroskopischen Untersuchung von PaATG24 zeigt sich, dass dieses Protein in P. anserina im Bereich der Vakuolen lokalisiert ist. Die Analyse der Vakuole-Autophagosomen-Fusion zeigt jedoch, dass dieser Mechanismus unabhängig von PaATG24 ist. Die Vakuolenmorphologie und Vakuolengröße ist in ΔPaAtg24 beeinträchtigt und dadurch kommt es zu dem beobachteten Defekt der nicht-selektiven und selektiven Autophagie.
Mutational analysis of ribosomal DNA and maturation-scheme analysis of ribosomal RNA in A. thaliana
(2022)
Ribosome biogenesis is a fundamental cellular process beginning with long precursor rRNA transcription from multi-copies of repetitive 45S ribosomal DNAs. At the subunit level, the primary pre-rRNA transcript encapsuled in 90S protein-RNA complex undergoes decisive splitting in two chief ways for further maturation into large (LSU) and small (SSU) ribosomal subunit. The usage of specific rDNA copies from defined chromosomes and their selective role during growth and development have been a topic of interest owing to its contribution to specialized ribosome theory which proposes non-monolithic functions for ribosomes and thereby their mRNA translation potential. Dual-guide CRISPR/Cas9 mediated disruption of rDNA regions resulted in stable disruption of up to 2.5% and 5% of all rDNA copies in hetero- and homozygous (ploop KD) conditions, respectively. At the RNA level, the mutation excised a critical structural element, P-loop on the LSU 25S rRNA. Mutation caused a dosage dependent defect with homozygosity leading to severe developmental defects through vegetative and reproductive growth phases which is manifested in their proteome by means of disregulation through both increase and decrease of several gene ontological categories of proteins in mutants. Interestingly, the mutation on chromosome 4 triggered dosage compensation through rRNA expression from chromosome 2 further compounded by ectopic rRNA biogenesis defects. The mutated copies however are not incorporated in the translating ribosomes and as a direct or indirect consequence led to elevated basal autophagic levels in the mutants.
The primary 35S transcript is known to undergo two modes of initial cleavages at the pre-rRNA level that aid in their subsequent maturation. Root cell culture (RCC) studies shows that these cells contain a novel ITS2-first cleaved precursor even under control growth conditions, P-C2 adding a third maturation means for the 35S pre-rRNA. This maturation path is further known to be triggered under elevated growth temperature forming a novel adaptive response in Arabidopsis and two other crop plants, tomato, and rice. Taken together, the pulse-chase labeling analysis of control and stressed tissues uncovers the fine-tuned pre-rRNA schematics with crossovers between multiple maturation paths.
A promising strategy to reduce the dependency from fossil fuels is to use the yeast Saccharomyces cerevisiae to bioconvert renewable non-food feedstocks or waste streams, like lignocellulosic biomass, into bioethanol and other valuable molecule blocks. Lignocellulosic feedstocks contain glucose and significant fractions of the pentoses xylose and arabinose in varying proportions depending on the biomass type. S. cerevisiae is an efficient glucose consumer, but it cannot metabolize xylose and arabinose naturally. Therefore, extensive research using recombinant DNA techniques has been conducted to introduce and improve the biochemical pathways necessary to utilize these non-physiological substrates. However, any functional pathway capable of metabolizing D xylose and L arabinose in S. cerevisiae requires the transport of these sugars across the plasma membrane. The endogenous sugar transport system of S. cerevisiae can conduct a limited uptake of D-xylose and L-arabinose; this uptake enables only basal growth when the enzymatic pathways are provided. For this reason, the uptake of D xylose and L-arabinose has been recognized as a limiting step for the efficient utilization of these non-physiological substrates.
Gal2, a member of the major facilitator superfamily, is one of the most studied hexose transporters in S. cerevisiae. Although its expression is repressed in the presence of glucose, it also transports this sugar with high affinity when constitutively expressed. Recent efforts to engineer yeast strains for the utilization of plant biomass have unraveled the ability of Gal2 to transport non-physiological substrates like xylose and arabinose, among others. Improving Gal2 kinetic and substrate specificity, particularly for pentoses, has become a crucial target in strain engineering. The main goal of this study is to improve the utilization of xylose and arabinose by increasing the cell permeability of these non physiological substrates through the engineering of the galactose permease Gal2.
GAL2 gene expression depends on galactose, which acts as an inducer; nevertheless, even in the presence of galactose, glucose act as a strict repressor; consequently, GAL2 gene is usually placed under the control of a constitutive promoter. However, the presence of glucose additionally triggers the Gal2 degradation, which is mediated by the covalent attachment of the small 76 amino acid protein ubiquitin (Ub) to the targeted transporter; in a multi-step process called ubiquitination.
Ubiquitination of hexose permeases involves the activation of the Ub molecule by the E1 Ub-activating enzyme using ATP; then, the activated Ub is transferred to a specific Ub-conjugating enzyme E2, which donates the Ub indirectly through a specific HECT E3 enzyme (Rsp5) to a lysine residue of the substrate, with the aid of an adaptor protein which recognizes the target (Rsp5-adaptor). Ubiquitinated permeases are sent by membrane invagination to early endosomes, where they encounter ESCRTs (endosomal sorting complex required for transport). The targeted permeases are sorted in intralumenal vesicles (ILV) inside of the endosome, which after several cycles, turns into a multivesicular body (MVB) that subsequently fuses with the vacuole to expose the protein content of the ILVs to lumenal hydrolases for degradation.
Gal2 contains 30 lysine residues that may accept the ubiquitin molecule, which targets its degradation. It is known that mono-ubiquitination by Rsp5 on multiple lysine residues is necessary to internalize Gal2 (Horak & Wolf, 2001). However, the authors did not identify the specific lysine residues involved in the ubiquitination processes. This study screened several Gal2 variants where lysine residues were mutated or removed from the protein sequence to discover which lysine residues are likely involved in ubiquitination and consequent turnover of the transporter. The results of the screening showed that mutation of the N terminal lysine residues 27, 37, and 44 to arginine (Gal23KR) produced a functional transporter that, when fused with GFP (Gal23KR_GFP), showed an exclusive localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b).
This study furthermore evaluated upstream signals caused by phosphorylation which triggers ubiquitination and consequent turnover of the targeted protein; using similar screening approaches to assess the stabilization of Gal2 by lysine residue modifications, it was possible to identify that N terminal serine residues 32, 35, 39, 48, 53, and 55 are likely involved in the internalization of Gal2, since a Gal2 construct where all these serines were mutated to alanine residues and tagged with GFP (Gal26SA_GFP) exhibited practically complete localization at the plasma membrane in cells growing in galactose or glucose as a sole carbon source (Tamayo Rojas et al., 2021b)...
Die oxygene Photosynthese bildet den Grundpfeiler des heutigen Ökosystems unseres Planeten. Neben den gut untersuchten Landpflanzen bilden Mikroalgen eine äußerst bedeutende Organismengruppe der phototrophen Lebewesen. Zu den Mikroalgen zählen die Diatomeen, welche sich beispielsweise durch eine Silikatschale und spezielle Lichtsammelkomplexe auszeichnen und für einen Großteil der marinen Primärproduktion verantwortlich sind. Die stoffwechselphysiologischen Grundlagen des ökologischen Erfolgs der Kieselalgen sind bislang noch unzureichend erforscht. Ein Vertreter der zentrischen Diatomeen, Cyclotella, wurde bereits zur Jahrtausendwende zur biochemischen Charakterisierung der Diatomeen Photosynthese verwendet (Eppard und Rhiel, 1998; Eppard und Rhiel, 2000), das Genom des Organismus aber erst vor kurzem sequenziert (Traller et al., 2016). Die Sequenzierung des Genoms konnte einige Gene für Lichtsammelproteine identifizieren, die Homologie zu den LhcSR-Proteinen aus C. reinhardtii aufweisen, welche nachweislich eine photoprotektive Funktion besitzen (Peers et al., 2009). Diese sogenannten Lhcx-Proteine der Diatomeen sind in den zwei Gruppen der Kieselalgen, den zentrischen und pennaten Diatomeen zu finden, unterscheiden sich aber in ihren jeweiligen Lhcx-Kandidaten. So können in der pennaten Diatomee P. tricornutum vier lhcx-Gene ausgemacht werden, während die zentrische Kieselalge T. pseudonana sechs lhcx-Gene besitzt und C. cryptica vier verschiedene lhcx-Kandidaten genomisch aufweist (Armbrust et al., 2004; Bowler et al., 2008; Traller et al., 2016). Die beschriebenen Diatomeen weisen alle eine Homologie im Lhcx1 auf, während sich die übrigen Lhcx-Kandidaten zwischen pennaten und zentrischen Diatomeen unterscheiden. Ein zwischen T. pseudonana und C. cryptica konserviertes Lhcx ist das Lhcx6_1, welches 2011 das erste Mal massenspektrometrisch an Photosystemen von T. pseudonana nachgewiesen wurde (Grouneva et al., 2011) und in weiteren Massenspektrometrie-gestützten Untersuchungen in beiden zentrischen Diatomeen an Photosynthese-Komplexen gefunden werden konnte (Gundermann et al., 2019; Calvaruso et al., 2020). Die Funktion des Lhcx6_1 ist bislang unklar.
Diese Arbeit konnte das Lhcx6_1 aus C. meneghiniana charakterisieren und Antikörper-gestützt genauer lokalisieren, eine nicht dynamische Phosphorylierung der Thylakoidmembran-Proteine der zentrischen Diatomee nachweisen und die molekularbiologische Zugänglichkeit des Organismus optimieren. qRT-PCR gestützte Expressions-Analysen konnten eine unerwartete Expression des lhcx6_1-Gens aufdecken. Dieses weist, im Vergleich zum Lhcx1, keine Starklicht induzierte Expression auf. Die Expression des Gens konnte nach wenigen Stunden Schwachlicht als maximal bestimmt werden, während sie im Starklicht abnimmt. Das Muster der Genexpression glich im Schwachlicht eher der des lhcf1-Gens. Die Sequenzierung des lhcx6_1 aus C. meneghiniana identifizierte eine verlängerte N-terminale Sequenz des Proteins, welche Homologie zu den minoren Antennen aus A. thaliana besitzt und Teil des reifen Proteins ist. Mittels eines C-terminalen Epitops wurde ein Antikörper gegen das Lhcx6_1 entworfen, welcher das Protein in C. meneghiniana spezifisch nachweisen kann. Die Isolation von Thylakoidmembranen der zentrischen Diatomee und weitergehende Aufreinigung mittels Saccharosedichtegradienten und lpBN-PAGE konnten die Lokalisation des Lhcx6_1 eingrenzen. Das Protein zeigt dabei keine Unterschiede in seiner Lokalisation nach Inkubation in Schwach-, Stark- und Fernrot-Licht und ist vorrangig mit Photosystem I assoziiert. In geringerer Menge konnte es zudem an Photosystem II nachgewiesen werden, während der immunologische Nachweis in Lichtsammelkomplexen (FCPs) minimale Mengen erbrachte. Ferner konnte eine Phosphorylierung des Lhcx6_1 an Threonin-Resten nachgewiesen werden, während die meisten anderen Thylakoidmembran-Proteine mittels Phospho-Serin Antikörper detektiert werden konnten. Weder die Phosphorylierung des Lhcx6_1, noch der anderen Thylakoidmembran-Proteine, zeigt eine dynamische Regulation, im Stile einer state-transition ähnlichen Kinase auf. Die Qualität des Umgebungslichts führte zu keinerlei Unterschieden in Phosphorylierungsmustern. Weiterführende Untersuchungen der Lhcx6_1-Phosphorylierung mittels Phos-tag PAGE identifizieren eine unphosphorylierte und eine einfach phosphorylierte Form des Proteins. Dabei kann an PSI ausschließlich die phosphorylierte Version des Lhcx6_1 gefunden werden. Im Zuge der Arbeit konnte zudem erstmalig die Elektroporation und Konjugation für C. meneghiniana als Transformations-Methoden etabliert werden, während das Protokoll für die biolistische Transformation optimiert wurde. Die Elektroporation erbrachte die höchste Transformationseffizienz. Molekularbiologische Unterfangen eines Lhcx6_1-Knockdowns mittels Antisense-RNA erzielten zunächst, aufgrund der starken Gegenregulation der Diatomee, keinen Erfolg...
Regulatory required, classical toxicity studies for environmental hazard assessment are costly, time consuming, and often lack mechanistic insights about the toxic mode of action induced through a compound. In addition, classical toxicological non-human animal tests raise serious ethical concerns and are not well suited for high throughput screening approaches. Molecular biomarker-based screenings could be a suitable alternative for identifying particular hazardous effects (e.g. endocrine disruption, developmental neurotoxicity) in non-target organisms at the molecular level. This, however, requires a better mechanistic understanding of different toxic modes of action (MoA) to describe characteristic molecular key events and respective markers.
Ecotoxicgenomics, which uses modern day omic technologies and systems biology approaches to study toxicological responses at the molecular level, are a promising new way for elucidating
the processes through which chemicals cause adverse effects in environmental organisms. In this context, this PhD study was designated to investigate and describe MoA-characteristic
ecotoxicogenomic signatures in three ecotoxicologically important aquatic model organisms of different trophic levels (Danio rerio, Daphnia magna and Lemna minor).
Applying non-target transcriptomic and proteomic methodologies post chemical exposure, the aim was to identify robust functional profiles and reliable biomarker candidates with potential
predictive properties to allow for a differentiation among different MoA in these organisms. For the sublethal exposure studies in the zebrafish embryo model (96 hpf), the acute fish embryo toxicity test guideline (OECD 236) was used as conceptual framework. As different test compounds with known MoA, the thyroid hormone 3,3′,5-triiodothyronine (T3) and the thyrostatic 6-propyl-2-thiouracil (6-PTU), as well as six nerve- and muscle-targeting insecticides (abamectin, carbaryl, chlorpyrifos, fipronil, imidacloprid and methoxychlor) were evaluated. Furthermore, a novel sublethal immune challenge assay in early zebrafish embryos (48 hpf) was evaluated for its potential to assess immuno-suppressive effects at the gene expression level. Therefore, toxicogenomic profiles after an immune response inducing stimulus with and without prior clobetasol propionate (CP) treatment were compared. For the aquatic invertebrate D. magna, the study was performed with previously determined low effect concentrations (EC5 & EC20) of fipronil and imidacloprid according to the acute immobilization test in water flea (OECD 202). The aim was to compare toxicogenomic signatures of the GABA-gated chloride channel blocker (fipronil) and the nAChR agonist (imidacloprid). With similar low effect concentrations, a shortened 3 day version of the growth inhibition test with L. minor (OECD 221) was conducted to find molecular profiles differentiating between photosynthesis and HMG-CoA reductase inhibitory effects. Here, the biological interpretation of the molecular stress response profiles in L. minor due to the lack of functional annotation of the reference genome was particularly challenging. Therefore, an annotation workflow was developed based on protein sequence homology predicted from the genomic reference sequences.
With this PhD work, it was shown how transcriptomic, proteomic and computational systems biology approaches can be coupled with aquatic toxicological tests, to gain important mechanistic insights into adverse effects at the molecular level. In general, for the different investigated adverse effects for the different organisms, biomarker candidates were identified, which describe a potential functional link between impaired gene expressions and previously reported apical effects. For the assessed chemicals in the zebrafish embryo model, biomarker candidates for thyroid disruption as well as developmental toxicity targeting the heart and central nervous system were described. The biomarkers derived from nerve- and muscletargeting insecticides were associated with three major affected processes: (1) cardiac muscle cell development and functioning, (2) oxygen transport and hypoxic stress and (3) neuronal development and plasticity. To our knowledge, this is the first study linking neurotoxic insecticide exposure and affected expression of important regulatory genes for heart muscle (tcap, actc2) and forebrain (npas4a) development in a vertebrate model. The proposed immunosuppression assay found CP to affect innate immune induction by attenuating the response of genes involved in antigen processing, TLR signalling, NF-КB signalling, and complement activation ...
