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Microsporidia are a group of parasites that infect a wide range of species, many of which play important roles in agriculture and human disease. At least 14 microsporidian species have been confirmed to cause potentially lifethreatening infectious diseases in both immunocompromised and immunocompetent humans. Approximately 1,400 species of microsporidia have been described. Depending on their host and habitat they are classified into three groups, the aquasporidia, the terresporidia and the marinosporidia.
Microsporidia were originally classified as fungi by Naegeli (1857). However, their lack of typical eukaryotic components – such as mitochondria, Golgi bodies or peroxisomes – suggested to place the microsporidia together with other amitochondriate protists within the Archezoa kingdom. This "microsporidia-early" hypothesis was further supported by molecular phylogenies inferred from individual genes. Despite this evidence, the placement of microsporidia as an early branching eukaryote remained a topic for debate. The phylogeny of microsporidia is prone to suffer from biases in their reconstruction. The high evolutionary rate of microsporidian proteins tends to place these proteins together with other fast evolving lineages, a phenomenon known as long-branch attraction. In 1996, the first molecular phylogenetic studies placed the microsporidia inside the fungi.
Subsequently, several further studies located the microsporidia at different positions inside the fungal clade. Since then, microsporidia have been considered as members of the Ascomycota, Zygomycota, Cryptomycota, or as a sister group to the Ascomycota and Basidiomycota, or even as the sister group of all fungi.
The difficulties in determining the evolutionary origin of microsporidia are not only caused by their lack of several cellular components but also by their reduced genomes and metabolism. Being obligate intracellular parasites, microsporidia successfully reduced their genome sizes, down to the range of bacteria. As the smallest eukaryotic genome described so far, the genome of Encephalitozoon intestinalis is just 2.3 Mbp, about half the size of the one of Escherichia coli. Due to their low number of protein coding genes (less than 4,000), microsporidia are thought to retain only genes essential for their survival and development. Furthermore, several key metabolic pathways are missing in the microsporidia, such as the citric acid cycle, oxidative phosphorylation, or the de novo biosynthesis of nucleotides. As a result they are in an obligatory dependence on many primary metabolites from the hosts. However, the presence of hsp70 protein suggests a more complex genome of the microsporidian ancestor. Consequently, the small microsporidian genomes and the reduced metabolism would be consequences of a secondary loss process that molded the contemporary microsporidia from a functionally more complex ancestral species. However, it remains unclear whether the last common ancestor (LCA) of the microsporidia was already reduced, or whether the genome compaction was lineage-specific and started from a more complex LCA.
We investigated the evolutionary history of the contemporary microsporidia through the reconstruction and analysis of their LCA. As a first step in our analysis, we have developed and implemented a software facilitating an intuitive data analysis of the large presence absence-patterns resulting from the tracing of microsporidian proteins in gene sets of many different species. These so called phylogenetic profiles can now be dynamically visualized and explored with PhyloProfile. The software allows the integration of other additional information layers into the phylogenetic profile, such as the similarity of feature architecture (FAS) between the protein under study and its orthologs. The FAS score can be displayed along the presence-absence pattern, which can help to identify orthologs that have likely diverged in function. PhyloProfile closes the methodological gap that existed between tools to generate large phylogenetic profiles to delineate the evolutionary history and the contemporary distribution of large – and ultimately complete – gene sets, and the more function-oriented analysis of individual protein. In the next step we tackled the problem of how to transfer functional annotation from one protein to another. We have developed HamFAS that integrates a targeted ortholog search based on the HaMStR algorithm with a weighted assessment of feature architecture similarities (FAS) between orthologs. In brief, for a seed protein we identify orthologs in reference species in which proteins have been functionally annotated based on manually curated assignments to KEGG Ortholog (KO) groups. The FAS scores between the orthologs and seed proteins are calculated. Subsequently, we compute pairwise FAS scores for all reference proteins within a KO group. A group's mean FAS score serves then as cutoff that must be exceeded to warrant transfer of its KO identifier to the seed. A benchmark using a manually curated yeast protein set showed that HamFAS yields the best precision (98.5%) when compared with two state-of-the-art annotation tools, KAAS and BlastKOALA. Furthermore, HamFAS achieves a higher sensitivity. On average HamFAS annotates almost 50% more proteins than KAAS or BlastKOALA.
With this extended bioinformatics toolbox at hand, we aimed at reconstructing the evolutionary history of the microsporidia. We generated a robust phylogeny of microsporidia using a phylogenomics approach. As a data basis, we identified a set of microsporidian proteins encoded by 80 core genes with one-to-one orthologs. A maximum likelihood analysis of this data
with 48 fungi and additionally in 13 species from more distantly related such as animals and plants combined in a supermatrix strongly supported the hypothesis that microsporidia form the sister group of the fungi. We confirmed that the data explains this microsporidia-fungi relationship significantly better than any other of the previously proposed phylogenetic hypotheses.
On the basis of this phylogeny, and of the phylogenetic profiles of microsporidian proteins, we then focused on reconstructing the dynamics microsporidian genome evolution. Between 2% of the proteins in the compact microsporidia Encephalitozoon intestinalis and up to 49% of the proteins of Edhazardia aedis are private for individual microsporidian species. A comparison of the sequence characteristics of these proteins to that of proteins with orthologs in other microsporidian species revealed individual differences. Yet, without further evidences it remains unclear whether these private genes are indeed lineage-specific innovations contributing to the adaptation of each microsporidium to its host, or whether these are artifacts introduced in the process of gene annotation. A total of 14,410 microsporidian proteins could then be grouped into 1605 orthologous groups that can be traced back to the last common ancestor of the microsporidia (LCA set). We found that 94% of the microsporidian LCA proteins could be tracked back to the last eukaryotic common ancestor. The high evolutionary age of these proteins, together with the resistance against gene loss in the microsporidia suggests that the corresponding functions are essential for eukaryotic life. Further 3% of the LCA proteins could be dated to the common ancestor microsporidia share with the fungi. Only 3% of the LCA proteins appear as microsporidia specific inventions. These proteins are potentially of importance for the evolutionary of the obligate parasitic lifestyle nowadays shared by all microsporidia.
The functional annotation and metabolic pathway analysis of the microsporidian LCA protein set gave us more insight into the adaptation of the microsporidia to their parasitic lifestyle and the origin of the microsporidian genome reduction. The presence of E1 and E3 components of the pyruvate dehydrogenase complex and the mitochondrial hsp70 protein support an ancestral presence of mitochondria in the ancestral microsporidia. In addition, several ancient proteins that complement gapped metabolic pathways were found in the microsporidian LCA. They suggested a more complex genome and metabolism in the LCA. However, our reconstruction of the metabolic network of the microsporidian LCA still lacks many main pathways. For example, the TCA cycle for effective energy production, and key enzymes that are required for in vivo synthesis of critical metabolites like purines and pyrimidines appear absent. We therefore find that the parasitic lifestyle and the genome reduction already occurred in the microsporidian LCA. This ancestral state was followed by further losses and gains during the evolution of each individual microsporidian lineage.
In conclusion, I described for the first time the in vivo functions of PAK2 during cardiac development and its requirement for heart contractility
AIM1 – Characterization of Pak2a and Pak2b functions during cardiovascular system development: description of the phenotype triggered by the loss of expression of pak2b in the pak2a mutant Firstly, in addition to the confirmation of the published data regarding the pak2a mutant and morphant phenotype, I showed that pak2bbns159 mutant does not exhibit morphological defects, neither in the ISV formation nor in the brain vascular patterning. More importantly, I analyzed in more details the phenotypic consequences of pak2a and pak2b loss of expression in the trunk and brain vasculatures. Indeed, the lack of blood flow in the embryos, was associated with central arteries migration defects and reduced lumen in these central arteries and the ISVs. Moreover, pak2a and pak2b loss of expression resulted in cardiac failure.
AIM2 – Role of Pak2 on cardiac contractility From 40 -46 hpf, I found a weaker heart contractility in the pak2ami149/mi149;pak2bbns159/bns159. Although, the PAK proteins have been shown to impact the actin cytoskeleton organization, the heart morphological defects associated with the altered contractility, were not associated with acto-myosin filament reorganization. However, by analyzing in more details the structure of the sarcomeres, I was able to demonstrate that the proteins constituting the sarcomeres were strongly affected and showed an altered spatial organization. Then, I also described the effects of the loss of expression of both paralogs on the junctional protein localization. I demonstrated the loss of Pak2 function resulted in junction protein rearrangement in the cardiomyocytes in the pak2ami149/mi149;pak2bbns159/bns159 mutants at 40 and 46 hpf.
Thus, I was able for the first time to demonstrate in vivo PAK2 functions during cardiac development and its requirement for proper cardiac contractility activity.
AIM3 – Decipher mechanism of Pak2 signaling cascade involved during cardiac development Both pak2a and pak2b WT mRNAs were able to rescue the pak2ami149/mi149;pak2bbns159/bns159 mutant heart defects and the results indicated that these paralogs share overlapping function during cardiac development. Moreover, although I was not able to examine the control transgenic lines, myocardial and endothelial specific pak2a overexpression did not ameliorate the mutant cardiac deficiency. Thus,the absence of rescue by reactivating pak2a in cardiomyocytes indicates a non-cell autonomous function of Pak2a on cardiomyocytes.
