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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.