Lipopolysaccharide (LPS) is a major glycolipid component in the outer leaflet of the outer membrane of Gram-negative bacteria and known as endotoxin exhibited by the lipid A moiety, which serves as a membrane anchor. The effective permeability barrier properties of the outer membrane contributed by the presence of LPS in the extracellular layer of the outer membrane confer Gram-negative bacteria a high resistance against hydrophobic compounds such as antibiotics, bile salts and detergents to survive in harsh environments. The biogenesis of LPS is well studied in Escherichia coli (herewith E. coli) and the LPS transport (Lpt) is carried out by a transenvelope complex composed of seven essential proteins (LptABCDEFG), which are located in the three compartments of the cell such as the outer membrane, the inner membrane and the periplasm. The Lpt system also exists in Anabaena sp. PCC 7120 (herewith Anabaena sp.), however, homologues of LptC and LptE are still missing. BLAST search failed to identify a homologue of LptC, in contrast, the secondary structure analysis using the Pfam database based on the existing ecLptC secondary structure identified one open reading frame All0231 as the putative Anabaena sp. homologue of LptC, which is designated anaLptC. Despite the low sequence similarity, the secondary structure alignment between anaLptC and ecLptC using the HHpred server showed that both proteins share high secondary structural similarities. The genotypic analysis of the insertion mutant anaLptC did not identify a fully segregated genome and its phenotypic analysis revealed that it was sensitive against chemicals, suggesting that the analptC gene is essential for the growth of Anabaena sp. and involved in the outer membrane biogenesis. This is further supported by the observation of the small cell phenotype in the anaLptC mutant via transmission electron microscopy. Moreover, physical interactions between the anaLptC periplasmic domain with anaLptA as well as with anaLptF were established, indicating that the anaLptC periplasmic domain is correctly folded and alone functional and that the transmembrane helix is not required for the interaction with anaLptA and anaLptF. Furthermore, the reduction of the O-antigen containing LPS was observed in the insertion mutant anaLptC and the dissociation constant Kd of the anaLptC periplasmic domain for ecLPS was determined.The three-dimensional structure of the periplasmic domain of anaLptC was solved by X-ray crystallography with a resolution of 2.8 Å. The structural superposition between the ecLptC crystal structure (PDB number 3my2) and the crystal structure of anaLptC periplasmic domain obtained by this study showed the similarity in the folding of the two proteins with a Cα r.m.s.d value of about 1 Å and confirmed that the length of anaLptC is more than two times longer than that of ecLptC. The structural comparison also revealed that both structures share the typical β-jellyroll fold and conserved amino acids, which were shown in ecLptC to bind to LPS in vivo and found in anaLptC. Overall, these data strongly suggest that anaLptC is involved in the transport of LPS and support the model whereby the bridge spanning the inner membrane and the outer membrane would be assembled via interactions of the structurally conserved β-jellyroll domains shared by five (LptACDFG) out of seven Lpt proteins.
Mitglieder der ubiquitär verbreiteten Cryptochrom-Photolyase-Familie sind Blaulicht-absorbierende Flavoproteine mit hoher Sequenzhomologie aber diversen Funktionen. Photolyasen katalysieren die Reparatur UV-Licht-induzierter DNA-Schäden. Cryptochrome (CRYs) wirken als lichtunabhängige Transkriptionsrepressoren innerhalb des Kern-Oszillators der circadianen Uhr oder als primäre Photorezeptoren zur Synchronisation dieser mit dem äußeren Tag-Nacht-Rhythmus und steuern durch Regulation der Genexpression Wachstum und Entwicklung. Gemeinsames Strukturmerkmal aller CPF-Vertreter ist die Photolyase- homologe Region (PHR), die das Chromophor Flavinadenindinukleotid (FAD) bindet, das lichtabhängig zwischen den Redoxformen oxidiert (FADox), semireduziert (FAD●- bzw. FADH●) und vollreduziert (FADH-) wechseln kann und damit die CRY-Konformation und -Aktivität beeinflusst. Unterscheidungsmerkmale sind die spezifische C-terminale Erweiterung (CTE) sowie die Komposition der FAD-Bindetasche, die unterschiedliche FAD-Redoxformen stabilisiert. Die Mechanismen der CRY-Photosignaltransduktion sind nicht völlig erforscht.
CryP ist eines von vier CRYs in der Diatomee Phaeodactylum tricornutum und gehört zur bislang nicht charakterisierten Gruppe pflanzenähnlicher CRYs. In vorhergehenden Untersuchungen wurde für CryP eine nukleare Lokalisation und damit verbunden eine blaulicht- sowie dunkelabhängige Regulation der Transkription unterschiedlichster Gene gezeigt. Zudem reguliert CryP das Proteinlevel photosynthetischer Lichtsammelkomplexe. CryP interagiert mit bisher nicht charakterisierten Proteinen aus dem Bereich DNA und Regulation sowie Ribosomen und Translation. Heterolog exprimiertes und isoliertes CryP stabilisiert das Neutralradikal FADH● und das Antennenchromophor Methenyltetrahydrofolat (MTHF).
In vorliegender Dissertation wurde die Bedeutung des FAD-Redoxzustands und der C-terminalen Proteindomäne für Strukturänderungen hinsichtlich der Oligomerisierung und Konformation sowie für das CryP-Interaktionsverhalten untersucht. Hierzu wurden rekombinante CryP-Varianten heterolog isoliert, die Mutationen in für die FAD-Reduzierbarkeit entscheidenden Aminosäuren oder eine Deletion der CTE tragen.
Die Analyse der CryP-Oligomerisierungsstufe und Konformation erfolgte mittels Ko-Präzipitation, nativen und zweidimensionalen PAGEs sowie partieller Proteolyse. Dabei wurde heterolog isoliertes CryP in seinen drei Redoxformen oxidiert (mit FADox), semireduziert (mit FADH●) und vollreduziert (mit FADH-) sowie das um die CTE-verkürzte CryP-PHR verglichen. Für CryP wurde eine redoxunabhängige, PHR-vermittelte Di- und Tetramerisierung über elektrostatische Wechselwirkung der Monomere beobachtet. Die CTE bindet spezifisch und redoxunabhängig an die PHR in einem Bereich um die FAD-Bindetasche. Dies schließt eine großräumige Konformationsänderung zwischen PHR und CTE infolge einer FAD-Photoreduktion wie für pflanzliche und viele tierische CRYs als Aktivierungsmechanismus für CryP aus.
Interaktionsstudien mittels zweidimensionaler PAGE gaben Aufschluss über unterschiedliche Bindeverhalten der beiden betrachteten Interaktionspartner an CryP. Sowohl BolA, ein potentieller redoxregulierter Transkriptionsfaktor, als auch ID42612 mit unbekannter Funktion interagieren mit CryP unabhängig von der FAD-Redoxform. Dabei bindet BolA an die CTE des CryP-Dimers und -Monomers, während ID42612 einen Komplex mit dem CryP-Dimer bildet.
Mittels in vitro Absorptions- und Fluoreszenzspektroskopie wurde die FAD-Redoxchemie von CryP und CryP-PHR verglichen. Die beiden Varianten unterscheiden sich in der FAD-Photoreduzierbarkeit und -Oxidationskinetik. Das Volllängenprotein CryP kann ohne externes Reduktionsmittel zum semireduzierten FADH● phototreduziert werden, das im Gegensatz zu bekannten CRYs über Tage im Dunkeln stabil gegen aerobe Oxidation ist. Eine Belichtung mit Reduktionsmittel führt zur Bildung des vollreduzierten FADH-, das innerhalb von Minuten zu FADH● rückoxidiert. Das um die CTE verkürzte CryP-PHR kann nur mit externem Reduktionsmittel zu FADH● photoreduziert werden, der vollreduzierte Zustand wird nie erreicht. Die Stabilisierung von FADH● gegen aerobe Oxidation im CryP-Holoprotein ist vergleichbar zur FAD-Redoxchemie von Photolyasen. Verglichen mit sonstigen charakterisierten CRYs ist die Wichtigkeit der CTE für eine effiziente FAD-Photoreduktion und FADH●-Stabilisierung eine CryP-spezifische Charakteristik.
Neben der CTE trägt die zu FAD-N5 proximal gelegene Position zur FADH●-Stabilisierung bei, wie Absorptionsmessungen an CryP_N417C zeigten. CryP weist mit Asparagin die gleiche Konservierung an dieser Position wie Photolyasen auf und unterscheidet sich damit ebenfalls von klassischen CRYs.
Analysen zur cryp-Transkription mittels qRT-PCR zeigten eine rhythmische Expression mit maximalen Transkriptmengen in der Nacht und eine rasche photoinduzierte Herunterregulation der Transkription...
In welchen Situationen steht ein Tier unter Stress und wie beeinflusst Stress dessen Wohlbefinden? Dies sind die Kernfragen, mit denen Zoos konfrontiert sind, wenn es darum geht, den Bedürfnissen ihrer Tiere gerecht zu werden. Die Beantwortung dieser Fragen ist jedoch angesichts der großen individuellen Variabilität des Inputs, der Stress hervorrufen kann,und des Outputs, der das Wohlbefinden bestimmt, eine Herausforderung. Um diese Herausforderung zu meistern, brauchen Zoos Kenntnisse darüber, welche Haltungsbedingungen und Managementsituationen Verhaltens-, physiologische oder emotionale Veränderungen hervorrufen, sowohl positive als auch negative. Dies trifft insbesondere auf Arten zu, die aufgrund ihrer Biologie und des großen öffentlichen Interesses große Anforderungen an das Management in Menschenobhut stellen, wie den Afrikanischen Elefanten. Die vorliegende Arbeit hatte daher das Ziel, unter Berücksichtigung der individuellen Variation die Auswirkungen bestimmter Managementsituationen auf physiologischen Stress und das Wohlbefinden der Tiere zu evaluieren.
Für diese Arbeit wurden zehn Afrikanische Elefanten aus drei Zoos im Rahmen eines Experiments in 2016 und 2017 mehrmals untersucht. Dieses Experiment umfasste zum einen die Messung von physiologischem Stress auf der Basis der Konzentration des „Stresshormons“ Cortisol im Speichel der Elefanten. Zu diesem Zweck wurden an bestimmten Tagen und zu folgenden Zeitpunkten Speichelproben entnommen: morgens, nachmittags vor und mehrmals nach einer von zwei Managementsituationen (positives Verstärkungstraining [PRT] und neuartiges Enrichmentobjekt [NOV]). Zum anderen diente die Exposition gegenüber dem neuartigen Enrichmentobjekt als sogenannter Novel Object Test. Dieser Standardtest der Persönlichkeitsforschung bei Tieren deckte bei anderen Arten konsistente Verhaltensunterschiede zwischen Individuen auf. Um zu untersuchen, ob dies auch auf Afrikanische Elefanten zutrifft, wurden die individuellen Verhaltensreaktionen auf das neuartige Objekt aufgezeichnet. Darüber hinaus wurden unabhängig von dem Experiment vor und nach einem Transport jeweils morgens und nachmittags Speichelproben von dem transferierten Tier und von zwei Tieren im Bestimmungszoo gesammelt, um den Effekt dieses potenziellen Stressors auf die individuellen Cortisolspiegel zu untersuchen.
Publikation A zeigt, dass die Elefanten unter den Bedingungen des Routinemanagements (das heißt dem routinemäßigen Tagesablauf der Tierpflege) am Morgen signifikant höhere Cortisolwerte im Speichel aufwiesen als am Nachmittag. Diese diurnale Variation der Cortisolsekretion ist typisch für tagaktive Arten und wurde daher auch für die untersuchten Elefanten erwartet. Unter Stressbedingungen wurde weder ein signifikanter Unterschied zwischen den Cortisolspiegeln vor und nach dem Transport noch zwischen den Cortisolwerten am Morgen und am Nachmittag festgestellt. Der prozentuale Unterschied zwischen dem morgendlichen und nachmittäglichen Cortisolspiegel war jedoch beim transferierten Tier nach dem Transport wesentlich geringer als vor dem Transport, was möglicherweise auf eine Stressreaktion auf den Transport und die Eingewöhnung im neuen Zoo hindeutet. Darüber hinaus zeigten sich deutliche Cortisolanstiege unmittelbar nach der ersten Zusammenführung des transferierten Tiers mit dem Bullen im neuen Zoo. Dieses Ergebnis demonstriert zum einen, dass Cortisol physiologischen Stress widerspiegelt. Zum anderen zeigt es die Notwendigkeit, zeitnah nach einem Stressor Speichelproben zu entnehmen, was nach dem Transport nicht möglich war.
Die Studie in Manuskript B zeigt unterschiedliche durchschnittliche Zeitverläufe der Cortisolantworten im Speichel auf die Managementsituationen PRT und NOV. PRT könnte aufgrund des beobachteten cortisolsenkenden und damit potenziell stresspuffernden Effekts förderlich für das Wohlbefinden sein. NOV induzierte im Mittel eine moderate, kurzfristige Cortisolantwort. Dies deutet darauf hin, dass die Tiere geringem physiologischem Stress ausgesetzt waren, mit dem sie jedoch erfolgreich umgehen konnten. Außerdem bestand eine bemerkenswerte individuelle Variation in den Cortisolverläufen in derselben Situation. Die Unterschiede im Cortisolspiegel zwischen den Tieren hingen mit dem Alter (bei NOV) und dem Zoo (bei PRT) zusammen. Der Effekt des Geschlechts und des Haltungssystems auf den Cortisolspiegel war hingegen variabel. Die Ergebnisse der Studie zeigen, dass die individuelle Variation der Cortisolsekretion unbedingt berücksichtigt werden muss, um physiologischen Stress zuverlässig zu erkennen.
Die Studie in Manuskript C ergab, dass sich die untersuchten Tiere im Novel Object Test konsistent in ihrem Verhalten gegenüber einem neuartigen Objekt unterschieden. Dieses Ergebnis zeigt, dass der Novel Object Test auch bei Elefanten genutzt werden kann, um die Persönlichkeit der Tiere zu untersuchen...
Patients harboring mutations in the gene DEPDC5 often display variations of neurological diseases including epilepsy, autism spectrum disorders (ASD) and other neuro-architectural alterations. DEPDC5 protein has been identified as an amino acid sensor responsible for negatively regulating the mechanistic target of rapamycin (mTOR), a central regulator in cell growth and cell homeostasis. Often, mutations of the DEPDC5 protein result in mTOR hyperactivity leading to abnormal neuronal phenotypes and the generation of excitatory/inhibitory imbalances in animal models. Complete knockout (KO) of DEPDC5 results in death shortly after birth, while inhibition of mTOR activity recovers postnatal death (Marsan et al. 2016). However, heterozygous DEPDC5-KOs in animals have been variable in their disease phenotypes during adulthood indicating developmental differences between subspecies and early development mechanisms which could be impactful on the outcome of the diseases.
To understand the mechanisms underlying DEPDC5 mutations during early development, a novel primary human neural progenitor cell line extracted from fetal tissue was characterized during proliferation and differentiation. CRISPR-Cas9 induced mutations of the DEPDC5 gene resulted in hyperphosphorylation of mTOR signaling processes and rapid expansion of the neuronal population during differentiation. Analysis of transcriptome data identified deregulation amongst p53 signaling, ribosome biogenesis, nucleotide and lipid synthesis as well as protein degradation pathways due to loss of DEPDC5. Disease gene datasets identified a correlation between Tuberous Sclerosis mutations as being more closely associated with DEPDC5 mutations while also finding overlap with some ASD and epilepsy genes. By using the mTOR inhibitor rapamycin, a substantial amount of the deregulated gene network was recovered while also reversing rapid neuronal differentiation caused by loss of DEPDC5. Though we saw increased dendritic arborization and subsequent decreases in dendrite lengths and soma sizes, rapamycin failed to recover these effects suggesting mTOR independent processes produced by DEPDC5-KO. This study provides new insights on the relationship between mutations in DEPDC5 and the functional, genomic and deregulatory networks it intertwines in humans and highlights that the DEPDC5 associated pathomechanisms are not fully related to mTOR hyperactivation, but include independent processes. This also sheds light on the question why rapamycin treatment only partially restores DEPDC5 related phenotypes and gives insight on treatments for DEPDC5 patients.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
In Europe, the sugar refinery is largely based on sugar beets. This route for obtaining household sugar results in a large amount of biomass waste, consisting mainly of the insoluble beet resi-dues, e.g., cell wall fragments. To a vast moiety this debris consists of the polymer pectin (up to 20% in the dry total solids). The structure of pectin is based on a backbone of D-galacturonic acid units (GalA), but also contains various other sugar monomers, predominantly L-arabinose, D-galactose, L-rhamnose and D-xylose. The amount of GalA adds up to a moiety of up to 70% with-in this sugar cocktail. So far, this debris is only fed to cattle or simply burnt. In nature, pectin is a common substrate for various organisms. The degradation of pectin-rich biomass is often per-formed by filamentous fungi like Hypocrea jecorina (also known as Trichoderma reesei) and As-pergillus niger, which evolved pectinases to degrade the pectin backbone and pathways to con-sume the monomer GalA as a sole carbon source. The fungal catabolism of pectin residues starts with the reduction of GalA to L-galactonate (GalOA) by a GalA-reductase. Even though filamen-tous fungi are native hosts of the GalA-catabolism and certain engineering approaches have al-ready been demonstrated, this class of organisms remains challenging with regard to bioreactor cultivation and tedious genetic accessibility. In contrast, the yeast S. cerevisiae is well known in fermentation processes and easily modified by a versatile set of genetic tools. So far, first ap-proaches have already been conducted to transfer the GalA utilization pathways into S. cerevisiae, but these approaches indicated limitations regarding GalA-uptake and redox cofac-tor replenishment due to the relatively high oxidative state of GalA compared to other sugars like glucose and galactose. Furthermore, the generally strongly increased demand for redox co-factors must be met by GalA reduction by finding new cofactor sources or redirecting reactions of the core metabolism.
This work aimed at the production of GalOA, which is the first intermediate of the fungal GalA catabolism. This compound shows an interesting range of potential applications, for instance as a food and cosmetic additive. To overcome the oxidized character of GalA, the presence of a more reduced co-substrate as a redox donor and as a carbon and energy source was required. To further enhance the reduction of GalA, modulation of the redox-cofactor supply and enzyme engineering were performed.