For the first time, this study allowed to follow PAK2 in vivo functions during cardiovascular development. More importantly, its role on heart contractility regulation would enable further investigations to generate new tools for the treatment of cardiomyopathies.
The fungal interaction with plants is a 400 million years old phenomenon, which presumably assisted in the plants’ establishment on land. In a natural ecosystem, all plant-ranging from large trees to sea-grasses-are colonized by fungal endophytes, which can be detected inter- and intracellularly within the tissues of apparently healthy plants, without causing obvious negative effects on their host. These ubiquitous and diverse microorganisms are likely playing important roles in plant fitness and development. However, the knowledge on the ecological functions of fungal root endophytes is scarce. Among possible functions of endophytes, they are implicated in mutualisms with plants, which may increase plant resistance to biotic stressors like herbivores and pathogens, and/or to abiotic factors like soil salinity and drought. Also, endophytes are fascinating microorganisms in regard to their high potential to produce a great spectrum of secondary metabolites with expected ecological functions. However, evidences suggest that the interactions between host plants and endophytes are not static and endophytes express different symbiotic lifestyles ranging from mutualism to parasitism, which makes difficult to predict the ecological roles of these cryptic microorganisms. To reveal the ecological function of fungal root endophytes, this doctoral thesis aims at assessing fungal root endophytes interactions with different plants and their effects on plant fitness, based on their phylogeny, traits, and competition potential in settings encompassing different abiotic contexts. To understand the cryptic implication of nonmycorrhizal endophytes in ecosystem processes, we isolated a diverse spectrum of fungal endophytes from roots of several plant species growing in different natural contexts and tested their effects on different model plants under axenic laboratory conditions. Additionally,we aimed at investigating the effect of abiotic and biotic variables on the outcome of interactions between fungal root endophytes and plants.
In summary, the morphological and physiological traits of 128 fungal endophyte strains within ten fungal orders were studied and artificial experimental systems were used to reproduce their interactions with three plant species under laboratory conditions. Under defined axenic conditions, most endophytes behaved as weak parasites, but their performance varied across plant species and fungal taxa. The variation in the interactions was partly explained by convergent fungal traits that separate groups of endophytes with potentially different niche preferences. According to my findings, I predict that the functional complementarity of strains is essential in structuring natural root endophytic communities. Additionally, the responses of plant-endophyte interactions to different abiotic factors, namely nutrient availability, light intensity, and substrate’s pH, indicate that the outcome of plant-fungus relationships may be robust to changes in the abiotic environment. The assessment of the responses of plant endophyte interactions to biotic context, as combinations of selected dominant root fungal endophytes with different degrees of trait similarity and shared evolutionary history, indicates that frequently coexisting root-colonizing fungi may avoid competition in inter-specific interactions by occupying specific niches, and that their interactions likely define the structure of root-associated fungal communities and influence the microbiome impacts on plant fitness.
In conclusion, my findings suggest that dominant fungal lineages display different ecological preferences and complementary sets of functional traits, with different niche preferences within root tissues to avoid competition. Also, their diverse effects on plant fitness is likely host-isolate dependent and robust to changes in the abiotic environment when these encompass the tolerance range of either symbiont.
Colorectal cancer (CRC) has the third highest incidence and the fourth highest mortality rate worldwide and represents a substantial health care burden and affects the life of millions of people. CRC is a genetic disease caused by the stepwise accumulation of genetic alterations. The initiating event in colorectal carcinogenesis is the aberrant activation of the WNT pathway, but other pathways are also commonly deregulated, including the PI3K/AKT pathway. A number of previous studies using genetically engineered mouse models aimed at dissecting the exact role of PI3K/AKT pathway in CRC, but have yielded in rather conflicting results. Despite the inconsistent results, these studies already put forward the idea that PI3K/AKT signaling in combination with other genetic events might substantially contribute to tumor progression.
Since the PI3K/AKT pathway is frequently activated in CRC, it represents an ideal candidate for therapeutic intervention. Although extensive efforts had led to the development of numerous inhibitors targeting the PI3K/AKT pathway, the diversity of genetic alterations can challenge the identification of the most effective therapeutic targets. Therefore, the discovery of shared tumor-promoting mechanisms downstream of these genetic alterations might unravel new biomarkers and druggable targets. The aim of this study was to elucidate the precise role of PI3K/AKT pathway during the course of colorectal carcinogenesis and to decipher novel pro-tumorigenic molecular mechanisms downstream of PI3K/AKT activation that can be used for therapeutic intervention.
To obtain a better insight into the role of the PI3K/AKT pathway during colorectal carcinogenesis, mice expressing an oncogenic variant of AKT1 (AktE17K) specifically in the intestinal epithelial cells (IEC) were used. At the age of 6 months untreated AktE17K mice showed clearly perturbed intestinal homeostasis, but no tumor formation. To induce colonic tumorigenesis, AktE17K mice were subjected to treatment with the colonic carcinogen azoxymethane (AOM). In response to AOM, AktE17K mice developed invasive but nonmetastatic tumors, which showed strong nuclear accumulation of TP53. To investigate the role of PI3K/AKT signaling specifically in CRC progression, AktE17K mice were crossed to TP53- deficient mice (Tp53ΔIEC). Unlike AktE17K mice, untreated Tp53ΔIECAktE17K, developed highly invasive small intestinal tumors by the age of 6 months. To investigate the role of AKT hyperactivation in colonic tumor progression, Tp53ΔIECAktE17K mice were subjected to AOM treatment. AKT hyperactivation significantly enhanced tumor progression and induced metastatic dissemination.
To get a better insight how AKT signaling can promote tumor progression, whole tumor tissues from AOM-treated Tp53ΔIEC and Tp53ΔIECAktE17K mice were subjected to next generation mRNA sequencing and phospho-proteomic analysis by mass spectrometry. Both analyses indicated that AKT hyperactivation expands the inflammatory tumor microenvironment and upregulates pathways associated with invasion and metastasis. Importantly, Gene Set Enrichment Analysis revealed that AOM-induced colon tumors of Tp53ΔIECAktE17K animals, are highly similar in their gene expression profile to the CMS4 subtype of human CRC, which is associated with worse overall- and relapse-free survival7 . Gene expression analysis also suggested elevated NOTCH signaling in the Tp53ΔIECAktE17K tumors. Interestingly, while the expression of Notch3 mRNA was increased in the tumors of Tp53ΔIECAktE17K mice, the expression of the other NOTCH receptors was unaffected by AKT hyperactivation. In vitro experiments using TP53-deficient mouse tumor organoids with hyperactive AKT signaling confirmed the direct, tumor cell-intrinsic link between AKT activation and increased Notch3 expression. Moreover, inhibition of EZH2 mimicked the effect of AKT hyperactivation on Notch3 expression, suggesting that AKT regulates Notch3 via an epigenetic mechanism.
Knock-down of Notch3 in TP53-deficient mouse tumor organoids with hyperactive AKT signaling resulted in differential regulation of several pathways with potential role in invasion and metastasis and in cell death and survival. Subsequent in vivo experiments confirmed the role of NOTCH3 signaling in CRC progression. Treatment of AOM-induced Tp53ΔIECAkt E17K mice with a NOTCH3 antagonistic antibody or the γ-secretase inhibitor DAPT significantly reduced invasion and metastasis. Importantly, NOTCH3 expression was also found to be associated with human CRC progression, suggesting that NOTCH3 represent a valid target for the treatment of CRC. This work, using genetically engineered mouse models and advanced in vitro techniques, has demonstrated a strong tumor promoting role for PI3K/AKT signaling in CRC progression and has identified NOTCH3 signaling as a potential therapeutic target downstream of the PI3K/AKT pathway.
Bei Autismus-Spektrum-Störungen (ASS) handelt es sich um genetisch komplexe Störungen mit hoher Erblichkeit. Als zugrundeliegender Pathomechanismus von ASS werden unter anderem Veränderungen der neuronalen Entwicklung diskutiert. Der Phänotyp von ASS ist definiert durch Einschränkungen in der sozialen Interaktion und Kommunikation sowie repetitives und stereotypes Verhalten. Genkopiepolymorphismen (englisch „copy number variations“/CNVs), also Deletionen oder Duplikationen einer chromosomalen Region, wurden wiederholt in Probanden mit ASS identifiziert. Hierbei ist in ASS die Region 16p11.2 mit am häufigsten von CNVs betroffen. Einige Gene aus diesem chromosomalen Abschnitt wurden bereits funktionell charakterisiert. Dennoch können die Befunde der bisherigen Einzelgenstudien nicht alle Aspekte erklären, die durch 16p11.2 CNVs hervorgerufen werden. Ziel dieser Studie war es daher, ein weiteres neuronal assoziiertes Kandidatengen dieser Region zu identifizieren und im Anschluss funktionell im Kontext der neuronalen Differenzierung zu charakterisieren.
