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Krebs entsteht durch das Zusammenspiel von Keimbahn- und somatischen Mutationen sowie Umweltfaktoren. Für Fortschritte in Prävention, Früherkennung und Behandlung von Krebs ist es essenziell die phänotypischen Folgen dieser Mutationen und die Rolle von Umweltfaktoren bei der Steigerung des Krebsrisikos zu verstehen. In dieser Arbeit untersuche ich zunächst die Auswirkungen von Mutationen in einfachen Sequenzwiederholungsregionen (SSRs) auf das Zellwachstum in einem Hefemodell. In einem Mutationsakkumulationsexperiment in Stämmen mit defektem Mismatch-Reparatursystem zeige ich, dass die Störung von MutSβ zu einer erhöhten SSR-Mutationsrate führt, insbesondere bei SSR-Loci die länger als 8 bp sind. Meine Ergebnisse legen nahe, dass MutSβ hauptsächlich für die Reparatur an längeren Repeat-Loci verantwortlich ist. Schließlich zeige ich, dass SSR-Mutationen meist geringfügige, negative Auswirkungen auf das Zellwachstum haben. Als Nächstes untersuche ich die Auswirkungen von Zigarettenrauch auf das Transkriptom von zugänglichem Atemwegsgewebe am Beispiel des Nasenepithels von gesunden Freiwilligen und Patienten mit Verdacht auf oder diagnostiziertem Lungenkrebs. Ich stelle fest, dass Gene und biologische Funktionen, die durch das Rauchen beeinträchtigt werden, bei Patienten langsamer auf ein gesundes Ausgangsniveau zurückkehren. Zudem zeige ich, dass Patienten durch anhaltende, Rauch-assoziierte Immunveränderungen gekennzeichnet sind. Schließlich präsentiere ich einen innovativen Lungenkrebs-Klassifikator, der durch die Berücksichtigung der nasalen Genexpression von Rauch-assoziierten Genen eindeutig bessere Ergebnisse erzielt als ein Modell, das ausschließlich auf klinischen Informationen basiert. Damit belege ich das Potenzial der Genexpression des Nasenepithels zur Verbesserung der Risikostratifizierung auf Bevölkerungsebene anhand eines nicht-invasiven Tests. Cancer is initiated and sustained by the interplay of germline and somatic mutations and environmental factors. Understanding the phenotypic consequences of mutations and the role of environmental factors in increasing cancer risk is key to improving cancer prevention, early detection, and treatment. In this thesis, I first take advantage of a yeast model to investigate the effects of mutations in simple sequence repeat regions (SSRs) on cell growth. I describe a mutation accumulation experiment conducted in strains with a deletion of the MutSβ gene, a component of the mismatch repair system (MMR). I show that abrogating MutSβ function leads to an increased SSR mutation rate, with a bias towards deletions. I also report a drastic increase in mutation rate in SSR loci longer that 8-bp, suggesting MutSβ is primarily responsible for repair at longer repeat loci. Finally, I show that many SSR mutations have small deleterious effects on cell growth. Next, I investigate the effects of cigarette smoke on the transcriptome of an accessible airway tissue, nasal epithelium, in a cohort of healthy volunteers and patients with suspected or diagnosed lung cancer. I find that smoke injury response is strikingly different in healthy individuals and clinic patients, with genes and biological functions affected by smoking showing a slower reversal to healthy baseline level in clinic patients. I find persistent smoking-associated immune alterations to be a hallmark of the clinic patients. Finally, I show that a lung cancer classifier including nasal expression of smoke-injury-associated genes performs better than a model based exclusively on clinical information, providing evidence for the potential of nasal epithelial gene expression to improve population-level risk stratification with the use of a non-invasive test.