Generally speaking, protein import into mitochondria and chloroplasts is a post-translational process during which the precursor proteins destined for mitochondria or chloroplasts are translated with cytosolic ribosomes and targeted. The previous results showed that the isolated chloroplasts can import in vitro synthesized proteins and the absence of ribosomes in the immediate area around chloroplasts in electron microscopy (EM) images. However, none of the EM images were recorded in the presence of a translation elongation inhibitor. Also, the observation showed that ribosomes stably bind to purified liver mitochondria in vitro, and the first indication of chloroplast localization of mRNAs encoding plastid proteins in Chlamydomonas rheinhardtii, which challenge the post-translational import and support the co-translational process. Therefore, in this study, the association of the ribosomes to the isolated chloroplasts were analyzed, a binding assay was established and showed that naked ribosomes are not considerably bound to chloroplasts. Additionally, mRNA localize in close vicinity to mitochondria also challenged post-translation protein import. Global analysis of transcripts bound to mitochondria in yeast or human revealed that around half of the transcripts of mitochondrial proteins displayed a high mitochondrial localization. The observed association of mRNAs with chloroplast fractions and the in vivo analysis of the distribution of mRNAs was used as base to formulate the hypothesis that mRNA can bind to chloroplast surface. Therefore, in this study, the mRNA binding assay was established and revealed that mRNAs coding for the mitochondrial cytochrome c oxidase copper chaperone COX17 showed unspecific binding to the chloroplasts. The mRNA coding for chloroplast outer envelope transport protein OEP24 and mRNA coding for the essential nuclear protein 1 (ENP1) showed specific binding, and OEP24 has a 3-fold higher affinity than ENP1 mRNA. Moreover, the BY2-L (Nicotiana tabacum non-green cell culture) could confer the highest enhancement of OEP24 mRNA binding efficiency than the COX17 and ENP1 mRNA and the preparation of the BY2-L was optimized. Afterwards, the feasibility to fix the interaction between mRNA and the proteins on the surface of chloroplasts was confirmed. OEP24 mRNA showed more efficiency in the UV-crosslinking. Following, the pull-down with antisense locked nucleic acid (LNA)/DNA oligonucleotides was established which could be used for the further investigation of the proteins involved in the mRNA binding to the chloroplasts.
Plastic pollution is a pervasive problem. In the environment, both the physical and chemical aspects of the material contribute to pollution. For instance, discarded plastic is useless waste that is fragmented upon degradation and so-called microplastics <5 mm are formed. Besides, the chemicals added into plastics are usually customized for specific functions, but these can easily transfer from the polymer into an ambient medium. This work examined both of these aspects. Moreover, the question of whether ecotoxicological effects are more likely to appear because of the microparticle properties or the chemicals transferring from the microplastics was addressed. A special focus was laid on the UV-weathering-induced chemical release.
First, conventional and biodegradable plastics made from fossil and bio-based resources were chosen. The different materials (pre-production and recycled pellets as well as final products)were weathered and their leachates evaluated in vitro. The leachates were analyzed with nontarget screening in order to measure the number of transferred chemicals. Plastics identified as toxic were subjected to further investigations in vivo. A biodegradable shampoo bottle was processed to microplastics and the particles’ physical and chemical properties were assessed with the freshwater worm Lumbriculus variegatus. Here, commonly used endpoints such as mortality, reproduction and weight were tested via different exposure routes. Moreover, the freshwater shrimp Neocaridina palmata was exposed to microplastic beads and fragments to clarify if the shape of the particles affects the ingestion and egestion, respectively. Thereafter, two materials that displayed the strongest toxic responses in vitro within the first study were weathered and leached. Finally, the shrimps were exposed to the leachates and the locomotor behavior was used as an ecologically relevant but less frequently studied endpoint.
The results of the studies highlight that plastics are chemically complex mixtures, containing a wide range of chemicals in terms of the number and functionality. These chemicals induced oxidative stress, baseline toxicity and endocrine activities. This shows that pellets represent a processing state that comprises chemically heterogenous materials. Moreover, it was shown that a degradation initiator is not necessarily relevant to trigger inherent substances to leach out from plastics. Despite this, the UV-weathering resulted in increasingly released chemicals and exacerbated the in vitro toxicities. Even plastics assessed as toxicologically harmless prior to weathering released toxic chemical mixtures once they were weathered. One recycled and all of the biodegradable plastics were toxicologically most concerning. This means that such materials are currently not better than conventional, virgin plastics in terms of their toxicity.
To clarify the source of the microplastic toxicity, L. variegatus was exposed to biodegradable microplastics. The particles were ingested by the worms and adversely affected the examined endpoints. In comparison, microplastics that were depleted from their chemicals via a solvent treatment were less toxic. Kaolin as a natural particle control was evaluated alongside and positively affected the weight of the worms. This emphasizes the ecological relevance of fine-sized matter for the test species. The chemicals extracted from the microplastics induced a 100% mortality. A chemical analysis of the material revealed two ecotoxicologically relevant biocides. The physically-mediated effects of the microplastics seemed to be less of a concern for the worms, which is probably linked to their adaptation to high concentrations of naturally occurring particles in the environment. However, the effects related to the chemicals of plastic cannot be ignored, especially for materials that are claimed to be environmentally friendly.
In the third study, the role of the particle shape in the gut passaging of N. palmata was studied. While the particle size was a determinant factor for the ingestion, the ingestion and egestion of the beads and fragments did not differ, respectively. The shrimps ingested less fragments when food was provided than in the absence of food. As for the worms, the shrimps are known to ingest many naturally occurring particles. Their unselective feeding behavior towards the particle shape could indicate that microplastics as a physical pollutant are negligible for the shrimps. That is why the chemicals of the two most toxic in vitro materials were tested with N. palmata. However, no trend towards elevated or reduced movements of the shrimps was observed, even though the leachates contained baseline toxicants. This shows that the in vitro toxicities of plastics are not necessarily indicative for effects to occur at the in vivo level...
The European Community has set a milestone in the European water policy in 2000: all water directives and policies were united into one comprehensive document – the European Water Framework Directive (EU WFD). The EU WFD requires the monitoring of 45 priority substances, primarily in the water phase, which is not related to a substantial amount of chemicals available on the market worldwide (about 50,000). About 60% of these are human and environmentally toxic. Hence, the currently monitored 45 priority substances are not even close to being sufficient to provide a comprehensive picture of the actual chemical pollution in the aquatic environment.
Furthermore, the EU WFD in its original shape paid less attention to sediments as an important source and sink for chemical contamination. Under stable hydrological conditions, polluted old sediments are covered by less polluted younger sediments preventing erosion of deeper sediment layers and, therefore, the release of particle-bound contaminants. However, urbanization, deforestation, flooding, dredging, riverbed renaturation, and stormwater overflow basin releases can lead to an unpredictable release of particle-bound pollutants. Therefore, in 2008, sediments were added to the EU WFD as a monitoring matrix for substances that tend to accumulate there. As a result, after 18 years of the EU WFD, less than half of all European waterbodies reached a good ecological (40%) and chemical (38%) status.
One of the primary pollution sources in aquatic ecosystems are wastewater treatment plants (WWTPs). Advanced wastewater treatment by ozonation is promising to remove most micropollutants. However, the knowledge about the possible improvement of the receiving waterbody is rare. The latter aspects were the main reasons for the start of the DemO3AC project in 2014. The study area was located in the federal state of North Rhine-Westphalia (Germany). The study area included the Wurm River and its tributary, the Haarbach River. Both waterbodies act as receiving waterbodies for WWTPs. One of them is the Aachen-Soers WWTP (receiving waterbody: Wurm River), upgraded by full stream ozonation as an advanced effluent treatment. Therefore, the extensive investigation program within the DemO3AC project included an investigation of the ecological and chemical status of both receiving waterbodies and the investigation of a possible improvement of the Wurm River after implementing advanced effluent treatment.
The current study was a part of the DemO3AC project and covered the sediment toxicity and a possible impact of the ozonation on aquatic organisms in the receiving waterbody. Time-resolved sampling campaigns allowed investigations under different hydrological conditions, mainly determined by the weather. The first sampling campaign took place in June 2017 during a prolonged dry period with low water flow in the receiving waterbodies. The second sampling campaign was performed exactly one year later (June 2018) after a long rainy period and corresponding high-water levels. Full-stream ozonation at the Aachen-Soers WWTP had been in operation for half a year. Furthermore, a wide range of organic micropollutants was investigated in the effluent of the studied WWTPs to assess a possible hazard emerging from contaminants released into the receiving waterbody.
The study design was developed based on the holistic approach to assessing the ecotoxicological pollution of surface waterbodies. It included the detection of chemical compounds combined with effect-based methods to identify possible drivers of toxicity. The sediment's ecotoxicological assessment included studies on endocrine-disrupting activity, genotoxic and embryotoxic potentials. These endpoints were evaluated using in vitro and in vivo bioassays. In addition, sediments’ chemical profiling was performed using modern analytical chemistry techniques.
The genotoxic potential was investigated using the Ames fluctuation assay with Salmonella typhimurium bacterial strains TA98, TA100, YG1041, and YG1042, sensitive to different classes of compounds, and the Micronucleus assay as a eukaryotic assay with mammalian cells. A unique feature of the present study was the implementation of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay. Moreover, a comprehensive genotoxicity ranking of chemical compounds identified in sediments was used and combined with statistical analysis to identify the drivers of genotoxicity. The results of this study were published in Shuliakevich et al. (2022a) (see also Annex 1), describing the mutagenic potential of all sampling sites, which was primarily driven by polycyclic aromatic hydrocarbons, nitroarenes, aromatic amines, and polycyclic heteroarenes. In addition, the rainwater overflow basin was identified as a significant source for particle-bound pollutants from untreated wastewater, suggesting its role as a possible source of genotoxic potential. The present study showed high sensitivity and applicability of non-standard Salmonella typhimurium bacterial strains YG1041 and YG1042 in the Ames fluctuation assay to assess the different classes of mutagenic compounds. A combination of effect-based methods and a chemical analysis was shown as a suitable tool for a genotoxic assessment of freshwater sediments.
The sediments' endocrine-disruptive activity was investigated using the cell-based reporter gene CALUX® assay. A simultaneous launch of the full-scale effluent ozonation at the Aachen-Soers WWTP was used for investigation of the entrance of the ozonated effluent into the Wurm River and the endocrine-disrupting activity in the water phase. A particular focus of the present study was the unique investigation of PAHs as possible drivers of the endocrine-disrupting activity in sediments of the Wurm River. The results of this study were laid down in the publication by Shuliakevich et al. (2022b) (see also Annex 2), describing variations in endocrine-disrupting activity in the Wurm River under different weather conditions. Briefly, under stable hydrological conditions in June 2017, the estrogenic and the antiandrogenic activities in sediments of the Wurm River were within the range of 0.03-0.1 ng E2 equivalents (eq.)/g dry weight sediment equivalents (dw SEQ) and 3.0-13.9 µg Flu eq./g dw SEQ, respectively. After extensive rain events in June 2018, the sediments' estrogenic and antiandrogenic activities were detected within the range of 0.06-0.2 ng E2 eq./g dw SEQ and 1.7-39.2 µg Flu eq./g de SEQ, respectively. Increased endocrine-disruptive activity (up to 0.2 ng E2 eq./g dw SEQ in ERα- and 39.2 µg Flu eq./g dw SEQ in anti-AR-CALUX® assays) in sediments downstream of the rainwater overflow basin suggested it as a possible source of pollution. A unique result of the second study was finding a positive correlation between measured particle-bound antiandrogenic activity and detected polyaromatic hydrocarbons (PAHs) ...
Interleukin-11 signaling is a global molecular switch between regeneration and scarring in zebrafish
(2022)
The two diametrically opposing outcomes after tissue damage are regeneration and fibrotic scarring. After injury, adult mammals predominantly induce fibrotic scarring, which most often leads to patient lethality. Fibrotic scarring is the deposition of excessive extracellular matrix that matures and hinders tissue function. The scarring response is mainly orchestrated by myofibroblasts, which arise only upon tissue damage, from various cellular origins, including tissue resident fibroblasts, endothelial cells and circulating blood cells. On the contrary, species like zebrafish, possess the remarkable capacity to regenerate their damaged tissues. After injury, instead of inducing a myofibroblast-mediated fibrogenic gene program, cells in these species undergo regenerative reprogramming at the transcriptional level to activate vital cellular processes needed for regeneration, including proliferation, dedifferentiation, and migration. Several pro-regenerative mechanisms have been identified to date. Most of them, if not all, are also important for tissue homeostasis and hence, are not injury specific. Therefore, the central aim of this study is to identify injury-specific mechanisms that not only induce regeneration, but also limit fibrotic scarring.
To test the notion that fibrotic scarring limits regeneration, I first compared the scarring response in the regenerative zebrafish heart after cryoinjury with what is known in the non-regenerative adult mouse heart. I found that zebrafish display ~10-fold less myofibroblast differentiation compared to adult mouse after cardiac injury. With these findings, I hypothesized that zebrafish employ mechanisms to actively suppress scarring response. Using a novel comparative transcriptomic approach coupled with genetic loss-of-function analyses, I identified that Interleukin-6 (Il-6) cytokine family-mediated Stat3 is one such pro-regenerative pathway in zebrafish.
Il-6 cytokine family consists of Il-6, Interleukin-11 (Il-11), Ciliary neurotrophic factor, Leukemia inhibitory factor, Oncostatin M, and Cardiotrophin-like cytokine factor 1. Il-6 family ligands signal through their specific receptors and a common receptor subunit (Il6st or Gp130). Using gene expression analyses after adult heart and adult caudal fin injuries in zebrafish, I identified that both the Il-11 cytokine encoding paralogous genes (il11a and il11b) are the highest expressed and induced among the Il-6 family cytokines. Hence, I chose Il-11 signaling as a candidate pathway for further analysis. To investigate the role of Il-11 signaling, I generated genetic loss-of-function mutants for both the ligand (il11a and il11b) and the receptor (il11ra) encoding genes. Using various tissue regeneration models across developmental stages in these mutants, I identified that Il-11/Stat3 signaling is indispensable for global tissue regeneration in zebrafish.
To investigate the cellular and molecular mechanisms by which Il-11 signaling promotes regeneration, I performed transcriptomics comparing the non-regenerative il11ra mutant hearts and fins with that of the wild types, respectively. I identified that Il-11 signaling orchestrates both global and tissue-specific aspects of regenerative reprogramming at the transcriptional level. In addition, I also found that impaired regenerative reprogramming in the il11ra mutant hearts and fins resulted in defective cardiomyocyte and osteoblast repopulation of the injured area, respectively.
On the other hand, by deep phenotyping the scarring response in il11ra mutant hearts and fins, I identified that Il-11 signaling limits myofibroblast differentiation. Furthermore, I found that cardiac endothelial cells and fibroblasts are one of the major responders to injury-induced Il-11 signaling. Using lineage tracing, I found that both the endothelial and fibroblast lineages in the non-regenerative il11ra mutants commit to a myofibroblast fate, spearheading the scarring response. In addition, using cell type specific manipulations, I showed that Il-11 signaling in cardiac endothelial cells allows cardiomyocyte repopulation of the injured area. Finally, using human endothelial cells in culture, I uncovered a novel feedback mechanism by which Il-11 signaling limits fibrogenic gene expression by inhibiting its parent activator and a master regulator of tissue fibrosis, TGF-β signaling.
Overall, I identified Interleukin-11/Stat3 signaling as the first global regulator of regeneration in zebrafish. Briefly, I showed that Interleukin-11 signaling promotes regeneration by regulating two crucial cellular aspects in response to injury – (1) it promotes regenerative reprogramming, thereby allowing cell repopulation of the injured area and (2) it limits mammalian-like fibrotic scarring by inhibiting myofibroblast differentiation and TGF-β signaling. Altogether, these zebrafish data, together with the contradicting mammalian data strongly indicate that the secrets of tissue regeneration lie downstream of IL-11 signaling, in the differences between regenerative and non-regenerative species. Furthermore, I establish the non-regenerative il11ra mutant as an invaluable zebrafish model to study mammalian tissue fibrosis.
The heart is the first functional organ that develops in the embryo. To become a functional organ, it undergoes several morphogenetic processes. These morphogenetic events involve different cell types, that interact with each other and respond to the surrounding extracellular matrix, as well as intrinsic and extrinsic mechanical forces, assuming different behaviors. Additionally, transcription factor networks, conserved among vertebrates, control the development.
To have a better understanding of cell behavior during development, it is necessary to find a model system that allows the investigation in vivo and at single-cell resolution. Thanks to the common evolutionary origin of the different cardiac structures, together with the conserved molecular pathways, the two-chambered zebrafish heart offers many advantages to study cell behavior during cardiac morphogenesis. Here, using the zebrafish heart as a model system, I uncovered the cell behavior behind two of the main cardiac morphogenetic events: cardiac wall maturation and cardiac valve formation.