Das SH-SY5Y Neuroblastom-Zellmodell wurde auf Transkriptom- und morphologischer Ebene auf seine Eignung als Modell für neuronale Differenzierung untersucht und bestätigt. Eine Analyse der Expressionen aller Gene der 16p11.2-Region zeigte, dass das Gen Quinolinat-Phosphoribosyltransferase (QPRT) eine vergleichsweise hohe Expression mit der stärksten und robustesten Regulierung über die Zeit aufwies. Eine de novo Deletion der 16p11.2-Region wurde in einem Patienten im Vergleich zu seinen Eltern validiert. In Patienten-spezifischen lymphoblastoiden Zelllinien derselben Familie konnten wir eine Gendosis-abhängige Expression von QPRT auf RNA-Ebene bestätigen. In SH-SY5Y-Zellen korrelierte die Expression von QPRT signifikant mit der Entwicklung von Neuriten während der Differenzierung. Um QPRT funktionell zu charakterisieren, benutzten wir drei verschiedene Methoden zur Reduktion der QPRT-Gendosis: (i) knock down (KD) durch siRNA, (ii) chemische Inhibition durch Phthalsäure und (iii) knock out (KO) über CRISPR/Cas9-Geneditierung. Eine Reduktion von QPRT durch siRNA führte zu einer schwachen Veränderung der neuronalen Morphologie differenzierter SH-SY5Y-Zellen. Die chemische Inhibition sowie der genetische KO von QPRT waren letal für differenzierende aber nicht für proliferierende Zellen. Eine Metabolitenanalyse zeigte keine Veränderungen des QPRT-assoziierten Tryptophanstoffwechsels. Gene, welche auf Transkriptomebene im Vergleich zwischen KO- und Kontrollzellen differenziell reguliert vorlagen, waren häufig an Prozessen der neuronalen Entwicklung sowie an der Bildung, Stabilität und Funktion synaptischer Strukturen beteiligt. Die Liste differenziell regulierter Gene enthielt außerdem überdurchschnittlich viele ASS-Risikogene und ko-regulierte Gengruppen waren assoziiert mit der Entwicklung des dorsolateralen präfrontalen Cortex, des Hippocampus sowie der Amygdala.
In dieser Studie zeigten wir einen kausalen Zusammenhang zwischen QPRT und der neuronalen Differenzierung in vitro sowie einen Einfluss von QPRT auf die Regulation von ASS-assoziierten Genen und Gen-Netzwerken. Funktionell standen diese Gene im Kontext mit synaptischen Vorgängen, welche durch Veränderungen zu einem Exzitations-Inhibitions-Ungleichgewicht und letztendlich zum Zelltod von Neuronen führen können. Unsere Ergebnisse heben in Summe die wichtige Rolle von QPRT in der Krankheitsentstehung von ASS, insbesondere in Trägern einer 16p11.2 Deletion, hervor.
This dissertation aimed to shed light on changes of the epigenetic landscape in heart and skeletal muscle tissue of the turquoise Killifish N. furzeri, a novel, short-lived animal model for aging research. The following results could be obtained:
1. A global trend towards closed chromatin conformation could be observed; histone markers for H3K27me3, H3K9me3 and H4K20me3 accumulated in skeletal muscle tissue from old N. furzeri. Markers for open chromatin conformation such as H3K4me3, H3K9ac and H4K16ac decreased in old skeletal muscle tissue. In old hearts from N. furzeri an accumulation of H3K27me3 could be detected while H3K9ac was found to increase with age as well. mRNA expression levels of methylating enzymes were higher in skeletal muscle tissue from old N. furzeri when compared to expression levels in skeletal muscle tissue from young N. furzeri.
2. The shift of epigenetic pattern was accompanied by a change of gene expression. Via mRNA sequencing in collaboration with the MPI, Bad Nauheim it could be shown that genes associated with cell cycle and DNA repair were lower expressed in skeletal muscle tissue from old N. furzeri than in tissue from young N. furzeri. Genes, associated with inflammatory signaling and glycolysis, displayed increased mRNA levels in skeletal muscle tissue from old N. furzeri. These results could be confirmed by Western blot and qRT-PCR analyses.
3. Markers for DNA damage and senescence increased in skeletal muscle tissue from old N. furzeri.
4. Cells derived from young and old N. furzeri skeletal muscle could be isolated and cultured for many passages. These cells were a mix of different cell types with properties and features of the native tissue. They could be used for treatment with drugs and/small compounds modulating the epigenetic landscape via specific interference with methylating enzymes.
5. DNA methylation and hydroxy-methylation were found to go in different directions in skeletal muscle and heart tissue from N. furzeri: while increasing in skeletal muscle tissue, a both DNA modifications declined in heart tissue with age.
6. In the heart of N. furzeri microRNA expression changes with age were assed with sequencing in collaboration with the FLI, Jena. It could be demonstrated that miRNA expression is age-dependent. Particular focus was on miR-29 and its target genes: miR-29 was highly upregulated in heart and skeletal muscle tissue, while target genes such as collagens and dnmts were reduced with age in the heart of N. furzeri.
7. Cardiac function remained stable with age and no accumulation of collagens could be found when comparing hearts of young and old N. furzeri despite the increase of markers for oxidative stress.
8. Cell culture experiments with human cardiac fibroblasts revealed that miR-29 is upregulated with increasing age of the donor. In addition to that, it could be shown that miR-29 is positively regulated by oxidative stress.
9. A zebrafish mutant with modified expression of miR-29 that was created in collaboration with the SNS, Pisa, presented a severe hypoxic phenotype and an altered mRNA expression profile compared to wild type control zebrafish. Cardiac dysfunction and hypertrophy were observed as well as an increase in DNA methylation and collagens.
Taken together, it could be shown that the aging process in skeletal muscle and heart tissue from N. furzeri leads to a series of changes on epigenetic levels. It remains to be elucidated whether these changes are result or cause for further changes of mRNA expression, protein levels and pathophysiology, yet the N. furzeri represents a promising research model for further aging studies.
Lizards of Paraguay: an integrative approach to solve taxonomic problems in central South America
(2018)
Paraguay is located in the center of South America with drier and warmer climatic conditions in the western part of the country, and more temperate and humid in the eastern region. Biogeographically, Paraguay is a key spot in South America, where several ecoregions converge. In my study, I sampled most of the ecoregions of Paraguay. The main objective of my work is to solve taxonomic problems, identified through genetic barcoding analyses, in the central region of South America. To achieve this objective, I used selected taxa of the Paraguayan Squamata as models taking into consideration the crucial geographic position of the country, plus the scarce available genetic data of Paraguayan reptiles.
The collecting activities were performed in the framework of a barcoding inventory project of the Paraguayan herpetofauna and carried out mostly in rural areas searching for animals in different types of habitats using active search as the sampling technique.
For genetics, the extraction of DNA was performed with DNeasy® Blood & Tissue Kit of Qiagen® for sets of few samples, and the fiber glass plate protocol for sets of 96 samples. I assessed the quality of sequences after amplification in agarose gel electrophoresis. The first marker sequenced was 16S mtDNA, used for barcoding analysis. A DNA barcode is a genetic identifier for a species. Once a taxonomic problem was detected, I generate more gene sequences to target the issue.
All the analyses to test phylogenetic hypotheses (based on single genes or concatenated datasets) were performed under Maximum Likelihood and Bayesian approaches. To root the phylogenetic trees, I chose the available taxon (or taxa) most closely related to the respective studied group as outgroups. For the general tree of Paraguayan Squamata, based on barcodes of 16S, I chose Sphenodon punctatus.
I generated a total of 142 sequences of 64 species of Squamata from Paraguay (Appendix I). The final alignment of 615 bp comprised 249 samples. The best substitution model for the Barcoding dataset based on the gene 16S was GTR+G, according to the BIC.
To complement molecular evidence generated with the ML grouping of 16S barcodes, I took a morphological approach based on voucher specimens collected during fieldwork (usually the same specimens that I used for genetic analysis), supplemented by the revision of museum collections.
Summarizing my results, samples of Colobosaura exhibit large genetic distances, and accordingly I revalidated Colobosaura kraepelini (Appendix II). Tropidurus of the spinulosus group show two clades and among them there is little genetic and morphological variation, I synonymized T. tarara and T. teyumirim with T. lagunablanca, and T. guarani with T. spinulosus (Appendix III). I detected the presence of candidate species of Homonota, and I restricted the name H. horrida for Argentina, and described two new species of Homonota (Appendices IV and V), and a new species of Phyllopezus also in the Family Phyllodactylidae (Appendix VI).
In this work I present the most comprehensive analysis of genetic samples of Squamata from Paraguay. The results obtained here will be useful to help to clarify further taxonomic issues regarding the squamate fauna from the central region of South America. Moreover, the data generated for this study will have a positive impact in a larger geographic context, beyond Paraguayan borders.
Regarding the conservation of the Paraguayan reptiles, and considering the taxonomic changes accomplished here, it is important to note that many species lack legal protection. In Paraguay, the major problem for conservation is habitat loss due to extensive crop farming. Thus, currently, the protected areas are the best strategy for conservation of biodiversity in the country. However, many such areas face legal problems (e.g., lack of official measurements, management plans, forest guards, infrastructure, etc.) so that the maintenance of their biodiversity over time is not guaranteed.