In the first part of this study, I investigated how the cardiac wall is maintained at the molecular level. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required for myocardial wall integrity. Global loss of snai1b leads to the extrusion of CMs away from the cardiac lumen, a process we show is dependent on cardiac contractility. Examining CM junctions in snai1b mutants, we observed that N-cadherin localization was compromised, thereby likely weakening cell-cell adhesion. In addition, extruding CMs exhibit increased actomyosin contractility basally, as revealed by the specific enrichment of canonical markers of actomyosin tension - phosphorylated myosin light chain (active myosin) and the α-catenin epitope α-18. By comparing the transcriptome of wild-type and snai1b mutant hearts at the early stages of CM extrusion, we found the dysregulation of intermediate filament genes in mutants including the upregulation of desmin b. We tested the role of desmin b in myocardial wall integrity and found that CM-specific desmin b overexpression led to CM extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 is a critical regulator of intermediate filament gene expression in CMs and that it maintains the integrity of the myocardial epithelium during embryogenesis, at least in part by repressing desmin b expression.
In the second part of this study, I focused on the behavior of valve cells during cardiac development. Using the zebrafish atrioventricular valve, I focus on the valve interstitial cells which confer biomechanical strength to the cardiac valve leaflets. We find that initially AV endocardial cells migrate collectively into the cardiac jelly to form a bilayered structure; subsequently, the cells that led this migration invade the extracellular matrix (ECM) between the two EC monolayers, undergo an endothelial-to-mesenchymal transition as marked by loss of intercellular adhesion, and differentiate into VICs. These cells proliferate and are joined by a few neural crest-derived cells. VIC expansion and a switch from a pro-migratory to an elastic ECM drive valve leaflet elongation. Functional analysis of Nfatc1 reveals its requirement during VIC development. Zebrafish nfatc1 mutants form significantly fewer VICs due to reduced proliferation and impaired recruitment of endocardial and neural crest cells during the early stages of VIC development. Analysis of downstream effectors reveals that Nfatc1 promotes the expression of twist1b, a well-known regulator of epithelial-to-mesenchymal transition. This study shows for the first time that Nfatc1 regulates zebrafish VICs formation regulating valve EMT in part by regulating twist1b expression. Moreover, it proposes the zebrafish valve as an excellent model to study the cellular and molecular process that regulate VIC development and dysfunction.
In conclusion, my work: 1) identified an unsuspected role of Snai1 in maintaining the integrity of the myocardial epithelium, opening new avenues in its role in regulating cellular contractility; 2) uncovered the function of Nfatc1 in the establishment of the VIC, establishing a new model to study valve development and function.
Despite all advancements in cancer research and clinical practice, cancer remains a life- threatening disease with an increasing incidence. According to a 2018 WHO forecast, cancer incidence will double to approximately 37 million new cancer cases by 2040. Today, clinical management of cancer is based on a "one-fits-all" strategy. Most cancers are still treated by surgical therapy followed by adjuvant or neoadjuvant chemotherapy based on rather strict guidelines (S3 guidelines in Europe) which are based on studies of large cohorts of patients with the same tumor entity. While this approach has led to substantial increases in progression-free survival and overall patient survival, most patients do not benefit from the administered treatment regimen. One reason for this is intra-tumor heterogeneity, which results from clonal evolution between cancer cells and their environment. This means that cancer patients may respond differently to a particular drug due to the different mutation patterns of their tumor cells. Therefore, patients should be screened in advance for reliable cancer biomarkers that definitively predict whether they will respond to a particular therapy. This would increase the probability of a successful treatment.
Colorectal cancer (CRC) is the third most diagnosed cancer and the second leading cause of cancer deaths worldwide. The main cause of death in CRC is a metastatic disease, which is presented in 20 % of patients and eventually develops in more than 30 % of early-stage patients. Despite the significant increase (to more than 30 months) in median survival with the development of cytotoxic agents and the introduction of targeted therapy, the progression-free survival in the first-line setting has remained largely unchanged over the past decade.
The heterogeneity in CRC is characterized by alterations in multiple signaling pathways that affect cellular functions such as cell proliferation or apoptosis. Commonly affected signaling pathways include the mitogen-activated protein kinase (MAPK)- and the transforming growth factor-β/bone morphogenetic protein (TGF-β/BMP)-pathway. Alterations in the TGF-β/BMP pathway, due to mutations in the SMAD4 gene (mothers against decapentaplegic homolog 4), are associated with different drug response and promote resistance to chemotherapy. In addition, they are associated with a higher recurrence rate.
SMAD4 is one of the most common cancer driver genes, and mutations occur in up to 15 % of CRC cases. Therefore, there is an urgent need for therapeutic agents that can specifically target SMAD4-mutated tumors.
The aim of the present study was the identification of the clinical relevance of the SMAD4 gene and the investigation of its suitability as a potential biomarker in CRC.
For this purpose, I investigated sibling patient-derived organoids (PDOs) derived from different regions of a chemo-naïve CRC tumor. PDOs are 3D cell cultures that reliably recapitulate the architecture of the tissue of origin, as well as preserve the genomic background and intra-tumor heterogeneity. The sibling PDOs (R1R361H and R4wt) shared the most common CRC mutations, such as KRASG12D (kirsten rat sarcoma), PIK3CAH1047R (phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha), and TP53C242F (tumor protein 53), but differed in a SMAD4R361H mutation and showed a different drug response. The single nucleotide variant R361H of the SMAD4 gene is among the most common pathogenic alterations in various cancers, including CRC.
The sibling PDOs showed significant differences in response to the MEK-inhibitors cobimetinib, trametinib, and selumetinib. MEK-inhibitors are antineoplastic agents that inhibit the function of MEK1 and MEK2, preventing phosphorylation of transcription factors, which leads to inhibition of tumor cell proliferation. MEK-inhibitors are approved for the treatment of malignant melanoma. Currently, they are in phase-III clinical trials for the treatment of patients with metastatic CRC.
To investigate whether SMAD4R361H is responsible for sensitivity to MEK-inhibitors, Iestablished three syngeneic PDOs harboring a SMAD4R361H mutation using the CRISPR/Cas9 genome editing system. All CRISPR-PDOs were significantly more sensitive to the MEK-inhibitors, compared to R4wt. I have shown that the SMAD4R361H mutation is responsible for sensitivity to MEK inhibition in CRC models and may be a predictive biomarker.
To test this hypothesis, I examined 62 CRC PDO models and treated them with the MEK-inhibitors cobimetinib, trametinib, and selumetinib. All models that had a pathogenic mutation or deletion in the SMAD4 gene (15 %) were sensitive to cobimetinib, 10 % of models were sensitive to trametinib, and 8 % were sensitive to selumetinib.
I performed transcriptome (RNA sequencing) and proteome analyses using the DigiWest® method to investigate the mechanism underlying MEK-inhibitor sensitivity.
DigiWest® is a Luminex® bead-based analysis that allows the simultaneous analysis of over 100 (phospho-)proteins. The transcriptome and proteome data support the observation that MEK inhibition primarily affects SMAD4R361H PDOs. Furthermore, I have shown that activation of the BMP signaling pathway in organoids with wild-type SMAD4 appears to be responsible for resistance to MEK-inhibitors. Thus, a genetic alteration in the BMP signaling pathway, beyond SMAD4, could lead to sensitivity to MEK-inhibitors.
I identified four genes involved in the TGF-β/BMP signaling pathway that are frequently mutated in CRC and grouped them into the so-called SFAB-signature (SMAD4, FBXW7 (F-box/WD repeat-containing protein 7), ARID1A (AT-rich interactive domain-containing protein 1A), or BMPR2 (Bone morphogenetic protein receptor type II). Clinical data show that approximately 36 % of CRC patients have at least one pathogenic mutation in these genes.
I tested all 62 CRC PDO models and found a significant positive prediction for sensitivity to cobimetinib (95 %) and selumetinib (70 %) for the SFAB-signature. Trametinib and the newly approved MEK-inhibitor binimetinib showed a similar trend. Therefore, the SFAB-signature has high predictive power for response to MEK-inhibitors and could be used as a predictive biomarker panel.
The current clinically used biomarkers for CRC are based on the mutation status of driver genes KRAS and BRAF, which are present in up to 50 % and 10 % of CRC, respectively. Investigation of molecular alterations in CRC revealed that mutations in the KRAS gene, which is downstream of EGFR (epidermal growth factor receptor) in the MAPK-pathway, interfere with an anti-EGFR-antibody therapy (e.g., cetuximab). Therefore, cetuximab is only relevant for RAS wild-type tumors. However, approximately 40 % of patients with RAS wild-type status do not respond to this treatment.
About 53 % of CRC PDO models carry a pathogenic RAS mutation, about 10 % harbor a pathogenic BRAF mutation. Both, the RAS and RAF status alone as well as the combination of RAS and RAF status with SFAB-signature did not provide a better prediction of sensitivity to MEK inhibition.
Eine große Gruppe von Aptameren sind die Guanosintriphosphat (GTP) Aptamere. Diese zeigt sehr eindrücklich, wie RNA unterschiedliche Strategien nutzt, um denselben Liganden zu erkennen. Die komplette Struktur des GTP Klasse II Aptamers wird in der ersten Publikation gezeigt. Interessanterweise zeichnet die Struktur ein stabil protoniertes Adenine unterhalb der GTP-Bindestelle aus. Dieses wurde durch eine Kombination aus weiterführenden NMR- und ITC-Experimente untersucht und charakterisiert. Es zeigte sich, dass die protonierte Base einen pKs-Wert hat, der weit von der Neutralität verschoben ist. Die Protonierung ist auch noch bei sehr basischen Puffern stabil.
Eine Art der funktionellen Protonierung wird von den zyklischen di-Nukleotiden (CDN) bindenden Riboswitches genutzt, um zwei CDN mit ähnlicher Affinität zu binden. c-di-GMP Riboswitches wurden als regulatorische Einheit beschrieben und deren Kristallstruktur aufgeklärt. Mutationsexperimente führten dazu, dass bei einer G-zu-A Mutation an der Gα-Bindestelle die Selektivität des Riboswitches verändert wurde. Die Mutante bindet sowohl c-di-GMP als auch cGAMP mit ähnlichen Bindungsaffinitäten. Riboswitche, die cGAMP binden wurden auch in der bakteriellen Genomen gefunden. Hierbei ist die Promiskuität unterschiedlich stark ausgeprägt. Die Untersuchung des Bindungsmodus und der damit verbundenen Promiskuität ist in der zweiten Publikation beschrieben. Hier wurde gezeigt, dass die Riboswitche beide Liganden nur binden können, wenn zur Bindung von c-di-GMP das Ligand bindende A protoniert vorliegt. Auch diese Protonierung konnte mit weiterführenden NMR- und ITC-Experimenten charakterisiert werden. Die Untersuchungen einer solch großen RNA sind mit NMR Spektroskopie herausfordernd. Hierbei wurde ausgenutzt, dass die Kristallstruktur bereits bekannt war, welche allerdings die Protonierung nicht zeigte. Auch diese Protonierung zeigt einen pKs-Wert, der weit von der Neutralität verschoben ist und außerdem bei unterschiedlichen pH stabil ist.
In den beiden untersuchten Beispielen wurden zwei verschiedene Arten von Protonierung gezeigt: eine strukturelle und eine funktionelle. Das GTP Klasse II Aptamer benutzt die Protonierung als strukturelle Basis für die Basis der Ligandenbindungsstelle. Hierbei werden durch die Protonierung des Adenines mehr nutzbare Wasserstoffbrücken ausgebildet und damit die Tertiärstruktur stabilisiert. Im Unterschied dazu nutzen die promiskuitiven CDN Ribsowicthes die Protonierung, um verschiedene Liganden binden zu können und es kommt damit zu einer Verschiebung der Funktionalität. Der regulatorische Nutzen dafür ist allerdings noch unbekannt.
Auch bei den SAM Riboswitches wurde ein promiskuitiver Vertreter beschrieben. SAM Riboswitches gehören zu den am längsten bekannten Klassen der Riboswitches. Bis heute sind hier die meisten unterschiedlichen Klassen bekannt. SAM wird häufig als Donor für funktionelle Gruppen benutzt, besonders häufig als Methlygruppendonor für die Methylierung einer Reihe unterschiedlicher Substrate (z.B. DNA, Proteine, Metabolite etc.). Bei dieser Reaktion entsteht SAH als Nebenprodukt. Zusätzlich ist SAH zelltoxisch, da es affin an Methyltransferasen bindet und damit diese essenzielle Reaktion inhibiert. Eine enge Kontrolle der SAH-Konzentration ist daher kritisch. SAM bindende Riboswitches haben zu SAM eine bis zu 1000-fach höhere Bindungsaffinität im Vergleich zu SAH. Die Beschreibung eines translationalen OFF-Riboswitches, der SAM und SAH mit ähnlicher Affinität bindet, ist daher überraschend. Zumal seine Genassoziation fast ausschließlich zu SAM Synthetasen ist, deren Regulation durch SAH wenig sinnvoll erscheint. Um ein besseres Verständnis für die Funktion des SAM/SAH Riboswitches zu erhalten, wurde seine 3D-Struktur mittels NMR-Spektroskopie aufgeklärt, wie in der vierten Publikation beschrieben. Dafür mussten zunächst alle Resonanzen der Sequenz und dem Liganden zugeordnet werden, wie in der dritten Publikation beschrieben. Dabei wurde als Ligand SAH gewählt, da dieser chemisch stabiler und damit für die teils tagelangen NMR-Messungen besser geeignet ist. Zusätzlich wurden Mutanten bzw. verwandte Liganden mittels ITC Experimente auf ihre Bindungseigenschaften untersucht, um die Bedeutung der Linkerlänge, einzelner Basenpaare und funktionelle Gruppen des Liganden zu untersuchen. Bei anderen bekannten SAM Riboswitches umschließt die RNA den Liganden fast komplett. Dabei wird zum einem das Sulfoniumion spezifisch durch die Carboxylgruppen verschiedener Uracil-Nukleotide erkennt und koordiniert. Außerdem bildet sich eine Bindetasche aus, die genug Platz für die stabile Bindung der Methylgruppe hat. Beim SAH Riboswitch wird die Selektivität für SAH dadurch erreicht, dass die Bindetasche sterisch keinen Platz für die Methylgruppe von SAM bereitstellt.
Zusammenfassend wurden in dieser Arbeit drei verschiedene Ligand bindende RNA-Strukturen untersucht, die alle sehr unterschiedliche Strategien zur Bindung der Liganden nutzen. Obwohl Portionierungen bei Aptameren und Riboswitches selten beschrieben wurden, haben sie eine maßgebliche Funktion in den beiden zuerst untersuchten Strukturen. Obwohl bisher im Hinblick auf alle bekannten RNA Strukturen eher selten beschrieben, gibt es doch neben den genannten zwei, einige Beispiele für strukturelle oder funktionelle Protonierungen. Auch in Hinblick auf zukünftige bzw. Verbesserung bestehender RNA-Strukturvorhersage-Programme ähnlich wie sie für Proteine schon lange nutzt werden, müssen protonierte Nukleobasen ernsthaft in Betracht gezogen werden. Außerdem konnte gezeigt werden, dass zwei der untersuchten Riboswitches zwei Liganden mit ähnlicher Affinität binden. Die genutzte Strategie ist hierbei unterschiedlich. Während bei den promiskuitiven CDN Riboswitches der regulatorische Nutzen noch unbekannt ist, konnte für den SAM/SAH Ribsowitch gezeigt werden, dass SAH nur zufällig aufgrund der wahrscheinlich sehr niedrigen intrazellulären Konzentration gebunden wird und dieser daher wahrscheinlich später in der evolutionären Entwicklung entstanden ist. Riboswitches halten es weiterhin spannend.
The intensive use of the North Sea area through offshore activities, sand mining, and the spreading of dredged material is leading to increasing pollution of the ecosystem by chemicals such as hydrophobic organic contaminants (HOCs). Due to their toxicological properties and their ability to accumulate in the environment, HOCs are of particular concern. The contaminants partition between aqueous (pore water, overlying water) and solid phases (sediment, suspended particulate matter, and biota) within these systems. The accumulated contaminants in the sediment are of major concern for benthic organisms, who are in close contact with sediment and interstitial water. It is thus particularly important to better understand how contaminants interact with biota, as these animals may contribute to trophic transfer through the food web. Furthermore, sediments are a crucial factor for the water quality of aquatic systems. They not only represent a sink for contaminants but also determine environmental fate, bioavailability, and toxicity. The Marine Strategy Framework Directive (MSFD) was introduced to protect our marine environment across Europe and includes the assessment of pollutant concentrations in the total sediment, which, however, rarely reflects the actual exposure situation. The consideration of the pollutant concentrations in the pore water is not implemented, although this is needed for the evaluation of bioavailability and risk assessment. For this reason, special attention is given to further development, implementation, and validation of pollutant monitoring methods that can determine the bioavailable fraction in sediment pore water. For risk assessment purposes, it is furthermore important to use biological indicators in addition to classical analytics to determine the effect of pollutants on organisms. The main objective of this thesis was to gain insight into the pollution load and the potential risk of hydrophobic organic chemicals (HOCs) in the sediment of the North Sea and to evaluate these results with regard to possible risks for benthic organisms and the ecosystem. The following five aims are covered within these studies to gain a holistic assessment of sediment contamination:
1. Assessment of the pore water concentrations of PAHs and PCBs
2. Determination of the bioturbation potential by macrofauna analysis
3. Application of the SPME method on biological tissue
4. Assessment of recreated environmental mixtures in passive dosing bioassays
5. Development of SPME method for DDT in sediments
The thesis is comprised of three main studies supported by three additional studies ...
Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.