In conclusion, in this study I present contributions on the taxonomy of mostly lizards from Paraguay. Due to lack of samples, I was not able to deal with a deep taxonomic revision of the country's snakes. Based on my results, I can argue that analyses of Xenodontini and Pseudoboini are currently a pressing research issue. This barcoding project may continue since some colleagues in Paraguay are interested in collaboration. Given that the sequenced specimens are yet a small portion of the actual diversity of Paraguay, it will be of utmost importance to continue and expand these studies that will further improve our taxonomic knowledge. Furthermore, it is desirable to have Paraguayan scientists not only involved, but to see them taking the lead of high quality taxonomic research.
The metabolome of any live cell consists of several hundred, if not thousands of different molecules at any given moment, be it a relatively small bacterial cell or a whole multicellular organism. Although there are continuous attempts to differentiate between primary and secondary metabolites, the borders often blur in the eye of almost perfect interconvertability of all such matter. With chemistry and physics dominating this domain of biology it is an interdisciplinary endeavor to tackle the questions surrounding the workings of the metabolic pathways involved, searching for answers that ultimately help us to better understand life and find solutions to problems that affect us humans. One area of biochemistry that serves as a formidable example of the intertwined primary and secondary metabolic pathways are fatty acids, essential components of bacterial membranes, sources of energy and carbon but also important building blocks of several natural products. The second area to be mentioned is the metabolism of amino acids, the basic components of proteins and enzymes, which also serve as precursors to a diverse set of metabolites with many biological purposes.
This work focuses on these two areas of biochemistry, as several intermediates of their metabolism serve as building blocks for complex secondary metabolites whence many interesting and bioactive natural products are derived. The powerful and relatively novel tool of click-chemistry is employed to track azide-labeled precursors of primary and secondary metabolism in various bacterial strains to observe biochemistry at work and adds to the knowledge gained through other methods. The methods presented in this work serve the observation of fatty acid biosynthesis, degradation, modification and transport through direct ligation of azido fatty acids with cyclooctynes on one hand, leading to a revision of fatty acid transport in general. On the other hand a cleavable azide-reactive resin is devised to generally track the fate of azidated compounds through the myriads of metabolic pathways offered by entomopathogenic bacteria possessing a rich secondary metabolism. The resulting findings led to the identification of several antimicrobial peptides, amides and other compounds of which many had remained so far undetected in the strains that underwent investigation, underlining the worth of this method for future metabolomic research and beyond.
The role of the homeobox transcription factor Meis2b in zebrafish heart development and asymmetry
(2018)
Zebrafish heart development: The heart of the zebrafish is the first organ to form and function during embryonic development, and is composed by one atrium and one ventricle. Between 5-17 somites stage, the cardiomyocyte precursors form the bilateral cardiac fields in the anterior lateral plate mesoderm (ALMP); where the endocardial precursors are located anterior to the cardiac fields (Zeng, Wilm et al. 2007). Then, the pools of endocardial andmyocardial precursors fuse at the midline and form the heart disc; where atrial cardiomyocytes are located around, the ventricular cardiomyocytes are located in the centerof the heart disc, and the future endocardium is located in a ventral position relative to the cardiomyocytes (Bakkers 2011). After the heart disc is formed, the cardiomyocyte progenitors start to migrate and rotate asymmetrically to form the heart tube (de Campos-Baptista, Holtzman et al. 2008, Rohr, Otten et al. 2008, Smith, Chocron et al. 2008). This process is followed by a rightward bending of the heart tube, and the arterial and venous poles rotate at different speed and directions (a process known as heart looping) (Smith, Chocron et al. 2008). The heart looping process results in a ventricle located on the right side and a more posterior atrium located on the left side with respect to the midline; at this point the atrium and ventricle are separated by a fine segment called the atrioventricular canal, where the valves will be formed (Staudt and Stainier 2012). The second heart field (SHF) is a pool of cardiac progenitors that are specified later during the formation of the heart disc and until the heart looping stages. The SHF contributes withcells to the distal side of the ventricle, the outflow and inflow tracts, and is important for the specification of the cardiac conduction system (de Pater, Clijsters et al. 2009, Hami, Grimes et al. 2011, Zhou, Cashman et al. 2011, Witzel, Jungblut et al. 2012, Guner-Ataman, Paffett-Lugassy et al. 2013)....
Heat stress transcription factors (Hsfs) are required for transcriptional changes during heat stress (HS) thereby playing a crucial role in the heat stress response (HSR). The target genes of Hsfs include heat shock proteins (Hsps), other Hsfs and genes involved in protection of the cell from irreversible damages due to exposure to elevated temperatures. Among 27 Hsfs in Solanum lycopersicum, HsfA1a, HsfA2 and HsfB1 constitute a functional triad which regulates important aspects of the HSR. HsfA1a is constitutively expressed and described as the master regulator of stress response and thermotolerance. Activation of HsfA1a under elevated temperatures leads to the induction of HsfA2 and HsfB1 which further stimulate the transcription of HS-responsive genes by forming highly active complexes with HsfA1a. Despite the well-established role of these three Hsfs in tomato HSR, information about functional relevance of other Hsfs is currently missing.
The heat stress inducible HsfA7 belongs alongside with HsfA2 to a phylogenetically distinct clade. Thereby the two proteins share high homology and a functional redundancy has been assumed. However, HsfA7 function and contribution to stress responses have not been investigated into detail in any plant species.
Tomato HsfA7 protein accumulates already at moderately elevated temperatures (~35°C) while HsfA2 becomes dominant at higher temperatures (>40°C). HsfA7 pre-mRNA undergoes complex and temperature-dependent alternative splicing resulting in several transcripts that encode for three protein isoforms. HsfA7-I contains a functional nuclear export signal (NES) and shows nucleocytoplasmic shuttling while HsfA7-II and HsfA7-III have a truncated NES which leads to the strong nuclear retention of the protein. Differences in the nucleocytoplasmic equilibrium have a major impact on the stability of protein isoforms, as nuclear retention is associated with increased protein turnover. Consequently, HsfA7-I shows a higher stability and can be detected even after 24 hours of stress attenuation, while HsfA7-II is rapidly degraded. The degradation of these factors is mediated by the ubiquitin-proteasome pathway.
HsfA7 can physically interact with HsfA1a and HsfA3 and form co-activator (“superactivator”) complexes with a very high transcriptional activity as shown on different HS-inducible promoters. In order for the complex to be successfully transferred to the nucleus and confer its activity it needs a functional nuclear localization signal (NLS) of HsfA7. In contrast, the activator (AHA) motif of HsfA7 is not essential for its co-activator function. Interestingly, while interaction of HsfA7 with either HsfA3 or HsfA1a stabilizes HsfA7 isoforms, concomitantly this leads to an increased turnover of HsfA1a and HsfA3. In contrast, HsfA2 has a stabilizing effect on the master regulator HsfA1a.
Thus, HsfA7 knockout mutants generated by CRISPR/Cas9 gene editing, show increased HsfA1a levels and a stronger induction of HS-related genes at 35°C compared to wild-type plants and HsfA2 knockout mutants. Consequently, HsfA7 knockout seedlings exhibit increased thermotolerance as shown by the enhanced hypocotyl elongation under a prolonged mild stress treatment at 35°C. In summary, these results highlight the importance of HsfA7 in regulation of cellular responses at elevated temperatures. Under moderately elevated temperatures, the accumulation of HsfA7 and its subsequent interaction with HsfA1a, leads to increased turnover of the latter, thereby ensuring a milder transcriptional activation of temperature-responsive genes like Hsps. In turn, in response to further elevated temperatures, HsfA2 becomes the dominant stress-induced Hsf. HsfA2 forms co-activator complexes with HsfA1a which in contrast to HsfA7, allows the stabilization of the master regulator, leading to the stronger expression of HS-responsive genes required for survival. Thereby, this study uncovers a new regulatory mechanism, where the temperature-dependent competitive interaction of HsfA2 and HsfA7 with HsfA1a control the fate of the master regulator and consequently the activity of temperature-responsive networks.
Echolocation allows bats to orientate in darkness without using visual information. Bats emit spatially directed high frequency calls and infer spatial information from echoes coming from call reflections in objects (Simmons 2012; Moss and Surlykke 2001, 2010). The echoes provide momentary snapshots, which have to be integrated to create an acoustic image of the surroundings. The spatial resolution of the computed image increases with the quantity of received echoes. Thus, a high call rate is required for a detailed representation of the surroundings.
One important parameter that the bats extract from the echoes is an object’s distance. The distance is inferred from the echo delay, which represents the duration between call emission and echo arrival (Kössl et al. 2014). The echo delay decreases with decreasing distance and delay-tuned neurons have been characterized in the ascending auditory pathway, which runs from the inferior colliculus (Wenstrup et al. 2012; Macías et al. 2016; Wenstrup and Portfors 2011; Dear and Suga 1995) to the auditory cortex (Hagemann et al. 2010; Suga and O'Neill 1979; O'Neill and Suga 1982).