Weltweit werden etwa 17% aller Infektionskrankheiten von Vektoren auf den Menschen übertragen. Dabei dienen meist blutsaugende Arthropoden wie Stechmücken, Zecken oder Sandfliegen als Überträger von Bakterien, Viren oder einzelligen Parasiten. Zur letzteren Gruppe gehört auch der protozoische Erreger der Chagas-Krankheit Trypanosoma cruzi. Er wird von hämatophagen Triatominae, einer Unterfamilie der Raubwanzen (Hemiptera: Reduviidae) während der Blutmahlzeit an einem infizierten Säugerwirt aufgenommen, durchläuft komplexe Entwicklungsschritte im intestinalen Trakt der triatominen Insekten und wird anschließend über den Fäzes und Urin der Wanzen abgegeben. Die Infektion des nächsten Wirts erfolgt dann durch das versehentliche Einreiben der Erreger in die Stichwunde oder auf Schleimhäute. Auch eine Infektion über die orale Aufnahme von kontaminierter Nahrung, Mutter-Kind-Infektionen und die Übertragung durch Blutkonserven und Organtransplantate sind möglich. Die Chagas‑Krankheit, oder auch Amerikanische Trypanosomiasis, ist insbesondere in Mittel- und Südamerika verbreitet und betrifft nach Schätzungen der WHO 6 bis 7 Millionen Menschen. Infolge von globaler Immigration und erhöhtem Reiseverkehr treten jedoch in den letzten Jahrzehnten auch vermehrt Fälle in Europa, den USA, Kanada und den westlichen Pazifikstaaten auf. Da dort bislang geeignete Vektoren fehlen, kommt es außerhalb des lateinamerikanischen Kontinents nicht zu vektorübertragenen Infektionen. Dies könnte sich jedoch im Zuge des Klimawandels und einer voranschreitenden Globalisierung ändern, sollte der Ausbreitung der Chagas-Krankheit eine Ausbreitung ihrer triatominen Vektoren folgen.
Inwieweit Triatominae unter heutigen Bedingungen klimatisch geeignete Habitate außerhalb des amerikanischen Kontinents finden, wurde innerhalb des ersten Projekts der vorliegenden Dissertation untersucht. Dazu wurde mit Hilfe der ökologischen Nischenmodellierung und Vorkommensdaten verschiedener vektorkompetenter Raubwanzenarten sowie klimatischer Umweltvariablen die klimatische Eignung verschiedenster Lebensräume modelliert und global projiziert. Es zeigte sich, dass insbesondere tropische und subtropische Gebiete Afrikas sowie Ost- und Südostasiens zwischen 21° nördlicher Breite und 24° südlicher Breite für viele triatomine Vektorarten geeignete Bedingungen aufweisen. Auffällig ist dabei insbesondere die Art Triatoma rubrofasciata, welche nachweislich bereits in Südchina, Vietnam und weiteren Ländern Afrikas und Asiens gefunden wurde. Die Modellierung
offenbarte, dass weitere ausgedehnte Teile der Küstenregionen Afrikas und Südostasiens als für T. rubrofasciata klimatisch geeignet angesehen werden müssen. Eine weitere Ausbreitung dieser Art ist demnach äußerst wahrscheinlich und stellt bislang das größte Risiko autochthon übertragener Chagas-Infektionen außerhalb des amerikanischen Kontinents dar. Es konnten außerdem zwei triatomine Arten identifiziert werden, namentlich T. infestans und T. sordida, welche in gemäßigten Klimazonen geeignete Habitate finden. Zu diesen gehören beispielsweise Neuseeland und Teile Australiens, aber auch südeuropäische Länder wie Spanien, Italien, Griechenland und Portugal. Da mit einer Ausweitung der klimatisch geeigneten Gebiete infolge des sich verändernden Klimas zu rechnen ist, wäre ein Monitoring der Vektoren, wie es bereits in Südchina etabliert ist, aber insbesondere die Einführung der Meldepflicht für Amerikanische Trypanosomiasis in diesen Regionen sinnvoll. Die Ergebnisse der Studie zeigen deutlich, dass die bisher vernachlässigte Tropenkrankheit Chagas nicht allein ein Problem des lateinamerikanischen Kontinents ist, sondern deren Erforschung vielmehr weltweit Beachtung finden sollte.
So konzentrierten sich die folgenden Forschungsprojekte der Promotion verstärkt auf die Mechanismen, welche die Entwicklung und Transmission des Parasiten und die Interaktion mit seinen Vektoren betreffen. Von besonderem Interesse waren dabei die ökologischen Prozesse, welche bei der Kolonisation des Darmtrakts der Vektoren durch T. cruzi ablaufen und essentiell für die Proliferation und damit die Übertragung des Parasiten sind. Eine entscheidende Rolle spielen dabei die mit dem Vektor assoziierten Mikroorganismen und ihre funktionellen Fähigkeiten – zusammengefasst als Mikrobiom bezeichnet. Dieses erfüllt wichtige physiologische Funktionen des Insekts und kann beispielsweise das Immunsystem und die Detoxifikation beeinflussen. Um die Veränderungen der organismischen Zusammensetzung und der funktionellen Kapazitäten, welche die Infektion mit dem Pathogen im Darmtrakt der Vektoren auslösen, zu untersuchen, wurde ein metagenomischer Shotgun Sequenzierungsansatz gewählt. Die daraus resultierenden Datensätze wurden anschließend bioinformatisch ausgewertet und auf ihre mikrobielle Zusammensetzung und metabolischen Fähigkeiten hin untersucht. Es zeigte sich zunächst, dass das Bakterium Rhodococcus rhodnii, welches lange als alleiniger echter Symbiont des untersuchten Vektors Rhodnius prolixus galt, in seiner Funktionalität nicht einzigartig im Mikrobiom des Insekts ist. ...
In Zeiten der globalen Klimaerwärmung und des Klimawandels werden Strategien zur Vermeidung, Reduzierung oder Wiederverwertung von CO2-Emissionen sowie die Abkehr von fossilen Energieträgern immer wichtiger. Aus diesem Grund finden Technologien zur Bindung, Speicherung und Wiederverwertung von CO2 immer größere Aufmerksamkeit und diverse chemische als auch biologische Ansätze werden verfolgt. Eine dieser Möglichkeiten umfasst die Reduktion von CO2 mit Hilfe von molekularem Wasserstoff. Im Prozess der direkten Hydrogenierung von CO2 zu Ameisensäure bzw. Formiat wird nicht nur CO2 gebunden, sondern ebenfalls H2 in flüssiger Form gespeichert. Die Ameisensäure weist gegenüber dem hochflüchtigen Wasserstoffgas verschiedene Vorteile auf und zählt zu der Gruppe der flüssigen, organischen Wasserstoffspeicherverbindungen. Daneben ist das Einsatzgebiet von Ameisensäure als Ausgangstoff für Chemikalien oder als mikrobielle Kohlenstoffquelle sehr vielseitig und die Verbindung erfreut sich zunehmenden Interesses.
Die Natur hält biologische Katalysatoren (Enzyme) für die Reduktion von CO2 bereit. Die Gruppe der obligat anaeroben, acetogenen Bakterien verwendet so genannte Formiatdehydrogenasen als CO2-Reduktasen, um CO2 im Wood-Ljungdahl-Weg (WLP) der Bakterien fixieren zu können. Diese Enzyme katalysieren die reversible 2-Elektronen Reduktion von CO2 zu Ameisensäure. Kürzlich konnte aus den beiden Vertretern A. woodii (mesophil) und T. kivui (thermophil) ein neuartiger, cytoplasmatischer Enzymkomplex isoliert werden. Dieser Enzymkomplex koppelt die Reduktion von CO2 direkt an die Oxidation von H2 und wird deshalb als Wasserstoff-abhängige CO2-Reduktase bezeichnet (engl. hydrogen-dependent CO2 reductase, HDCR). Die HDCR katalysiert dabei die reversible Hydrogenierung von CO2 zu Formiat mit annähernd gleicher Kinetik und gleichen Umsatzraten. Die bei der CO2 Reduktion erreichten Umsatzraten übertrafen dabei bisherige chemische als auch biologische Katalysatoren um mehre Größenordnungen.
Im Hinblick auf die besonderen katalytischen Eigenschaften der HDCRs wurde in dieser Arbeit die biotechnologische Anwendbarkeit der Enzyme als Biokatalysatoren zur Speicherung und Sequestrierung von H2 und CO2 in Form von Ameisensäure untersucht. Im Speziellen wurde ein HDCR-basiertes Ganz-Zell-System für das thermophile Bakterium T. kivui entwickelt. Um eine Ganz-Zell basierte Umwandlung von H2 und CO2 zu Formiat zu gewährleisten, wurde zuvor die Weiterverwertung des Formiats zu Acetat im WLP gestoppt. Durch eine Reduktion des zellulären ATP-Gehalts konnte eine weitere Prozessierung des aus der HDCR-Reaktion gebildeten Formiats im Zellstoffwechsel des Bakteriums unterbunden werden. Die Formiatbildung aus H2 und CO2 wurde in Zellsuspensionen von T. kivui untersucht und charakterisiert. Hier zeigten T. kivui Zellen die höchste spezifische Formiatbildungsrate, die bis dato in der Literatur genannt wurde. Ebenfalls wurde in dieser Arbeit die Umwandlung von Synthesegas (H2 + CO2 und CO) und CO zu Formiat geprüft. Bioenergetisch entkoppelte und auf CO-adaptierte T. kivui Zellen konnten in der Tat Synthesegas exklusiv zu Formiat umsetzen. Um die CO-Verwertung zu Acetat und Formiat im Stoffwechsel der Rnf- (A. woodii) und Ech-Acetogenen (T. kivui) verstehen zu können, wurden Mutanten von Δhdcr, ΔcooS, ΔhydBA, Δrnf and Δech2 von A. woodii und T. kivui zur Hilfe genommen. In beiden Organismen war die CO-basierte Formiatbildung vom Vorhandensein eines funktionalen HDCR-Enzymkomplexes abhängig.
Für eine mögliche biotechnologische Anwendung wurde die Maßstabsvergrößerung des Ganz-Zell-Systems angestrebt und hin zum Bioreaktormaßstab mit kontrollierten Prozessbedingungen skaliert. Diese Arbeit demonstriert die effiziente Umwandlung von H2 und CO2 zu Formiat und vice versa unter Verwendung eines Rührkesselreaktors. Der Prozess zeigte eine Effizienz von 100% für die Umwandlung von CO2 zu Formiat und spezifische Raten von 48.3 mmol g-1 h-1 wurden von A. woodii Zellen erreicht. Die spezifische H2-Produktionsrate (qH2) aus der Ameisensäureoxidation betrug 27.6 mmol g-1 h-1 und mehr als 2.12 M Ameisensäure konnte über einen Zeitraum von 195 h oxidiert werden. Wichtige Parameter der Enzymkatalyse wie Wechselzahl (engl. turnover frequency, TOF) und katalytische Produktivität (engl. turnover number, TON) wurden ebenfalls im Versuch bestimmt. Basierend auf dem generierten Prozessverständnis und der effizienten Reversibilität der katalysierten Reaktionen wurde abschließend ein Ganz-Zell-basierter Bioreaktoraufbau gewählt, der die vielfache Speicherung und Freisetzung von H2 in einem einzigen Rührkesselreaktor und unter Verwendung des gleichen Katalysators ermöglicht. Über eine Prozesszeit von 2 Wochen und 15 CO2 Reduktions-/Formiat Oxidations-Zyklen konnte so im Mittel 330 mM Formiat produziert und oxidiert werden.
Zusammenfassend thematisiert diese Arbeit die biotechnologische Anwendbarkeit eines Ganz-Zell-Systems zur Speicherung und Sequestrierung von H2 und CO2 in Form von Formiat und vice versa. Die katalytische Aktivität der betrachteten Organismen fußt dabei auf der Aktivität eines neuartigen Enzymkomplexes, der erstmals in der Gruppe der acetogenen Bakterien entdeckt wurde. Der als Wasserstoff-abhängige CO2-Reduktase bezeichnete Enzymkomplex könnte die zukünftige Konzipierung Enzym-inspirierter und effizienter chemischer Katalysatoren vorantreiben. Auch der Einsatz des Enzyms/der Zellen in so genannten Hydrogelen oder die Etablierung elektrochemischer Prozesse sind vorstellbar. Diese Arbeit stellt somit eine Basis für mögliche zukünftige Anwendungen des etablierten Ganz-Zell-Systems von A. woodii und T. kivui im Bereich der Wasserstoffökonomie dar.
The increasing demand of the high value ω-3 fatty acids due to its beneficial role for human health, explains the huge need for alternative production ways of ω-3 fatty acids. The oleaginous alga Phaeodactylum tricornutum is a prominent candidate and has been investigated as biofactory for ω-3 fatty acids, e.g. the synthesis of eicosapentaenoic acid (EPA). In general, the growth and the lipid content of diatoms can be enhanced by genetic engineering or are influenced by environmental factors, e.g. nutrients, light or temperature.
In this study, the potential of P. tricornutum as biofactory was improved by heterologously expressing the hexose uptake protein 1 (HUP1) from the Chlorophyte Chlorella kessleri.
An in situ localization study revealed that only the full length HUP1 protein fused to eGFP was correctly targeted to the plasma membrane, whereas the N-terminal sequence of the protein is only sufficient to enter the ER. Protein and gene expression data displayed that the gene-promoter combination was relevant for the expression level of HUP1, while only cells expressing the protein under the light-inducible fcpA promoter showed a significant expression. In these mutants an efficient glucose uptake was detectable under mixotrophic growth condition, low light intensities and low glucose concentrations leading to an increased cell dry weight.
In a second approach, the growth and lipid content of wildtype cells were analyzed in a small 1l photobioreactor. Here, a commercial F/2 medium and a common culture medium, ASP and modified versions were compared. There was neither a significant impact on the growth and lipid content in P. tricornutum cells due to the supplemention of trace elements nor due to elevated salt concentrations in the media. In a modified version of ASP medium, with adapted nitrate and phosphate concentration a constantly high biomass productivity was achieved, yielding the highest value of 82 mg l-1 d-1 during the first three days. This was achieved even though light intensity was reduced by 40%. The differences in biomass productivity as well as the lipid content and the lipid composition underlined the importance of the choice of culture medium and the harvest time for enhanced growth and EPA yields in P. tricornutum.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
Sleep is one of the fundamental requirements of all animals from nematodes to humans. It appears in different formats with shared features such as reduced muscle activities and reduced responsiveness to the environment. Despite the long history of sleep research, why a brain must be taken offline for a large portion of each day remains unknown. Moreover, sleep research focused on mammals and birds reveals two stages, rapid-eye-movement (REM) and slow-wave (SW) sleep, alternating during sleep. Whether these two stages of sleep exist in other vertebrates, particularly reptiles, is debated, as is the evolution of sleep in general.
Recordings from the brain of a lizard, the Australian bearded dragon Pogona vitticeps, indicate the presence of two electrophysiological states and provides a better picture of their sleep. Local field potential (LFP) signals, head velocity, eye movements, and heart rate during sleep match the pattern of REM and SW sleep in mammals. The SW and REM sleep patterns that we observed in lizards oscillated continuously for 6 to 10 hours with a period of 80-100 seconds when the ambient temperature was ~27°C. Lizard SW dynamics closely resemble those observed in rodent hippocampal CA1, yet originated from a brain area, the dorsal ventricular ridge (DVR), that does not correspond anatomically or transcriptomically to the mammalian hippocampus. This finding pushes back the probable evolution of these dynamics to the emergence of amniotes, at least 300 million years ago.
Unlike mammals and birds, REM and SW sleep in lizards occupy an almost equal amount of time during sleep. The clock-like alternation between these two sleep states was found initially by measuring the power modulation of two frequency bands, delta and beta. I recorded the full-band LFP and found an infra-slow oscillation (ISO) in the frequency range between 5 and 20 milli-Hz during sleep. The magnitude of ISO increased during sleep and decreased during both wakefulness and arousal during sleep. The up- and down-states of ISO were synchronized with the sleep state alternating rhythm but with a significant time lag dependent on the locations of the recording electrodes. Multi-site LFP recordings indicated that this ISO is a putative propagation wave sweeping extremely slowly, 30-67 µm/sec, from the posterior-dorsal pole to the anterior-ventral pole of the DVR.
Previous studies in other animals showed that brainstem areas such as the locus coeruleus, laterodorsal tegmentum, and periaqueductal gray are involved in sleep states regulation. It is sadly impossible to carry out in vivo recordings in the lizard brainstem without severely affecting them and their quality of life. I thus carried out ex vivo recordings in both DVR and brainstem. Pharmacological stimulation of the brainstem could reversibly silence one distinct EEG pattern characteristic of SW sleep, the sharp-wave and ripple complex, in DVR. An ISO could be recorded simultaneously in both DVR and brainstem. From data collected in both intact and split ex vivo brains, I concluded that there are independent ISO generators in at least two areas, the brainstem and the telencephalon. Their signals may normally be synchronized by long-range connections. The DVR ISO leads the brainstem ISO by ~29 sec. Optogenetic stimulation of brainstem neurons was able to disrupt the ISO in DVR reversibly.
In conclusion, the lizard brain offers a relatively simple model system to study sleep. Despite a diversity of results in different lizard species, my results revealed a number of new findings. Relevant for sleep research in general: 1) REM and SW sleep exist in a reptile. Since they also exist in birds and mammals, they probably existed in their common amniote ancestor, if not earlier. 2) REM and SW occupy equal amounts of time during sleep (50% duty cycle), a unique feature among all described sleep electrophysiological patterns, suggesting the possible existence of a simple central pattern generator of sleep, possibly ancestral. 3) I discovered the existence, in the local field potential, of an infra slow oscillation with extremely slow propagation, locked to the SW-REM alternating rhythm. The causes and mechanisms of this ISO remain to be understood. To my knowledge, the correlation between sleep states and a slow rhythm has only been reported in human scalp EEG recordings so far.
Genetic engineering of Saccharomyces cerevisiae for improved cytosolic isobutanol biosynthesis
(2021)
The finite nature of fossil resources and the environmental problems caused by their excessive usage requires alternative approaches. The transformation from a fossil based economy to one based on renewable biomass is called a “bioeconomy”. To substitute fossil resources, various microorganisms have already been modified for the biosynthesis of valuable chemicals from biomass. However, the development of such efficient microorganisms at an industrial scale, remains a major challenge. The most prominent and robust microorganism for industrial production is the yeast Saccharomyces cerevisiae, which is known to produce ethanol that is used as renewable biofuel. However, S. cerevisiae is also naturally able to produce isobutanol in small amounts. Isobutanol is favoured as a biofuel compared to ethanol due to its higher octane number and lower hygroscopicity, which makes it more suitable for application in conventional combustion engines. In S. cerevisiae, the biosynthesis of isobutanol is permitted by the combination of mitochondrial valine synthesis (catalysed by Ilv2, Ilv5 and Ilv3) and its cytosolic degradation (catalysed by Aro10 and Adh2). The different compartmentalisation of the two pathways limit isobutanol biosynthesis. Thus, Brat et al. (2012) were able to increase the isobutanol yield up to 15 mg/gGlc by cytosolic re localisation of the enzymes Ilv2Δ54, Ilv5Δ48 and Ilv3Δ19 (cyt-ILV), with simultaneous deletion of ilv2. This corresponds to approximately 3.7% of the theoretical yield of 410 mg/gGlc, implying existing limitations in isobutanol biosynthesis, which have been investigated in this work.
For yet unknown reasons, isobutanol was only produced by S. cerevisiae in a valine free medium, according to Brat et al. (2012). This work shows that this can be attributed to the catalytic activity of Ilv2Δ54, which acted as growth inhibitor to S. cerevisiae. By this logic, a negative selection on the ILV2∆54 gene was exerted, which made the ilv2 deletion and simultaneous valine exclusion necessary to maintain the functional expression of toxic ILV2∆54. Furthermore, it was shown that valine exclusion is not mandatory due to the feedback regulation of Ilv2, permitted by Ilv6. Rather, increased isobutanol yield was observed when cytosolic Ilv6∆61 was expressed in the valine free medium, which is explained by the enhanced regulation of Ilv2Δ54 by Ilv6∆61 when BCAA are absent. Isobutanol biosynthesis is neither redox nor NAD(P)H co factor balanced. It was seen that co factor imbalance could be mitigated by the expression of an NADH oxidase (NOX), but not by expression of the NADH dependent ilvC6E6, since the latter showed low in vivo activity. Furthermore, it was seen that NAD(H) imbalance did already limit isobutanol biosynthesis, but the NADP(H) imbalance did not. Another limitation of cytosolic isobutanol biosynthesis is the secretion of the intermediate 2‑dihydroxyisovalerate, which then no longer is taken up by S. cerevisiae, causing a reduced isobutanol yield. This is attributed to insufficient Ilv3∆19 activity, due to poor iron sulphur cluster apo protein maturation. Therefore, it was aimed to replace Ilv3∆19 by heterologous dihydroxyacid dehydratases. Even though some of the enzymes were functionally expressed, none showed better in vivo activity than Ilv3∆19. Therefore, the Ilv3∆19 apo protein maturation was improved. This was achieved by the genomic deletion of fra2 or pim1 as well as by the cytosolic expression of Grx5∆29.
In addition to the isobutanol pathway, S. cerevisiae was optimised for isobutanol biosynthesis by rational and evolutionary engineering. For this purpose, the genes which are necessary for isobutanol production were integrated into the ilv2 locus, and the resulting strain was evolved in a medium containing the toxic amino acid analogue norvaline. Evolved single colonies were isolated, which presented improved growth and increased isobutanol yields (0.59 mg/gGlc) in a valine free medium, as compared to the initial strain. This is explained by a gene dosage effect which occurred during the evolutionary engineering experiment. In collaboration with Dr. Wess, the genes ilv2, bdh1/2, leu4/9, ecm31, ilv1, adh1, gpd1/2 and ald6 were cumulatively deleted in CEN.PK113 7D to block competing metabolic pathways. The resulting strain JWY23 achieved isobutanol yields up to 67.3 mg/gGlc, when expressing the cyt ILV enzymes from a multi copy vector. The most promising approaches of this work, namely the deletion of fra2 and the expression of Grx5∆29, Ilv6∆61, and NOX, were confirmed in this JWY23 strain. The highest isobutanol yield from this work was observed at 72 mg/gGlc for Ilv6∆61 and cyt ILV enzymes expressing JWY23, which corresponds to 17.6% of the theoretical isobutanol yield.
Isobutyric acid (IBA) is a by product of isobutanol biosynthesis, but it is also considered a valuable platform chemical. Therefore, the approaches that improved isobutanol biosynthesis were applied to the biosynthesis of IBA in S. cerevisiae. The highest IBA yield of 9.8 mg/gGlc was observed in a valine free medium by expression of cyt ILV enzymes, NOX and Ald6 in JWY04 (CEN.PK113 7D Δilv2; Δbdh1; Δbdh2; Δleu4; Δleu9; Δecm31; Δilv1). This corresponded to an 8.9 fold increase compared with the control and is, to our best knowledge, the highest IBA yield reported to date for S. cerevisiae.
In recent years, several neuronal differentiation protocols were published that circumvent the requirement of embryoid body (EB) formation under serum-deprivation and simplified medium conditions. But a neuronal default model to establish an approach that works efficiently for all pluripotent cells and neuronal precursors is still lacking. Whether such a default neural mechanism exist and how this is implemented across a broad spectrum of cell source, is addressed in several studies and still controversially discussed. It was proposed that the default neuronal fate is initiated in the absence of extrinsic signals and is achieved by eliminating extracellular inhibitors of neuroectodermal fate and suppressing cell-cell signalling through limited cell density. Previous studies reported that ESC and ECC grown at low density and in absence of exogenous factors or feeder layers die within 24 h but acquire a neural identity as indicated by expression of the neural marker Nestin. Thus, this application is not suitable for generating neural cultures. Furthermore, it was reported that P19 cells survive and express neuroectodermal marker genes in serum-free DMEM/F12 medium containing transferrin, insulin, and selenite, although no neurites were identified.
Based on this background, in this study, a novel approach to induce neuronal differentiation in vitro was developed that implements a nutrient-poor environment, which, in contrast to previous studies, ensures the survival of neuronally differentiated cells over a long period of time and allows normal formation of neurites. Neither the formation of free-floating aggregates nor supplementation of growth factors or known inducers was required to establish a reliable neuronal differentiation protocol. A simple medium, consisting of DMEM/F12+N2 that was highly diluted in salt solution, was sufficient to drive a fast neuronal differentiation in monolayer cultures. Serum deprivation and strong dilution of DMEM/F12+N2 medium cause a nutrient-poor environment in which the influence of growth factors and inducers is minimized. This medium creates a metabolically defined environment that is presumably free of extrinsic signals that prevent the decision of neuronal fate. Analysis of the medium components discovered no actual inducer. Hence, it was suggested that the metabolic composition of the medium exclusively covers specific cell requirements of neurons, therefore ensures their survival, and drives the switch from pluripotent cells to neurons. The self-developed method was established by usage of the murine embryonal carcinoma cell line P19 and could be transferred to murine ESC. Consequently, the method could provide a feasible protocol for a generally valid neuronal default model.
The established protocol provides several advantages such as the possibility to generate stable pure neuronal cultures by a fast, simple, and highly reproducible one-step induction under defined medium conditions with a minimum of exogen effectors. The method is characterised by clear and steady medium conditions that makes the investigation of specific cell requirements during differentiation accessible. It is therefore expected to be a useful tool to investigate the molecular basis of neuronal differentiation as well as for high throughput screenings. The phenotype of mature postmitotic neurons was arising within one week and cultures were shown to stay stable at least for three weeks. The neuronal identity was confirmed by expression of neuronal markers through immunofluorescence staining and mass spectrometry analysis. Furthermore, increased levels of axon markers were detected in early neuronal differentiation and functionality of the synapses of the P19-derived neurons was ascertained by detection of calcium activity. Axonal laser ablation, immediately followed by fast regrowth of connections in the neuronal network, revealed a strong regeneration potential under the given conditions. Furthermore, the generated neurons showed a morphologically distinct phenotype and the formation of neural rosettes. Immunofluorescence staining demonstrated the generation of pure and homogeneous neuronal cultures, free of glial cells.
Retinoic acid (RA) plays an essential role in cell signalling during embryogenesis and efficiently induces neuronal differentiation in vitro in a concentration dependent manner. Neither retinol nor retinoic acid was included in any of the components of the self-prepared medium in this work. However, I observed, dependence on RARβ- and/or RARγ-regulated RA signalling in serum-free monolayer cultures. Nevertheless, neuronal differentiation in serum-free monolayer cultures was assumed to be RARα-independent because (i) RARα was slightly downregulated after neuronal induction, (ii) the truncated RARα of the RAC65 mutant had no effect on induction efficiency, and (iii) a pan-RAR inhibitor suppressed neuronal differentiation. In contrast to serum-free monolayer cultures, the truncated RARα prevented neuronal differentiation by application of the conventional protocol where cells are grown in free floating cell aggregates in serum-containing medium. Proteome analysis of P19 cells, treated by the self-developed differentiation protocol over five days showed increased levels of cellular RA binding proteins that mediate the cellular RA transport and are involved in canonical as well as non-canonical RA signalling.
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1. Das Wachstum und die Fähigkeit zur Butyratproduktion von E. callanderi KIST612 wurde in geschlossenen Batch-Kulturen mit den Substraten Glukose, Methanol, Formiat, H2 + CO2 und CO untersucht. E. callanderi KIST612 zeigte sich nur bei Wachstum auf 20 mM Glukose oder 20 mM Methanol in der Lage, Butyrat in größeren Mengen (3,7 – 4,3 mM) zu produzieren. Das Hauptprodukt bei allen untersuchten Wachstumssubstraten war jedoch Acetat.
2. In bioinformatischen Analysen des Genoms von E. callanderi KIST612 konnte nur eine A1AO-ATP-Synthase gefunden werden, welche eine V-typ c-Untereinheit bestehend aus 4 TMH‘s mit nur einer Na+-Bindestelle aufweist. Diese konnte aus gewaschenen Membranen von E. callanderi durch Saccharose-Dichtegradientenzentrifugation, Anionenaustausch-Chromatographie (DEAE) sowie einer Größenausschluss-Chromatographie (Superose 6) bis zur apparenten Homogenität gereinigt werden. Nach Produktion einzelner Untereinheiten (A, B, C, D, E, F und H) in E. coli und Generierung von Antikörpern, konnten alle Untereinheiten (A, B, C, D, E, F, H, a sowie c) in der gereinigten Enzympräparation immunologisch oder mittels „Peptide-Mass-Fingerprinting“ nachgewiesen werden. Es konnte somit erstmals eine A1AO-ATP-Synthase aus einem mesophilen Organismus ohne Verlust von Untereinheiten gereinigt werden.
3. Der Gesamtkomplex wies unter nativen Bedingungen eine molekulare Masse von ca. 670 kDa auf. In elektronenmikroskopischen Aufnahmen zeigte sich anhand der hantelförmigen Strukturen, dass die A1AO-ATP-Synthase als intakter Gesamtkomplex gereinigt werden konnte.
4. Die gereinigte A1AO-ATP-Synthase wurde zunächst anhand ihrer ATP-Hydrolyse-Aktivität biochemisch charakterisiert. Die ATP-Hydrolyse-Aktivität hatte ein pH-Optimum von 7 – 7,5 und ein Temperaturoptimum bei 37 °C. Durch Messung der ATPase-Aktivität in Abhängigkeit von verschiedenen Mengen an Na+ konnte die vorhergesagte Na+-Abhängigkeit des Enzyms nachgewiesen werden. Zudem zeigten Hemmstoffexperimente mit DCCD, dass dieser Inhibitor mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert. Dies bestätigte nochmals, dass das Enzym funktionell gekoppelt gereinigt werden konnte.
5. Zur weiteren Untersuchung der Ionenspezifität wurde der an die ATP-Hydrolyse gekoppelte Ionentransport durch Rekonstitution des Enzyms in Liposomen und anschließender Messung des Na+- oder H+-Transports gemessen. In den Proteoliposomen konnte mit Hilfe von 22Na+ gezeigt werden, dass das Enzym Natriumionen translozieren kann. Während in Anwesenheit des Natriumionophors ETH 2120 kein 22Na+-Transport beobachtet werden konnte, führte die Anwesenheit des Protonophors TCS zu einer geringfügigen Stimulation der 22Na+-Translokation. Insgesamt konnte ein primärer Na+-Transport nachgewiesen werden, welcher von der A1AO-ATP-Synthase aus E. callanderi katalysiert wird.
6. Durch Rekonstitution der A1AO-ATP-Synthase aus E. callanderi in Liposomen konnte erstmals biochemisch nachgewiesen werden, dass ein solches Enzym trotz seiner V-Typ c-Untereinheit in der Lage ist, ATP zu synthetisieren. Durch die Zugabe von Ionophoren (ETH 2120 und TCS) konnte der elektrochemische Ionengradient aufgehoben werden, wodurch keine ATP-Synthese beobachtet werden konnte. Der erstmalige Nachweis der ATP-Synthese wurde bei einem ΔµNa+ von 270 mV erbracht.
7. Die ATP-Synthese zeigte sich ebenfalls abhängig von der Na+-Konzentration. Der KM-Wert lag bei 1,1 ± 0,4 mM und war vergleichbar mit dem für die ATP-Hydrolyse ermittelten Wert. Ebenso konnte für die ATP-Synthese-Richtung gezeigt werden, dass DCCD mit Na+ um die gemeinsame Bindestelle in der c-Untereinheit konkurriert.
8. Um den biochemischen Nachweis zu erbringen, dass die A1AO-ATP-Synthase auch unter physiologisch relevanten Potentialen zur ATP-Synthese befähigt ist, wurde der energetische Schwellenwert der ATP-Synthese bestimmt. Dieser betrug 87 mV als Triebkraft für ΔpNa, 94 mV als Triebkraft für Δψ und 90 mV als Triebkraft für ΔµNa+. Erstaunlicherweise konnte die ATP-Synthese der A1AO-ATP-Synthase aus E. callanderi KIST612 sowohl durch Δψ als auch ΔpNa angetrieben werden. Unterschiedliche Kombinationen von Δψ und ΔpNa führten zu dem gleichen energetischen Schwellenwert; Δψ und ΔpNa waren im Enzym aus E. callanderi KIST612 äquivalente Triebkräfte.
9. Der energetische Schwellenwert der A1AO-ATP-Synthase aus E. callanderi KIST612 wurde mit dem der F1FO-ATP-Synthasen aus A. woodii, E. coli und P. modestum verglichen. Dazu wurden die Enzyme im ATP-Synthase-defizienten E. coli-Stamm DK8 produziert und anschließend durch Ni2+-NTA-Affinitätschromatographie gereinigt. Nach Einbau der Enzyme in Liposomen waren alle Enzyme in der Lage, ATP als Reaktion auf ΔµNa+ (A. woodii und P. modestum) oder ΔµH+ (E. coli) zu synthetisieren. Im Vergleich zum Enzym aus E. callanderi zeigten sich zwei auffällige Unterschiede. Erstens war keine der F1FO-ATP-Synthasen in der Lage, ΔpNa/ΔpH als alleinige Triebkraft zu nutzen. Während die ATP-Synthese in den Enzymen aus E. coli und P. modestum nur durch ΔµH+ bzw. ΔµNa+ angetrieben werden konnte, konnte das Enzym aus A. woodii zusätzlich auch durch Δψ als einzige Triebkraft angetrieben werden.
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This work comprises the investigation of four different biosynthesis gene clusters from Xenorhabdus. Xenorhabdus is an entomopathogenic bacterium that lives in mutualistic symbiosis with its Steinernema nematode host and together they infect and kill insect larvae. Xenorhabdus is well known for the production of so-called specialised metabolites and many of these compounds are synthesised by non-ribosomal peptide synthetases (NRPSs) or NRPS-polyketide synthase (PKS)-hybrids. These enzymes are organised in a modular manner and produce structurally very diverse molecules, often with the help of modifying domains and tailoring enzymes. In general, the genes involved in the biosynthesis are organised in so-called biosynthetic gene clusters (BGCs) in the genome of the producing strain. Exchanging the native promoter with an inducible promoter, e.g. PBAD, allows the targeted activation of the BGC and in turn the analysis of the biosynthesis product via LC-MS analysis.