Electrophysiological studies usually characterize neuronal processing by using artificial and simplified versions of the echolocation signals as stimuli (Hagemann et al. 2010; Hagemann et al. 2011; Hechavarría and Kössl 2014; Hechavarría et al. 2013). The high controllability of artificial stimuli simplifies the inference of the neuronal mechanisms underlying distance processing. But, it remains largely unexplored how the neurons process delay information from echolocation sequences. The main purpose of the thesis is to investigate how natural echolocation sequences are processed in the brain of the bat Carollia perspicillata. Bats actively control the sensory information that it gathers during echolocation. This allows experimenters to easily identify and record the acoustic stimuli that are behaviorally relevant for orientation. For recording echolocation sequences, a bat was placed in the mass of a swinging pendulum (Kobler et al. 1985; Beetz et al. 2016b). During the swing the bat emitted echolocation calls that were reflected in surrounding objects. An ultrasound sensitive microphone traveling with the bat and positioned above the bat’s head recorded the echolocation sequence. The echolocation sequence carried delay information of an approach flight and was used as stimulus for neuronal recordings from the auditory cortex and inferior colliculus of the bats.
Presentation of high stimulus rates to other species, such as rats, guinea pigs, suppresses cortical neuron activity (Wehr and Zador 2005; Creutzfeldt et al. 1980). Therefore, I tested if neurons of bats are suppressed when they are stimulated with high acoustic rates represented in echolocation sequences (sequence situation). Additionally, the bats were stimulated with randomized call echo elements of the sequence and an interstimulus time interval of 400 ms (element situation). To quantify neuronal suppression induced by the sequence, I compared the response pattern to the sequence situation with the concatenated response patterns to the element situation. Surprisingly, although the bats should be adapted for processing high acoustic rates, their cortical neurons are vastly suppressed in the sequence situation (Beetz et al. 2016b). However, instead of being completely suppressed during the sequence situation, the neurons partially recover from suppression at a unit specific call echo element. Multi-electrode recordings from the cortex allow assessment of the representation of echo delays along the cortical surface. At the cortical level, delay-tuned neurons are topographically organized. Cortical suppression improves sharpness of neuronal tuning and decreases the blurriness of the topographic map. With neuronal recordings from the inferior colliculus, I tested whether the echolocation sequence also induced neuronal suppression at subcortical level. The sequence induced suppression was weaker in the inferior colliculus than in the cortex. The collicular response makes the neurons able to track the acoustic events in the echolocation sequence. Collicular suppression mainly improves the signal-to-noise ratio. In conclusion, the results demonstrate that cortical suppression is not necessarily a shortcoming for temporal processing of rapidly occurring stimuli as it has previously been interpreted.
Natural environments are usually composed of multiple objects. Thus, each echolocation call reflects off multiple objects resulting in multiple echoes following the calls. At present, it is largely unexplored how neurons process echolocation sequences containing echo information from more than one object (multi-object sequences). Therefore, I stimulated bats with a multi-object sequence which contained echo information from three objects. The objects were different distances away from each other. I tested the influence of each object on the neuronal tuning by stimulating the bats with different sequences created from filtering object specific echoes from the multi-object sequence. The cortex most reliably processes echo information from the nearest object whereas echo information from distant objects is not processed due to neuronal suppression. Collicular neurons process less selectively echo information from certain objects and respond to each echo.
For proper echolocation, bats have to distinguish between own biosonar signals and the signals coming from conspecifics. This can be quite challenging when many bats echolocate adjacent to each other. In behavioral experiments, the echolocation performance of C. perspicillata was tested in the presence of potentially interfering sounds. In the presence of acoustic noise, the bats increase the sensory acquisition rate which may increase the update rate of sensory processing. Neuronal recordings from the auditory cortex and inferior colliculus could strengthen the hypothesis. Although there were signs of acoustic interference or jamming at neuronal level, the neurons were not completely suppressed and responded to the rest of the echolocation sequence.
The fruit fly Drosophila melanogaster is one of the most important biological model organisms, but only the comparative approach with closely related species provides insights into the evolutionary diversification of insects. Of particular interest is the live imaging of fluorophores in developing embryos. It provides data for the analysis and comparison of the threedimensional morphogenesis as a function of time. However, for all species apart from Drosophila, for example the red flour beetle Tribolium castaneum, essentially no established standard operation procedures are available and the pool of data and resources is sparse. The goal of my PhD project was to address these limitations. I was able to accomplish the following milestones:
- Development of the hemisphere and cobweb mounting methods for the non-invasive imaging of Tribolium embryos in light sheet-based fluorescence microscopes and characterization of most crucial embryogenetic events.
- Comprehensive documentation of methods as protocols that describe (i) beetle rearing in the laboratory, (ii) preparation of embryos, (ii) calibration of light sheet-based fluorescence microscopes, (iv) recording over several days, (v) embryo retrieval as a quality control as well as (vi) data processing.
- Adaption of the methods to record and analyze embryonic morphogenesis of the Mediterranean fruit fly Ceratitis capitata and the two-spotted cricket Gryllus bimaculatus as well as integration of the data into an evolutionary context.
- Further development of the hemisphere method to allow the bead-based / landmark-based registration and fusion of three-dimensional images acquired along multiple directions to compensate the shadowing effect.
- Development of the BugCube, a web-based computer program that allows to share image data, which was recorded by using light sheet-based fluorescence microscopy, with colleagues.
- Invention and experimental proof-of-principle of the (i) AGameOfClones vector concept that creates homozygous transgenic insect lines systematically. Additionally, partial proof-of-principle of the (ii) AClashOfStrings vector concept that creates double homozygous transgenic insect lines systematically, as well as preliminary evaluation of the (iii) AStormOfRecords vector concept that creates triple homozygous transgenic insect lines systematically.
- Creation and performance screening of more than fifty transgenic Tribolium lines for the long-term imaging of embryogenesis in fluorescence microscopes, including the first Lifeact and histone subunit-based lines.
My primary results contribute significantly to the advanced fluorescence imaging approaches of insect species beyond Drosophila. The image data can be used to compare different strategies of embryonic morphogenesis and thus to interpret the respective phylogenetic context. My technological developments extend the methodological arsenal for insect model organisms considerably.
Within my perspective, I emphasize the importance of non-invasive long-term fluorescence live imaging to establish speciesspecific morphogenetic standards, discuss the feasibly of a morphologic ontology on the cellular level, suggest the ‘nested linearly decreasing phylogenetic relationship’ approach for evolutionary developmental biology, propose the live imaging of species hybrids to investigate speciation and finally outline how light sheet-based fluorescence microscopy contributes to the transition from on-demand to systematic data acquisition in developmental biology.
During my PhD project, I wrote a total of ten manuscripts, six of which were already published in peer-reviewed scientific journals. Additionally, I supervised four Master and two Bachelor projects whose scientific questions were inspired by the topic of my PhD work.
The continuous conversion of natural wildlife habitats into agricultural areas, as well as the fragmentation of the last wildlife refuges, is increasing the interface between people and wildlife. When wildlife negatively impacts on people and vice versa, we speak about human-wildlife conflicts (HWCs). This definition includes losses on both sides and takes into consideration the rooting of most of these conflicts between different groups of interest, such as advocates for nature conservation and economic groups. The centres of highest biodiversity are located in developing countries, which are also characterized by poverty. In African and Asian countries, people living in the vicinity of national parks and other conservation areas mostly receive only little support through the government or conservation organisations. Especially for those people who are dependent on agriculture, damage to fields and harvests can have catastrophic consequences. If the species causing damage is protected by national or even international law, the farmer is not allowed to use lethal methods, but has to approach the authority in charge. If this agency, however, cannot offer appropriate support, resentment, anger or even hate develops, and the support for wildlife conservation activities declines. For this reason, HWCs were declared as one of the most important conservation topics today, being particularly relevant for large and threatened species such as the African and Asian elephant, hippopotamus and the greater one-horned rhino, as well as for large predators. Up to today, no general assessment scheme has been recommended for damage caused by protected wildlife species.
In my study, HWCs in Asia and Africa are compared, focussing on all herbivorous species identified which damaged crops. For the French NGO Awely, des animaux et des hommes, I developed a detailed assessment scheme suitable for all terrestrial ecosystems, and any type of HWCs and any species (Chapter 2). This HWC assessment scheme was used in four different study areas located in two African countries (South Luangwa/Zambia (SL), Tarangire/Tanzania (TA)) and two Asian countries (Bardia/Nepal (BA) and Manas/India (MA)). This scheme ran for six consecutive years (2009 to 2014) for Zambia, Nepal and India and two years (2010 to 2011) for Tanzania. To carry out the assessments, I trained local HWC officers (Awely Red Caps) to assess HWCs by field observations (measurement of damage, identification of species through signs of presence, landscape attributes etc.) and interviews with aggrieved parties (socio economic data). Results of this assessment are presented in Chapters 2-4.
To determine whether elephants prefer or avoid specific crop species, two field experiments were carried out, one in SL and one in BA (Chapter 5 and 6). For this, two test plots were set up and damage by elephants (and other herbivores) were quantified.
Within this doctoral thesis, 3306 damage events of 7408 aggrieved parties were analysed. In three out of the four study areas (SL, BA, MA), elephants caused the highest number of damage events compared to all other wildlife species, however, in TA, most fields were damaged by zebra. Furthermore, the greater one-horned rhino, hippopotamus, wild boar, bushpig, deer and antelope, as well as primates, caused damage to fields and harvests. Damage to houses and other property were nearly exclusively caused by elephants.
With this doctoral thesis I was able to show that season, crop availability, type and the phenological stage of the crop played an important role for crop damaging behavior of herbivores (Chapter 2). Elephants especially damaged rice, maize and wheat and preferred all crop types in a mature stage of growth. In contrast, rhinos preferred wheat to rice and similar to antelope and deer, they preferred crops at earlier stages of growth, before ripening. Crop damage by wildlife species varied strongly in size; most damages fell below 40% of the total harvest per farmer, but in several cases (3 to 8% depending on the study area), harvests were completely destroyed. Interestingly, during times of low nutritional availability in the natural habitat (dry season), crop damages in all four study areas were significantly less than during other seasons.
In all four study areas, crop protection strategies, such as active guarding in the fields, chasing wildlife with noise or fire torches or erecting barriers, were used. In some cases protection strategies were combined. Analysis of data revealed that traditional protection strategies did not reduce the costs of damage (Chapter 3). In some cases, costs of damage, on protected fields were even higher than for unprotected fields. Only in MA did strategic and cohesive guarding significantly reduce crop damage by wildlife species.
Besides damage in the fields, elephants also caused damage to properties in the villages. In search for stored staple crops, they damaged houses, grain stores and kitchens. Such damage was analysed in three study areas (SL, BA, MA) (Chapter 4). Although property damage occurred less frequently compared to crop damage in the fields, the mean cost of this damage was found to be double in BA/MA and four times higher in SL, compared to the costs of crop damage in the fields. It is further remarkable that property damage significantly increased towards the dry season, when the harvest was brought into the villages.
The findings of this study underpin the assumption that wildlife herbivores, especially elephants, are lured to fields and crops because the highly nutritional food (crop) being readily available. Traditional crop protection is cost and labour intensive and does not reduce the costs of damage. For this reason, crop types, which are thought to be not consumed by elephants were systematically tested on their attractiveness in field experiments in SL and BA (Chapter 5 and 6). In SL, lemon grass, ginger and garlic were proven to be less attractive to African elephants than maize and in BA, basil, turmeric, chamomile, coriander, mint, citronella and lemon grass were found to be less attractive to Asian elephants than rice.
The results of this doctoral thesis are relevant for the management of wildlife conservation as they can lead to new approaches to the mitigation of HWCs in African and Asian countries. Finally, specific needs for more scientific research in this field have been identified.
Characterizing the hologenome of Lasallia pustulata and tracing genomic footprints of lichenization
(2017)
The lichen symbiosis – consisting of fungal mycobionts and photoautotroph photobionts (green algae or cyanobacteria) – is globally successful. It covers an estimated 6% of the global surface with habitats ranging from deserts to the arctic. This success is reflected in the diversity of the mycobionts, with around 21% of all fungal species participating in lichen symbioses that can be facultative or obligate. Lichenization is furthermore evolutionary old, with fossil evidence for lichens reaching back 415 million years. For an individual fungal lineage, the Lecanoromycetes, the lichenization happened around 300 million years ago. This longstanding symbiotic relationship and the diversity of observed symbiotic dependency make them promising models to study the genomic consequences that follow the establishment of symbioses. Despite this, only little is known about the genomic effects of lichenization and extreme symbiotic dependency. To fill this gap we sequenced the hologenome of the lichen Lasallia pustulata, where the mycobiont could so far not been cultivated, suggesting that it might be more dependent on its symbionts.
As the poor culturability of lichen symbionts renders their genomes inaccessible to standard sequencing practices, we evaluated the extent to which different metagenome sequencing- and de novo assembly-strategies can be used to sequence and reconstruct the genomes of the individual symbionts. We find that the abundances of individual genomes present in the L. pustulata hologenome vary substantially, with the mycobiont being most abundant. Using in silico generated data sets and real Illumina sequencing data for L. pustulata we observe that the skewed abundances prevent a contiguous assembly of the underrepresented genomes when using only short-read sequencing. We conclude that short-read sequencing can offer first insights into lichen hologenomes. The fragmentation of the reconstructions hinders downstream analyses into the genomic consequences of lichenization though, as these are focused on identifying the gain and loss of genes.
We thus demonstrate a hybrid genome assembly strategy that is based on both short- and long-read sequencing. We show that this strategy is capable of creating highly contiguous genome reconstructions, not only for the L. pustulata mycobiont but also its photobiont Trebouxia sp., along with substantial amounts of the bacterial microbiome. A subsequent analysis of the microbiome of L. pustulata – performed over nine different samples collected in Germany and Italy – showed a stable taxonomic composition across the geographic range. We find that Acidobacteriaceae, which are known to thrive in nutrient poor habitats, are the dominant taxa. These would make them well adapted for the co-habitation with L. pustulata, which largely grows on rocks. Whether the Acidobacteriaceae are functionally involved in the lichen symbiosis is unclear so far.
As further comparative genomic studies rely on comprehensive genome annotations, we evaluate the completeness and fidelity of the gene annotations for the mycobiont L. pustulata as well as four further Lecanoromycetes. This reveals that un- and mis-annotated genes impact all evaluated genomes, with artificially joined genes and unannotated genes having the largest impact. In addition to these factors we find that the sequence composition – especially G/C-rich inverted repeats – lead to sequencing errors that interfere with the gene prediction. We minimize the effects of these artifacts through a rigorous curation.
Given the extremely sparse taxon sampling of available green alga genomes, we focus our search for the genomic footprints of lichenization on the mycobionts. We compare the genomes of the Lecanoromycetes to their closest relatives, the Eurotiomycetes and Dothideomycetes. This reveals that the last common ancestor of the Lecanoromycetes has lost around 10% of its genes after they split from the non-lichenized ancestor they share with the Eurotiomycetes. These losses are furthermore enriched, showing an excessive loss of genes involved with the degradation of polysaccharides. The loss of these genes fits a change from an ancestral saprotrophic lifestyle that depends on degrading complex plant matter, to the symbiotic lifestyle that relies on simpler nutrients provided by the photobionts. While the last common ancestor of the Lecanoromycetes additionally gained around 400 genes these could so far not be further characterized due to a lack of functionally annotated reference data.
As the mycobiont L. pustulata could so far not been grown in axenic culture, we initially expected to find an extensive genomic remodeling compared to the other mycobionts that easily grow in culture. We do not find evidence for this. Analyzing both the contraction of gene families and the loss of genes, we observe that L. pustulata and Umbilicaria muehlenbergii – its close relative that is easily grown in culture – share most of these. Furthermore, L. pustulata does not show an excessive loss of evolutionary old and well-conserved genes. These effects are mirrored on the functional level, as neither gene family contractions nor gene losses show a functional enrichment. This is partially due to the lack of functional reference data, analogous to the genes gained in the Lecanoromycetes, rendering their characterization hard. Thus, further studies on the genomic consequences of lichenization and differences in symbiotic dependence will have to be conducted, including larger taxon sets. This will be even more important for the photobionts, as the Chlorophyta are even more sparsely sampled today, hindering an effective functional and evolutionary study.
Tissue size regulation is critical for the normal functioning of the organ as well as to prevent unwanted pathogenesis such as cancer. The Hippo signaling pathway is well known for its robust regulation of tissue growth by the negative regulation of its nuclear effectors YAP1 and WWTR1. In this study, I have described the role of Yap1/Wwtr1 in zebrafish development, with a primary emphasis on the cardiovascular system.
I have generated zebrafish yap1 and wwtr1 mutants by CRISPR/CAS9. The mutant alleles are likely to be nonfunctional due to a premature stop codon and they show evidence of nonsense-mediated decay. Given that Yap1 and Wwtr1 are closely related proteins and have overlapping functions, I am given the opportunity to perform combinatorial analysis of the mutations on zebrafish development. Together with molecular probing tools, high-throughput sequencing and high-resolution imaging, I showed that
1. Double yap1;wwtr1 mutants exhibit severe posterior elongation phenotype, but somitogenesis appears to proceed as usual.
2. Yap1 and Wwtr1 may play an important role in PCV development and secondary angiogenic sprouting. However, key experiments will be needed to elucidate the direct role of Yap1 and Wwtr1 on these processes.
3. wwtr1-/- larvae hearts have a reduction in trabeculation, but in mosaic WT hearts, mutant cardiomyocytes prefer to populate the trabecular layer. My studies revealed that the mutant compact wall could not support trabeculation, which explains the hypotrabeculation phenotype of wwtr1-/- hearts. Additionally, Wwtr1 is required for myocardial Notch activity and can inhibit compact wall cardiomyocytes from entering the trabecular layer.