The first BGC investigated in this work is responsible for the biosynthesis of xenofuranones. Based on gene deletions, this work shows that the NRPS-like enzyme XfsA produces a carboxylated furanone intermediate which is subsequently decarboxylated by XfsB to yield xenofuranone B. The next step in xenofuranone biosynthesis is the O-methylation of xenofuranone B to yield xenofuranone A. A comparative proteomics approach allowed the identification of four methyltransferase candidates and subsequent gene deletions confirmed one of the candidates to be responsible for methylation of xenofuranone B. The proteome analysis was based on the comparison of X. szentirmaii WT and X. szentirmaii Δhfq because distinct levels of the methylated xenofuranone A were observed when the xfs BGC was activated in either WT or Δhfq strain. Hfq is a global transcriptional regulator whose deletion is associated with the down regulation of natural product biosynthesis in Xenorhabdus. The strong PBAD activation of the xfs BGC also allowed the detection of two novel xenofuranone derivatives which arise from incorporation of one 4-hydroxyphenylpyruvic acid as first or second building block, respectively.
PBAD based activation of the second BGC addressed in this work lead to the detection of a novel metabolite and compound purification allowed NMR-based structure elucidation. The molecule exhibits two pyrrolizidine moieties and was named pyrrolizwilline (pyrrolizidine + twin (German: “Zwilling”)). The BGC comprises seven genes and single gene deletions as well as heterologous expression in E. coli and NRPS engineering were conducted to investigate the biosynthesis. The first two genes xhpA and xhpB encode a bimodular NRPS and a monooxygenase which synthesise a pyrrolizixenamide-like structure, similar to PxaA and PxaB in pyrrolizixenamide biosynthesis. It is suggested that the acyl side chain incorporated by XhpA is removed by the α,β-hydrolase XhpG. The keto function is then reduced by two subsequent two electron reductions catalysed by XhpC and XhpD. One of these two reduced pyrrolizidine units most likely is extended with glyoxalate prior to non-enzymatic dimerisation with the second pyrrolizidine moiety. To finally yield pyrrolizwilline, L-valine is incorporated, probably by the free-standing condensation domain XhpF.
The third BGC investigated is responsible for the production of a tripeptide composed of β-D-homoserine, α-hydroxyglycine and L-valine and is referred to as glyoxpeptide. This work demonstrates that the previously observed glyoxpeptide derivative is derived from glycerol present in the culture medium. Furthermore, this work shows that the monooxygenase domain, which is found in an unusual position between motifs A8 and A9 within the adenylation domain, is responsible for the α-hydroxylation of glycine. It is suggested that the α-hydroxylation of glycine renders the tripeptide prone to hydrolysis via hemiacetal formation. Hence, the XgsC_MonoOx domain might be an interesting candidate for further NRPS engineering.
The fourth BGC addressed is responsible for the production of xildivalines and this work describes two additional derivatives which are detected only when the promoter is exchanged and activated in the X. hominickii WT strain but not in X. hominickii Δhfq. Deletion of the methyltransferase encoding gene xisE results in the production of non-methylated xildivalines. It remains to be determined when the N-methylation of L-valine takes place. It is discussed that the methyltransferase could act on the NRPS released product but also during the assembly. The peptide deformylase is not involved in the proposed biosynthesis as xildivaline production is detected in a ΔxisD strain. The PKS XisB features two adjacent, so-called tandem T domains. The inactivation of the first or the second T domain by point mutation causes decreased production titres of detected xildivalines in the respective mutant strain when compared to the wild type.
Clean water is fundamental to human health and ecosystem integrity. However, water quality deteriorates due to novel anthropogenic pollutants present at microgram per liter concentrations in urban water cycles (termed micropollutants). Wastewater treatment plants (WWTP) have been identified as major point sources for aquatic (micro-)pollutants. Chemical and ecotoxicological analyses have shown that conventional biological WWTPs do not fully remove micropollutants and associated toxicities, which is often because of mobile, polar and/or recalcitrant compounds and transformation products (TPs). To minimize possible environmental risks, advanced wastewater treatment (AWWT) technologies could be a promising mitigation measure. Multiple processes are therefore being developed and evaluated such as ozonation and ozonation followed by granulated activated carbon (GAC) or biological filtration. Assessing the performance of these combined AWWTs was the focus the TransRisk project. Within this project, this thesis accomplished four major goals.
Firstly, the preparation of (waste)water samples was optimised for in vitro bioassays. Acidification, filtration and solid phase extraction (SPE) were tested for their impact on environmentally relevant in vitro endocrine activities, mutagenicity, genotoxicity and cytotoxicity. Significantly different outcomes of these assays were detected comparing neutral and acidified samples. Sample filtration had a lesser impact, but in some cases retention of particle-bound compounds could have caused significant toxicity losses. Out of three SPE sorbents the Telos C18/ENV at sample pH 2.5 extracted highest toxicity, some undetected in aqueous samples. These results indicate that sample preparation needs to be optimised for specific sample matrices and bioassays to avoid false-positive or -negative detects in effect-based analyses.
Secondly, the above listed in vitro toxicities were monitored in a protected region for drinking water production in South-West Germany (2012-2015). Out of 30 sampling sites surface water and groundwater were the least polluted. Nonetheless, a few groundwater samples induced high anti-estrogenic activity that prompted further monitoring. The latter included a waterworks in which no toxicity was detected. Hospital wastewater also had elevated in vitro toxicities and hospitals are, thus, relevant intervention points for source control. The biological WWTPs were effective in removing most of the detected toxicity, and the selected bioassays proved to be pertinent tools for water quality assessment and prioritisation of pollution hotspots.
Thirdly, the in vivo bioassay ISO10872 based on Caenorhabditis elegans (C. elegans) was adapted for this thesis. Using this model, a median effect concentration (EC50) for reproductive toxicity of the polycyclic aromatic hydrocarbon β-naphthoflavone (β- NF) of 114 µg/L was computed which is slightly lower than reported in the scientific literature. β-NF induced cyp-35A3::GFP (a biomarker in transgenic animals) in a time and concentration dependent manner (≤ 21.3–24 fold above controls). β-NF spiked wastewater samples supported earlier hypotheses on particle-bound pollutants. Reproductive toxicity (96 h) and cyp-35A3 induction (24 h) of biologically treated and/or ozonated wastewater extracts and growth promoting effects of GAC/biologically filtered ozonated wastewater extracts were observed. This suggested the presence of residual bioactive/toxic chemicals not included in the targeted chemical analysis. It also highlighted the importance of integrating multiple (apical and molecular) endpoints in wastewater assessments.
Fourthly, five in vitro and the adapted C. elegans bioassay were integrated into a wastewater quality evaluation (developed within TransRisk). Out of the five AWWT options, ozonation (at 1 g O3,applied/g DOC, HRT ~ 18 min) combined with nonaerated GAC filtration was rated most effective for toxicity removal. All five AWWTs largely removed estrogenic and (anti-)androgenic activities, but not anti-estrogenic activity and mutagenicity, which even increased during ozonation. This has been observed in related studies and points towards toxic TPs. These results also emphasized the need for implementing an effective post-treatment for ozonation. The results from a parallel in vivo study with Lumbriculus variegatus and Potamopyrgus antipodarum conducted on site at the WWTP (using flow through systems) were in accordance with the C. elegans results. In this context, it is suggested to further implement C. elegans as sensitive, feasible and ecologically relevant model.
In conclusion, this thesis shows how optimised sample preparation, long-term (in vitro) environmental monitoring, sensitive and ecologically relevant (in vivo) bioassays as well as innovative evaluation concepts, are pivotal in improving the removal of micropollutants and their toxicities with AWWTs. Future research should further develop and evaluate measures at sewer systems, conventional biological, tertiary and other advanced treatment technologies, as well as sociopolitical strategies (e.g., source control or natural conservation) and restoration projects. The effect-based tools optimised in this thesis will support assessing their success.
Zur Evolution der Hirnmorphologie und Anpassungen an Extremhabitate im Taxon Poecilia (Teleostei)
(2020)
Diese Dissertation befasst sich mit den Auswirkungen kontrastierender Umweltbedingungen auf die Gehirnmorphologie von neotropischen Fischen der Gattung Poecilia, welche unterschiedlichen abiotischen sowie biotischen Stressoren ausgesetzt sind. Da das Gehirn der Teleostei ein energetisch kostspieliges Organ und viel plastischer ist als z. B. bei Säugetieren, stellt sich die Frage, wie die Gehirnanatomie durch divergierende ökologische Faktoren in verschiedenen Umgebungen geformt wird, die ´extreme´, ´ressourcenbeschränkte und günstige´ Umgebungen repräsentieren. Zur Beantwortung dieser Frage wurden intraspezifische Studien an freilebenden und Laborindividuen von Poecilia-Arten durchgeführt, um die evolutionäre und ökologische Formgebung des Gehirns besser verstehen zu lernen. Im ersten Teil der Arbeit wurden Gehirnvolumina verglichen zwischen reproduktiv isolierten Populationen des neotropischen Fisches Poecilia mexicana (Ntotal = 95), die in Dunkelheit leben (Cueva Luna Azufre), in einem nahegelegenen Oberflächenhabitat (El Azufre), welcher giftigen Schwefelwasserstoff enthält und einer Kombination aus beiden Stressoren Dunkelheit und H2S (Cueva del Azufre). In einer zweiten Studie wurde auf anatomische („konvergente“) Veränderungen im Teleost-Gehirn entlang eines natürlichen Gradienten von Sulfidkonzentrationen getestet. Hierfür wurden Gehirne (Ntotal = 100) von P. mexicana verglichen, die in drei Flusssystemen im Süden Mexikos unabhängig voneinander eine erhöhte Toleranz gegenüber Schwefelwasserstoff (H2S) entwickelt haben. Dazu gehörten eine phylogenetisch alte H2S-adaptierte Form (P. sulphuraria) und zwei P. mexicana Formen, welche frühere Stufen der Anpassung an H2S darstellen. Zur Überprüfung des Einflusses anderer abiotischer und biotischer Faktoren auf die Morphologie der Gehirnregionen wurde eine weitere Studie durchgeführt. Hierbei wurden die phänotypischen Variationen der Gehirnregionen und der Körpermorphologie von Poecilia vivipara-Populationen (Ntotal = 211) aus Lagunen des Restinga de Jurubatiba Nationalpark untersucht, die sich in abiotischen Umgebungsbedingungen, insbesondere in Salzgehalt, Wassertransparenz, Phosphat und Nitrat sowie biotischen Faktoren wie Prädatorendichte unterschieden. Die erste Studie zeigte lebensraumabhängige Unterschiede bei freilebenden Fischen. Bei Fischen, die in Dunkelheit ohne H2S (LA) oder in Oberflächenhabitaten mit H2S lebten, wurden vergrößerte telenzephale Lappen, kleinere Augen und optische Tekta gefunden. Fische aus der sulfidischen Höhle (CA) zeigten zusätzlich vergrößerte Corpus cerebelli. Der Vergleich mit den Gehirnen von Labor aufgezogenen weiblichen Fischen (Ntotal = 25) zeigt eine allgemeine Verringerung der Gehirngröße sowie eine geringe Abweichung der Gehirngröße zwischen Labor aufgezogenen und freilebenden Fischen. Auch in der zweiten Studie zeigten alle in H2S-haltigen Lebensräumen lebenden Fische kleinere Augen, ein kleineres optisches Tektum und ein kleineres Gehirnvolumen, jedoch größere Corpus cerebelli und Hypothalamusvolumen als Fische aus nicht-sulfidischen Lebensräumen. Flusssystem-spezifische Effekte wurden für die telenzephalen Lappen, das gesamte Gehirn und die Augengröße festgestellt, da die Geschlechter je nach Quelle des Flusssystems unterschiedlich auf das Vorhandensein von H2S reagierten. Die dritte Studie zeigt auch, dass andere Umwelteinflüsse bemerkenswerte Verschiebungen im Gehirn und in den Gehirnregionen verursachen können. Fische, die im Süßwasser leben, zeigten eine verringerte Gesamthirngröße, telenzephale Lappen, Corpus cerebelli und Hypothalamusvolumen. Darüber hinaus zeigten Fische aus Salzwasserlagunen (hypersalin), ein verringertes Volumen des optischen Tektum, während telenzephale Lappen, Corpus cerebelli und Hypothalamusvolumen im Vergleich zu Süßwasserfischen vergrößert waren. Im Brackwasser lebende Fische wiesen im Vergleich zu Süß- und Salzwasserfischen die größten Gehirnregion-Volumen auf. Darüber hinaus zeigten die Ergebnisse über die Lagunen hinweg auch Unterschiede in der Morphologie der Kopf- und Augendurchmesser. Bei Augengröße, Kopfgröße, optischem Tektum Volumen, Hypothalamusvolumen und dem Gesamthirnvolumen wurde ein sexueller Dimorphismus beobachtet. Die dargestellten Ergebnisse verdeutlichen, dass die gefundenen Muster nahezu mit denen von H2S-Fischen identisch sind. Die ausgeprägten Unterschiede in den Hirnregionen zwischen freilebenden Fischen können als Teil der Mosaikentwicklung interpretiert werden. Die Ergebnisse der Laborpopulation zeigen jedoch eine hohe phänotypische Plastizität. Diese Studie unterstreicht damit die Bedeutung der Kombination der Untersuchung von freilebenden mit im Labor lebenden Individuen zur Beantwortung von Fragen der Gehirnentwicklung. Kleinere Augen und ein kleineres optisches Tektum, aber größere telenzephale Lappen wurden auch bei Fischen aus einem sulfidischen Oberflächenhabitat in der Nähe einer der Höhlen gefunden und sind den Ergebnissen zufolge das Resultat begrenzter Sehkraft in trüben sulfidischen Lebensräumen.
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Ziel dieser Arbeit war es, einen genaueren Einblick in die Rolle von PaCLPXP für den Energiemetabolismus von P. anserina zu erhalten und mögliche Komponenten zu identifizieren, welche wichtig für die Langlebigkeit der PaClpP-Deletionsmutante sind. Folgende neue Erkenntnisse konnten hierbei gewonnen werden:
1. Die Substrat-Analyse durch eine Cycloheximid-Behandlung und anschließender Proteom-Analyse legte erfolgreich eine Reihe potentieller bisher nicht bekannter Substrate von PaCLPP offen. Interessanterweise waren unter den identifizierten Proteinen viele ribosomale Untereinheiten und Komponenten verschiedener Stoffwechselwege des Energiemetabolismus zu finden. Am auffälligsten unter diesen Substraten war die extreme Anreicherung eines Retikulon-ähnlichen Proteins, das einen neuen Aspekt der möglichen molekularbiologischen Rolle von PaCLPP in P. anserina andeutet.
2. Durch die Zugabe von Butyrat zum Medium, konnte erfolgreich die Autophagie sowohl im P. anserina Wildtyp als auch in der PaClpP-Deletionsmutante reduziert werden. Diese Verminderung der Autophagie sorgt bei ΔPaClpP für eine Verkürzung der Lebensspanne. Dieser Effekt ist spezifisch für die PaClpP-Deletionsmutante, während die Auswirkung von Butyrat auf den Wildtyp nur marginal ist. Dieses Ergebnis untermauert frühere Analysen dieser Deletionsmutante, welche besagen, dass die Langlebigkeit von ΔPaClpP Autophagie abhängig ist (Knuppertz und Osiewacz, 2017).
3. Die Metabolom-Analyse von ΔPaClpP im Vergleich zum Wildtyp zeigt, dass das Fehlen der PaCLPP zu Veränderungen in der Menge der Metaboliten der Glykolyse und des Citratzyklus kommt. Außerdem sind die Mengen der meisten Aminosäuren und der Nukleotide betroffen. Diese Analyse beweist, dass das Fehlen dieser mitochondrialen Protease weitreichende Folgen für die ganze Zelle hat. Durch die signifikante Verringerung von ATP und die Anreicherung von AMP in jungen ΔPaClpP-Stämmen und durch den Umstand der gesteigerten Autophagie in dieser Mutante, fiel das Augenmerk auf die AMPK. Dieses veränderte AMP/ATP-Verhältnis ist ein Indiz für eine gesteigerte AMPK-Aktivität und könnte auch den Umstand der gesteigerten Autophagie in ΔPaClpP erklären.
4. Das Gen codierend für die katalytische α-Untereinheit der AMPK (PaSnf1) konnte erfolgreich in P. anserina deletiert werden. Das Fehlen von PaSNF1 führt zu einer reduzierten Wuchsrate, eine beeinträchtige weibliche Fertilität und eine verzögerte Sporenreifung. Es konnte gezeigt werden, dass die Autophagie infolge einer PaSnf1-Deletion nicht gänzlich unterdrückt wird, PaSNF1 allerdings für die Stress-induzierte Autophagie notwendig ist. Überraschenderweise führt die Abwesenheit von PaSNF1 zu einer verlängerten Lebensspanne im Vergleich zum Wildtyp. Die meisten Effekte infolge einer PaSnf1-Deletion konnten durch die Einbringung eines FLAG::PaSNF1-Konstrukts komplementiert werden.