In summary, the Hippo signaling pathway, through Yap1/Wwtr1 has important regulatory functions in growth control. My work has revealed a surprising role for Yap1/Wwtr1 in tissue morphogenesis such as posterior tail morphogenesis and specific developmental processes of the cardiovascular system. It will be of interest to elucidate the regulation of Yap1/Wwtr1 in individual cells that translates into the complex cellular behaviors that drives morphogenesis.
Cardiovascular disease is the leading cause of death worldwide. Aging is among the greatest risk factors for cardiovascular disease. Cardiovascular disease comprises several diseases, for example myocardial infarction, elevated blood pressure and stroke. Many processes are known to promote or worsen cardiovascular disease and in the present study, cellular senescence and inflammatory activation were of special interest, as they have a strong association to aging and can be seen as hallmarks of cellular aging.
Long noncoding RNAs (lncRNAs) are noncoding RNAs with a length of more than 200 nucleotides. In recent years, numerous regulatory functions were shown for these transcripts and lncRNAs were shown to directly interact with DNA, RNA and proteins. The long noncoding RNA H19 was among the first described noncoding RNAs and was initially shown to act as a tumor suppressor. More recently, several studies showed oncogenic roles for H19. In regards to the cardiovascular system, H19 was not analyzed before.
We show that H19 is the most profoundly downregulated lncRNA in endothelial cells of aged mice compared to young littermates. Microarray analysis of human primary endothelial cells upon pharmacological H19 depletion revealed an involvement of H19 in cell cycle regulation. Loss of H19 in human endothelial cells in vitro led to reduced proliferation and to increased senescence. H19 depletion was shown to counteract proliferation before, but none of the described mechanisms applied to endothelial cells. We show that the reduction in proliferative capacity and the pro-senescent function of H19 is most probably mediated by an upregulation of p16ink4A and p21 upon H19 depletion.
When we compared the angiogenic capacity of aortic endothelial cells from young and aged mice in an aortic ring assay, rings from aged mice showed a reduced cumulative sprout length. Interestingly, pharmacological inhibition of H19 in aortic rings of young animals, where H19 is highly expressed, was sufficient to reduce the cumulative sprout length to levels we observed from aged animals. Furthermore, overexpression of human H19 in aortic rings of aged mice, where H19 is poorly expressed, rescued the impaired angiogenic capacity of aged endothelial cells.
We generated inducible endothelial-specific H19 knockout mice (H19iEC-KO) and subjected these animals to hind limb ischemia surgery followed by perfusion analysis in the hind limbs by laser-doppler velocimetry and histological analysis. Perfusion in the operated hind limb was increased in H19iEC-KO compared to Ctrl littermates, which was in contrast to a reduction in capillary density in the operated hind limbs of H19iEC-KO animals compared to Ctrl littermates and to our previous results. Analysis of arteriogenesis revealed an increase in collateral growth upon EC-specific H19 depletion in the ischemic hind limbs, which explains the increase in perfusion despite the reduction in capillary density. Further characterization of the animals revealed an increase in leukocyte infiltration into the tissue in the ischemic hind limbs upon endothelial-specific H19 depletion, indicating a potential role of H19 in inflammatory tissue activation.
Reanalysis of the microarray data from human primary endothelial cells upon H19 depletion revealed an association of H19 with inflammatory signaling and more specifically with IL-6/JAK2/STAT3 signaling. Analysis of cell surface adhesion molecule expression revealed an upregulation of ICAM-1 and VCAM-1 on mRNA level and an increase of the abundance of the two proteins on the cell surface of human primary endothelial cells. Consequently, adhesion of isolated human monocytes to human primary endothelial cells was increased upon H19 depletion in vitro. Interestingly, TNF-α mediated inflammatory activation of primary human endothelial cells repressed H19 expression. H19 did not function via previously described mechanisms. We excluded a competitive endogenous RNA (ceRNA) function for H19 in endothelial cells and showed that miR-675, which is processed from H19, does not play a role in the endothelium. Furthermore, H19 did not regulate previously described genes or pathways.
Analysis of transcription factor activity upon H19 depletion and overexpression revealed a differential activity of STAT3. STAT3 phosphorylation at TYR705 and thus activation was increased upon H19 depletion. Inhibition of STAT3 activation using a small compound inhibitor abolished the effects of H19 depletion on mRNA expression of p21, ICAM-1 and VCAM-1 and on proliferation, indicating that the effects of H19 are at least partially mediated via STAT3. STAT3 was shown to have positive effects on the cardiovascular system before, most likely due to upregulation of VEGF in a STAT3-dependent manner. We were not able to confirm previously described mechanisms for STAT3 in the present study and propose a new mechanism of action for the H19-dependent regulation of STAT3. Taken together, these results identify the long noncoding RNA H19 as a pivotal regulator of endothelial cell function. Figure 38 summarizes the described functions of H19 in endothelial cells.
Deciphering the ecological functions of fungal root endophytes based on their natural occurrence
(2017)
Plants are colonized by a large diversity of fungi, some residing on the surface and others penetrating the plant tissues, the latter referred to as fungal endophytes (endon Gr., within; phyton, plant; de Bary 1879). Despite the saprotrophic potential of fungal endophytes, they are not found to cause visible disease symptoms to the host. Plants are colonized simultaneously by various fungal species, which form rich and diverse endophytic assemblages. Although it is hypothesized that fungal endophytes contribute to the fitness of their hosts and to the functioning of ecosystems, the ecological function of fungal endophytic assemblages remains cryptic. The aims of this doctoral thesis are to gain insight to the ecological functions of root fungal endophytes, by deciphering their roles in ecosystems based on their natural occurrence and the structure of their assemblages. The thesis focuses on studying the diversity and structure of the endophytic mycobiome within roots of two annual and widespread plant hosts Microthlapsi perfoliatum and M. erraticum (Brassicaceae) in several locations across northern Mediterranean and central Europe. The thesis is composed by six Chapters, with a primary focus on Chapter 1, 2 and 3.
Chapter 1 (Glynou et al., 2016) aimed at characterizing the diversity of fungal endophytes in roots at a continental scale and at assessing the factors affecting the structure of endophytic assemblages with the use of cultivation-based methods. For that, root samples were collected from 52 plant populations, along with a collection of soil, bioclimatic, geographic and host data. Cultivation of surface-sterilized root samples on culture media and isolation of fungal colonies in pure culture generated 1,998 fungal colonies. Grouping of sequences into Operational Taxonomic Units (OTUs), based on the 97% similarity of the isolates’ rDNA Internal Transcribed Spacer (ITS) sequence, generated in total 296 OTUs, representing taxa mostly within the phylum Ascomycota with a minor representation of Basidiomycota. Endophytic assemblages were mostly correlated with variation in bioclimatic conditions. Interestingly, despite the large diversity revealed, the assemblages were dominated by only six OTUs related to the orders Hypocreales, Pleosporales and Helotiales, which had a widespread distribution across populations but with some following patterns of ecological preferences.
Chapter 2 aimed at characterizing the uncultivable fraction of the root fungal endophytic diversity, which was not possible to capture in Chapter 1. High-throughput sequencing via the
Illumina Miseq platform was implemented in 43 of the 52 original populations and mostly in the same root samples. In comparison with the cultivation-based approach, the HTS managed to cover the overall diversity within samples. It revealed a large non-cultivated endophytic diversity but the same cultivable fungi dominated assemblages. Moreover, the endophytic diversity was grouped mostly within fungal orders with demonstrated ability to grow in culture and taxonomically related groups were found to have divergent ecological preferences.
The genetic identity of the most abundant OTUs was further investigated in Chapter 3 (Glynou et al., 2017), aiming to unravel genotypic variability, which was possibly overlooked due to the use of lTS, as a universal genetic marker, and could explain their high abundance and widespread distribution. Multi-locus gene sequencing and AFLP profiling for the five most abundant OTUs suggested a low within-OTU genetic variability and show that these fungi have ubiquitous distribution and are not limited by environmental conditions within the ecological ranges of the study. A selection of endophytes frequently isolated in Chapter 1 was functionally characterized in Chapter 4 (Kia et al., 2017) based on the isolates’ traits and interactions with plants. In Chapter 5 (Cheikh-Ali et al., 2015) fungal cultures of Exophiala sp. with differential colony structure where investigated for their production of secondary metabolites. Moreover, Chapter 6 (Maciá-Vicente et al., 2016) comprises the description of the new species Exophiala radicis based on morphological and molecular characteristics.
Compilation of all results shows that the fungal endophytic diversity in roots of Microthlaspi spp. is high but few widespread OTUs dominate the assemblages, and have unlimited dispersal ability. These fungi seem also to have a wide niche breadth and are not affected by environmental filtering. The findings indicate that the local environment but also processes of competitive exclusion determine the structure of endophytic assemblages. In addition, the fungal endophytes associated with Microthlapsi spp. likely have saprotrophic activity however the interactions with plants are likely context-dependent. Further research is needed to assess the biotic interactions among endophytes and their effect on the structure of fungal endophytic assemblages. Ultimately, the findings of this thesis are useful to shed light on the processes underlying the structure of endophytic assemblages. They also upraise the need to describe diversity by combining genetic, metabolic and physiological data, in order to disentangle the elusive ecological roles of the endophytic mycobiome.
Savannas provide essential ecosystem services for human well-being in West Africa. Thus, ecosystem change not only directly affects biodiversity but also human livelihoods. Human land use considerably shaped these savanna ecosystems for millennia, particularly agriculture, livestock grazing, logging and the collection of non-timber forest products (NTFPs). NTFPs are wild plant products and comprise all organic matter from herbaceous plants, shrubs, and trees (excluding timber). Current increasing land use pressure through fast demographic changes is widely esteemed as a severe threat for savanna biodiversity and the socio-economy of rural communities. In consideration of the pivotal role of NTFP species for biodiversity and livelihoods, it is important to evaluate the effect of increasing land use change on savanna vegetation and on its provisioning service for human well-being. Thus, the major aim of this thesis is to investigate the impacts of land use intensification on vegetation composition, diversity and function and its consequences for provisioning ecosystem services (NTFPs) and human well-being in a West African savanna.
The research for this study was conducted in the North Sudanian vegetation zone of south-eastern Burkina Faso, where population growth exceeds the nationwide trend. Generally, Burkina Faso belongs to the worldwide poorest countries, where nearly one quarter of the population suffers from malnutrition (FAO 2014). The integration of NTFPs and particularly wild food species into rural household economies is, thus, an important measure in the national combat against poverty and food insecurity (FAO 2014). Against this background, I focus on vegetation changes, the economic importance of NTFPs as well as the decrease and substitution of wild food species in this study.
Vegetation resurveys of different vegetation types since the early 1990s showed that land use change led to more pronounced changes in the herbaceous than in the woody vegetation layer. Most woody vegetation types stayed stable in species composition and richness, even though some highly useful tree species (Vitellaria paradoxa, Parkia biglobosa) declined in some woody vegetation types. In contrast, in most herbaceous vegetation types species richness increased and species composition considerably changed. This change might be explained by a general ruderalisation process through a pronounced increase of wide-ranging herbaceous species. However, in spite of a general species increase in the herbaceous layer, a decrease of preferred herbaceous fodder species was found. Thus, the decline of useful species in both layers is alarming. Herbaceous vegetation types also showed more pronounced changes in plant functional trait characteristics in comparison to woody vegetation types. However, an increase of smaller plant species and species with a high diaspore terminal velocity (VTerm) was found in both vegetation layers. Since these two trait responses are generally related to grazing and browsing, the strong increase of livestock herds is likely to be responsible for the detected vegetation changes.
In addition to the vegetation study, interviews showed that all useful food species were widely considered to decline. The two economically most important tree species, the shea tree (Vitellaria paradoxa) and the locust bean tree (Parkia biglobosa) that contribute with 70% to wild food income, were considered among the most declining species of all cited wild food species. On this matter, local perceptions of species decline and results from field observations are in accordance. However, a wide range of cited substitutes indicated a great knowledge on alternative plant species in the area. Most wild food species are, however, substituted by other highly valued wild food species. Although our results suggest that rural communities are able to cope with the decrease or absence of wild food species, growing decline of one species would concurrently increase the pressure on other native food species. Therefore, the need to counteract the decrease of highly useful wild food species should be of high priority in management measures. In general, I showed that NTFPs are an essential component in rural households, since it contributed with 45 % to total household income. Significant differences in NTFP dependency between the two investigated villages and across the three main ethnic groups were detected, reflecting different traditional uses and harvesting practices. In general, it was shown that poorer households depend more on NTFP income than wealthier households. Against the background of this study, management strategies for agroforestry systems and poverty alleviation should consider local differences, and ethnicity-dependent NTFP-use patterns.
Overall, the combination of field studies on temporal and functional vegetation change with socio-economic and ethno-botanic interviews increases the knowledge on qualitative and quantitative vegetation changes and on the consequences for rural populations. This thesis gives a thorough insight into decreasing trends of economically valued plant species and thus gives evidence on the consequences of vegetation changes for ecosystem services of West African savanna ecosystems. Further, different NTFP-dependencies and use preferences according to socio-economic and cultural variables, such as ethnicity, present a valuable basis for specific decision-making and should be considered in management plans.
Research in cell and developmental biology requires the application of three-dimensional model systems that reproduce the natural environment of cells. Processes in developmental biology are therefore studied in entire systems like insects or plants. In cell biology, three-dimensional cell cultures (e.g. spheroids or organoids) model the physiology and pathology of cells, tissues or organs. In all systems, the cellular neighborhood and interactions, but also physicochemical influences, are realistically presented. The production and handling of these model systems is rather simple and allows for reproducible characterization.
Confocal and light sheet-based fluorescence microscopy (LSFM) enable the observation of these systems while maintaining their three-dimensional integrity. LSFM is applicable to imaging live samples at high spatio-temporal resolution over long periods of time. The quality of the acquired datasets enables the extraction of quantitative features about morphology, functionality and dynamics in the context of the complete system. This approach is referred to as image-based systems biology. Exploiting the potential of the generated datasets requires an image analysis pipeline for data management, visualization and the retrieval of biologically meaningful values.
The goal of this thesis was to identify, develop and optimize modules of the image analysis pipeline. The modules cover data management and reduction, visualization, reconstruction of multiview image datasets, the segmentation and tracking of cell nuclei and the extraction of quantitative features. The modules were developed in an application-driven manner to test and ensure their applicability to real datasets from three-dimensional fluorescence microscopy. The underlying datasets were taken from research projects in developmental biology in insects and plants, as well as from cell biology.
The datasets acquired in fluorescence microscopy are typically complex and require common image processing steps in order to manage, visualize, and analyze the datasets. The first module accomplishes automatic structuring of large image datasets, reduces the data amount by image cropping and compression and computes maximum projection images along different spatial directions. The second module corrects for intensity variations in the generated maximum projection images that occur as a function of time. The program was published as a part of an article in Nature Protocols. Another developed module named BugCube provides a web-based platform to visualize and share the processed image datasets.
In LSFM, samples can be rotated in-between two acquisitions enabling the generation of multiview image datasets. Prior to my work, Frederic Strobl and Alexander Ross acquired the complete embryogenesis of the red flour beetle, Tribolium castaneum, and the field cricket, Gryllus bimaculatus, with LSFM. I evaluated a plugin for the software FIJI as a module for the reconstruction of such datasets. The plugin was optimized for automation and efficiency. We obtained the first high quality three-dimensional reconstructions of Tribolium and Gryllus datasets.
Optical clearing increases the penetration depth into samples, thus providing endpoint images of entire three-dimensional objects with cellular detail. This work contributes a quantitative characterization module that was applied to endpoint images of optically cleared spheroids. A program for the generation of ground truth datasets was developed in order to evaluate the cell nuclei segmentation performance. The program was part of a paper that was published in BMC Bioinformatics. Using the program, I could show that the cell nuclei segmentation is robust and accurate. Approaches from computational topology and graph theory complete the segmentation of cell nuclei. Thus, the developed module provides a comprehensive quantitative characterization of spheroids on the level of the individual cell, the cell neighborhood and the whole cell aggregate. The module was employed in four applications to analyze the influence of different stress conditions on the morphology and cellular arrangement of cells in spheroids. The module was accepted for publication in Scientific Reports along with the results for one application. The cell nuclei segmentation further provided a data source for simulation models that used correlation functions to identify structural zones in spheroids. These results were published in Royal Society Interface.
The final part of this work presents a module for cell tracking and lineage reconstruction. In collaboration with Dr. Alexis Maizel, Dr. Jens Fangerau and Dr. Daniel von Wangenheim, I developed a module to track the positions of all cells involved in lateral root formation in Arabidopsis thaliana and used the extracted positions for extensive data analysis. We reconstructed the cell lineages and established the first atlas of all founder cells that contribute to the formation. The analysis of the retrieved data allowed us to study conserved and individual patterns in lateral root formation. The atlas and parts of the analysis presented in this thesis were published in Current Biology.
In this thesis, I developed modules for an image analysis pipeline in three-dimensional fluorescence microscopy and applied them in interdisciplinary research projects. The modules enabled the organization, processing, visualization and analysis of the datasets. The perspective of the image analysis pipeline is not restricted to image-based systems biology. With ongoing development of the image analysis pipeline, it can also be a valuable tool for medical diagnostics or industrial high-throughput approaches.
Die neuronalen Mechanismen, welche den meisten kognitiven Prozessen zu Grunde liegen, bestehen aus dem Zusammenspiel verschiedener Neuronen-Typen und deren spezifischen Funktionsmechanismen, sowohl in lokalen, als auch in globalen neuronalen Netzwerken. Eine funktionelle Interaktion mit diesen Netzwerken ist unumgänglich um das „kognitive“ Gehirn zu studieren, da neuronale Gruppen in einer hierarchischen, nicht linearen Weise miteinander interagieren, und dabei charakteristische raum-zeitliche Muster aufweisen. In dieser Arbeit untersuchten wir die Struktur und Funktion eines wichtigen Merkmals kortikaler Prozesse: Die neuronale gamma-Band Oszillation.