5. Eine gleichzeitige PaSnf1 und PaClpP-Deletion führt zu eine unerwarteten, extremen Lebenspannenverlängerung, die die Verlängerung der Lebensspanne bei der PaClpP-Deletionsmutante noch übertrifft. Interessanterweise geht dieser Phänotyp nicht mit einer erhöhten Autophagie einher. Des Weiteren konnte beobachtet werden, dass das Fehlen von PaSNF1 sowohl in ΔPaSnf1 als auch in ΔPaSnf1/ΔPaClpP zu einer veränderten Mitochondrien-Morphologie im Alter führt. Die Abwesenheit von PaSNF1 verursacht, dass die Stämme auch im Alter (20d) noch überwiegend filamentöse Mitochondrien aufweisen. Zudem zeigen die drei analysierten Deletionsstämme (ΔPaSnf1, ΔPaClpP und ΔPaSnf1/ΔPaClpP) massive Einschränkungen wenn sie auf die mitochondriale Funktion angewiesen sind.
6. Auffallend war, dass bei ΔPaSnf1, ΔPaClpP und bei ΔPaSnf1/ΔPaClpP die Stämme mit dem Paarungstyp „mat-“ langlebiger sind als die Stämme mit dem Paarungstyp „mat+“. Dieser Effekt ist bei der ΔPaSnf1/ΔPaClpP-Doppelmutante am stärksten ausgeprägt. Weitere Untersuchungen dazu ergaben, dass die Paarungstypen immer dann eine Rolle spielen, wenn die Stämme mitochondrialem Stress ausgesetzt, oder aber auf die mitochondriale Funktion angewiesen sind. Verantwortlich für diese Unterschiede sind zwei rmp1-Allele, die mit den unterschiedlichen Paarungstyp-Loci gekoppelt sind und mit dem jeweiligen Paarungstyp-Locus vererbt werden (rmp1-1 mit „mat-“; rmp1-2 mit „mat+“).
Seit den 1950er Jahren hat sich Plastik als unverzichtbare Ressource im menschlichen Alltag etabliert. Als negative Folge dieses Booms wird seit einigen Jahrzehnten jedoch eine zunehmende Belastung aquatischer Ökosysteme mit Plastikmüll bzw. dessen Degradationsprodukten, sogenanntes „Mikroplastik“ (MP, < 5 mm) bzw. „Nanoplastik“ (NP, < 1 µm), beobachtet. Ziel dieser Arbeit war die Untersuchung des aktuellen Vorkommens von MP in limnischen Gewässern, die Analyse der Interaktion zwischen MP und limnischen Wirbellosenarten und der daraus resultierenden Toxizität sowie eine erste Risikoabschätzung.
Das Vorkommen von Mikroplastik in limnischen Gewässern wurde exemplarisch anhand der Elbe als großes Fließgewässer in Deutschland untersucht. Durch die Auswertung von elf Probestellen entlang des Verlaufs der Mittel- und Unterelbe konnte gezeigt werden, dass die MP-Konzentrationen im Sediment (2,26x10^4 – 2,27x10^7 P m^-3) im Mittel fast 150.000-fach höher sind als in der Wasserphase (0,88–13,24 P m^-3). Sedimente sind somit eine Senke für MP. Die Zusammensetzung der Polymerarten sowie MP-Formen deuten zudem an, dass die Partikel sowohl aus diffusen wie auch aus Punktquellen (z.B. Industrieabwässer) stammen. Im globalen Vergleich können die MP-Konzentrationen in deutschen Fließgewässern z. Z. als durchschnittlich betrachtet werden. Allerdings muss insgesamt davon ausgegangen werden, dass die bisher bestimmten MP-Umweltkonzentrationen die realen Konzentrationen möglicherweise unterschätzen. So zeigte die Elbestudie, dass die Sedimentfeinfraktion < 100 µm einen bedeutenden Polymeranteil enthielt. Die meisten bisher durchgeführten Studien zur Bestimmung von MP-Partikeln in Flüssen haben Partikel < 100 µm jedoch nicht in ihrer Analyse berücksichtigt.
Die Interaktion von MP mit limnischer Biota wurde anhand der Artgruppen der Muscheln (Bivalvia), Schnecken (Gastropoda) sowie Krebstiere (Crustacea) näher untersucht. Die Intensität der Interaktion ist maßgeblich von der Aufnahme von MP durch die verschiedenen Arten abhängig. Anhand von zahlreichen Aufnahmestudien mit verschiedenen limnischen Arten, darunter den Muschelarten Dreissena polymorpha, Sinanodonta woodiana und Anodonta anatina, der Lungenschnecke Lymnaea stagnalis sowie der Amphipodenart Gammarus pulex, wurde nachgewiesen, dass die MP-Aufnahme von den Eigenschaften der exponierten Arten bzw. Individuen, den MP-Charakteristika sowie den Expositionsbedingungen abhängt. Die Experimente mit Muscheln verdeutlichten die rasche Aufnahme, aber auch Exkretion von MP-Partikeln innerhalb weniger Stunden. In allen drei Artgruppen war die Aufnahme konzentrationsabhängig mit zunehmender Aufnahme bei steigenden MP-Konzentrationen. Die Muschelexperimente zeigten jedoch auch, dass eine gleichzeitige Exposition mit anderen Partikeln (z.B. Nahrung) zu einer reduzierten Aufnahme führt. Auch die Größe der Testorganismen beeinflusste die Aufnahme: So nahmen im Fall der Muscheln und Krebse kleinere Individuen (bzw. im Fall der Muscheln auch Arten) relativ pro Körpermasse mehr MP-Partikel auf als größere Individuen bzw. Arten. Für alle untersuchten Arten wurde darüber hinaus gezeigt, dass die MP-Größe relevanten Einfluss auf die Menge an aufgenommenen Partikeln hat.
Ein Vergleich zwischen den Artgruppen zeigte, dass Muscheln als filtrierende Organismen in den Laboruntersuchungen bei gleicher Expositionskonzentration mehr MP aufnahmen als Krebse (Zerkleinerer) und Schnecken (Weidegänger). Im Gegensatz zu Muscheln nutzen Krebstiere und Schnecken allerdings die Grenzschicht zwischen Wasser- und Sedimentphase als Suchraum für ihre Nahrung und sind in der Umwelt (auf Grund des höheren MP-Vorkommens in Sedimenten) somit möglicherweise gegenüber höheren MP-Konzentrationen exponiert als Muscheln. Die Extrapolation der gewonnenen Labordaten sowie der Vergleich mit publizierten Umweltdaten legen allerdings nahe, dass das MP-Vorkommen in Individuen aller drei Artgruppen bisher auf einige wenige MP-Partikel begrenzt ist. Ausgeprägte Unterschiede zwischen den Artgruppen sind bisher nicht erkennbar.
MP-Toxizitätsstudien mit G. pulex, L. stagnalis sowie D. polymorpha konnten trotz der Berücksichtigung einer Vielzahl an Endpunkten (Mortalität, Reproduktion, Nahrungsaufnahme, oxidativer Stress, Energiereserven, Immunzellaktivität) und trotz des Einsatzes zum Teil sehr hoher MP-Konzentrationen weit oberhalb aktueller Umweltkonzentrationen nur sehr wenige MP-induzierte Effekte nachweisen, darunter eine Steigerung der Filtrationsaktivität (D. polymorpha) bzw. Veränderung der Immunfunktion von Hämolymphzellen (L. stagnalis).
Zur weiteren Risikoabschätzung wurden diese Studienergebnisse mit publizierten Daten für marine und limnische Muschel- und Krebsarten in Artenempfindlichkeitsverteilungen (Species Sensitivity Distributions, SSD) zusammengeführt und jeweils eine SSD für Muscheln und Krebstiere erstellt. Die Erstellung einer SSD nur für limnische Arten ist zum jetzigen Zeitpunkt auf Grund der geringen Datenlage noch nicht möglich.
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Human GLUTs represent a family of specialized transporters that facilitate the diffusion of hexoses through membranes along a concentration gradient. The 14 isoforms share high sequence identity but differ in substrate specificity and affinity, and tissue distribution. According to their structure similarity, GLUTs are divided into three classes, with class 1 comprising the most intensively studied isoforms GLUTs1 4. An abnormal function of different GLUT members has been related to the pathogenesis of various diseases, including cancer and diabetes. Hence, GLUTs are the subject of intensive research, and efforts concentrate on identifying GLUT-selective ligands for putative medical purposes and their application in studies aiming to further unravel the metabolic roles of these transporters.
The hexose transporter deficient (hxt0) yeast strain EBY.VW4000 is devoid of all its endogenous hexose transporters and unable to grow on glucose or related hexoses. This strain has proven to be a valuable platform to investigate heterologous transporters due to its easy handling, increased robustness, and versatile applications. However, the functional expression of GLUTs in yeast requires certain modifications. Single point mutations of GLUT1 and GLUT5 led to their functional expression in EBY.VW4000, whereas the native GLUT1 was actively expressed in EBY.S7, a hxt0 strain carrying the fgy1 mutation that putatively reduces the phosphatidylinositol-4-phosphate (PI4P) content in the plasma membrane. GLUT4 was only actively expressed in the hxt0 strain SDY.022, which also contains the fgy1 mutation and in which ERG4 is additionally deleted. Erg4 is one of the late enzymes in the ergosterol pathway, and therefore SDY.022 probably has an altered sterol composition in its membrane.
The goal of this thesis was to actively express GLUT2 and GLUT3 in a hxt0 yeast strain, providing a convenient system for their ligand screening. A PCR-derived amino acid exchange in the sequence of GLUT3 enabled its functional expression in EBY.VW4000 and the unmodified GLUT3 protein was active in EBY.S7. Functional expression of GLUT2 was achieved by rational design. The extracellular loop between the transmembrane regions 1 and 2 is significantly larger in GLUT2 than in other class 1 GLUTs. By truncating this loop by 34 amino acids and exchanging an alanine for a serine, a GLUT3-like loop was implemented. The resulting construct GLUT2∆loopS was functional in EBY.S7. With an additional point mutation in the transmembrane region 11, GLUT2∆loopS_Q455R was also actively expressed in EBY.VW4000. Inhibition studies with the known GLUT inhibitors phloretin and quercetin showed a reduced transporter activity for GLUT2 and GLUT3 in uptake assays and growth tests when inhibitors were present, demonstrating that both systems are amenable for ligand screening experiments.
The newly established GLUT2 yeast system was then used to screen a library of compounds pre-selected by in silico screening. Thereby, eleven identified GLUT2 inhibitors exhibited strong potencies with IC50 values ranging from 0.61 to 19.3 µM. By employing the other yeast systems, these compounds were tested for their effects on GLUT1, and GLUTs3-5, revealing that nine of the identified ligands were GLUT2-selective. In contrast, one was a pan-class 1 inhibitor (inhibiting GLUTs1-4), and one affected GLUT2 and GLUT5, the two fructose transporting isoforms. These compounds will serve as useful tools for investigations on the role of GLUT2 in metabolic diseases and might even evolve into pharmaceutical agents targeting GLUT2-associated diseases.
Due to the beneficial effect of the putatively changed sterol composition in SDY.022 (by ERG4 deletion) on the functional expression of GLUT4, it was hypothesized that the presence of the human sterol cholesterol, or cholesterol-like sterols, might have a beneficial effect on GLUT expression, too. Thus, it was attempted to generate hxt0 strains that synthesize these sterols by genetic modifications targeting the ergosterol pathway. In the scope of these experiments, several strains with different sterol compositions were generated. Drop tests on glucose medium with the different strains expressing GLUT1 or GLUT4 revealed that the deletion of ERG6 is clearly advantageous for a functional expression of GLUT1 (but not GLUT4). This indicates that the methyl group at the ergosterol side chain (introduced by Erg6 and reduced by Erg4) negatively influences GLUT1 activity. However, this effect on GLUT1 activity was less pronounced than the putative altered PI4P content in EBY.S7.
Additionally, in this thesis, a new tool to measure glucose transport rates of transporters expressed in the hxt0 yeast system was developed to facilitate their kinetic characterization. For this, the pH-sensitive GFP variant pHluorin was employed as a biosensor for the cytosolic pH (pHcyt) by measuring the ratio (R390/470) of emission intensities at 512 nm from two different excitation wavelengths (390 and 470 nm). Sugar-starved cells exhibit a slightly acidic pHcyt because ATP production is depleted, reducing the activity of ATP-dependent proton pumps.
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My PhD work employed genetic and pharmacological manipulations, coupled with highresolution live imaging, to understand intercellular communications during zebrafish cardiovascular development. The heart is the first organ to form, and it is composed of several tissues, among which interactions are crucial. I identified two important interactions between muscular and non-muscular tissues in poorly characterized contexts, and the molecules required for the signalling. First, I discovered an important cellular and molecular crosstalk orchestrating the development of the cardiac outflow tract (i.e., the aortic root in mammals).
Endothelial-derived TGF-beta signalling controls the generation of the local extracellular matrix (ECM). The ECM in turn affects endothelial proliferation as well as smooth muscle cell organization (Boezio et al, 2020; Bensimon-Brito*, Boezio* et al, 2020). In my second project, I investigated the crosstalk between the epicardial layer and the myocardial wall. By generating epicardial-impairment models, I identified a novel role for the epicardium in regulating cardiomyocyte volume during heart development (Boezio et al, 2021). Ultimately, this research contributed to our understanding of how paracrine signalling controls the multicellular interactions integral to organogenesis.
Tissue translocation, multigenerational and population effects of microplastics in Daphnia magna
(2021)
The last century saw the widespread adoption of plastic materials throughout nearly every aspect of our lives. Plastics are synthetic polymers that are made up of monomer chains. The properties of the monomer in conjunction with chemical additives allow plastics to have a sheer endless variety of features and use cases. They are cheap, lightweight, and extremely durable. Plastic materials are often engineered for single-use and in conjunction with high production volumes and insufficient waste management and recycling across the globe, this leads to a large number of plastics entering the environment. Marine ecosystems are considered sinks. However, freshwater ecosystems as entry pathways are highly affected by plastic waste as well. Throughout the past decade, the impact of plastic waste on human and environmental health has received a lot of attention from the ecotoxicological community as well as the public. Small plastic fragments (< 1 mm called microplastics) are a large part of this emerging field of research. Within this, the water flea Daphnia magna is probably the most common organism that is used to assess microplastics toxicity. As a filter-feeding organism, it indiscriminately ingests particles from the water column and is thus highly susceptible to microplastics. For this thesis, we identified some gaps in the available data on the ecotoxicity of microplastics to daphnids. To illuminate some of those gaps the present thesis was aimed at five main aspects:
(1) Tissue translocation of spherical microplastics in Daphnia magna
(2) Investigation of the toxicity of irregularly shaped microplastics
(3) Multigenerational and population effects of microplastics
(4) Comparison of the toxicity of microplastics and natural particles
(5) Effects of particle-aging on microplastics toxicity
The thesis is comprised of three peer-reviewed articles and one so-far unpublished study as “additional results”. The first study was aimed at understanding tissue translocation of spherical microplastics to lipid storage droplets of daphnids. The crossing of biological membranes is discussed as a prerequisite to eliciting tissue damage and an inflammatory response. Previously, researchers reported the translocation of fluorescently labeled spherical microplastics to lipid storage droplets of daphnids, even though no plausible biological mechanism to explain this occurrence. Therefore, in order to learn more about this process and potentially illuminate the mechanism we replicated the study. We were able to observe a fluorescence signal inside the lipid droplets only after increasing the exposure concentrations. Nonetheless, it appeared to be independent of particles. This led to the hypothesis, that the lipophilic fluorescent dye uncoupled from the particles and subsequently accumulated in lipid storage droplets. The hypothesis was further confirmed through an additional experiment with a silicone-based passive sampling device showing that the fluorescence occurred both independent of particles and digestive processes. Accordingly, we concluded that the reported findings were a microscopic artifact caused by the uncoupling of the dye from the particles. Therefore, a fluorescence signal alone is not a sufficient proxy to assume that particles have translocated. It needs to be coupled with additional methods to ensure that the observation is indeed caused by the translocation of particles.
It is still unclear whether the toxicity profile of microplastics is different from that of naturally occurring particles or if they are “just another particle”, as there are innumerable amounts in the natural environment surrounding an organism. The goal of the second study was to compare the toxicity of irregularly shaped polystyrene microplastics to that of the natural particle kaolin. The environment is full of natural non-food particles that daphnids ingest more or less indiscriminately and therefore are well adapted to deal with. Daphnids have a short generation time and usually experience food limitation in nature. Therefore, short-term studies only looking at acute toxicity with ad libitum food availability are not representative of the exposure scenario in nature. For a more realistic scenario, we, therefore, used a four-generation multigenerational design under food limitation to investigate how effects translate from one generation to the next. We observed concentration-dependent effects of microplastics but not of natural particles on mortality, reproduction, and growth. Some of the effects increased from generation to generation, leading to the extinction of two treatment groups. Here, microplastics were more toxic than natural particles. At least part of this difference can be explained by physical properties leading to the quick sedimentation of the kaolin, while microplastics remained in the water column. Nonetheless, buoyancy and sedimentation would also affect exposure in the environment and are likely different for most microplastics than for most naturally occurring particle types.
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