Der mitteleuropäische Wasserfroschkomplex umfasst Pelophylax lessonae (Genotyp LL), Pelophylax ridibundus (RR) und deren Hybriden Pelophylax esculentus (LR), der sich hemiklonal durch Rückkreuzen mit LL in lessonae-esculentus (L-E)-Populationen oder RR in ridibundus-esculentus (R-E)-Populationen reproduziert. Außerdem können Hybriden in reine Hybridpopulationen (E) bilden, in denen triploide Individuen die Elternarten funktionell ersetzen. Bislang ist wenig über die molekularen Mechanismen bekannt, die der klonalen Gametenbildung in der Keimbahn der Hybridform zugrunde liegen. In dieser Studie wurden erstmalig 160 Gene der Elternarten untersucht, die an der Gametenbildung beteiligt sind. Zusätzlich wurden 131 SNPs von 52 dieser Gene von 652 Wasserfröschen aus 26 Populationen analysiert. Im Ergebnis wurden 14 SNPs von 10 Genen entdeckt, deren Frequenzen mit dem Populationssystem assoziiert waren. In Übereinstimmung mit ihren Funktionen könnten diese Gene im Zusammenheng mit den system-spezifischen hybridogenetischen Reproduktionsmodi stehen. Sowohl transkriptomische als auch SNP-Daten lieferten Hinweise auf genetische Introgression, d. h. einen Transfer lessonae-spezifischer Allelen in den ridibundus-Genpool oder umgekehrt. Außerdem wurde bei P. lessonae eine kryptische genetische Diversität beobachtet. SNP-Analysen ergaben auch, dass LR-Individuen aus E-Populationen eine höhere genetische Ähnlichkeit mit LR-Individuen aus R-E als aus L-E-Populationen aufweisen. Diese Ergebnisse werfen die Frage nach dem Ursprung der E-Populationen auf, von denen bisher angenommen wurde, dass sie aus L-E-Populationen hervorgegangen sind. Die neuen molekularen Daten stehen mit den hemiklonalen Fortpflanzungsmodi von P. esculentus im Einklang und unterstreichen, dass diese wahrscheinlich auf komplexen Wechselwirkungen zwischen verschiedenen Genen und Faktoren basieren. The Central European water frog complex comprises Pelophylax lessonae (genotype LL), Pelophylax ridibundus (RR), and their hybridogenetic hybrid Pelophylax esculentus (LR), which reproduces by backcrossing with LL in lessonae-esculentus (L-E) populations or RR in ridibundus-esculentus (R-E) populations. In addition, hybrids are able to reproduce in all-hybrid (E) populations in which triploid individuals functionally replace the parental species. To date, little is known about the molecular mechanisms underlying clonal gamete formation in the hybrid germline. In this study, 160 genes from P. lessonae and P. ridibundus known to be involved in gametogenesis were characterized for the first time. In addition, 131 single nucleotide polymorphisms (SNP) from 52 of these genes were analyzed, derived from 652 water frogs of 26 populations. As a result of logistic regressions, 14 SNPs of 10 genes were detected whose frequencies were associated with the population system. Consistent with their functions during gametogenesis, these genes can be considered as candidates associated with the system-specific hybridogenetic modes, i.e. clonal inheritance of the L and/or R genomes. Both transcriptomic and SNP data provided evidence for genetic introgression, i.e. a transfer of lessonae-specific alleles into the ridibundus gene pool or vice versa. In addition, cryptic genetic diversity was observed in P. lessonae. SNP analysis also revealed that LR individuals from E populations exhibit higher genetic similarity to LR individuals from R-E than L-E populations where triploid frogs do not occur. These findings raise the question of the origin of E populations, which were previously thought to have arisen from L-E populations after the emergence of triploid hybrids. The new molecular data are consistent with the reproductive modes of P. esculentus and emphasize that clonal inheritance is most likely caused by complex interactions of different genes and genetic factors.

Malignant melanoma is an aggressive skin tumour with increasing incidence and, in advanced stages, limited therapeutic results. The major oncogenic alteration in melanoma is mutational activation of the B-Raf protein, with the predominant mutation V600E, which occurs in 60% of cases and hyperactivates the ERK signaling pathway. This cascade of Raf, MEK and ERK protein kinases is an integral part of an evolu- tionarily conserved signaling network that allows eukaryotic cells to sense a variety of extracellular signals. These protein kinases sequentially activate each other and translate extracellular signals into cellular responses such as proliferation, di↵erentiation, cell cycle changes, apoptosis or cell migration. The central role of the ERK pathway in oncogen- esis has made it one of the targets for therapeutic intervention, and inhibitors targeting mutant Raf, such as vemurafenib or dabrafenib, have been approved for the treatment of melanoma. These molecules specifically target B-Raf(V600E) and have led to a revo- lution in melanoma treatment. Unfortunately, such targeted therapy is complicated by the ability of cancer cells to acquire resistance. The aim of this work is to study at the transcriptional level the emergence of this resistance in patients who respond well to treatment and in...

In dieser Dissertation modellieren und analysieren wir scRNA-Seq-Daten, um Mechanismen, die biologischen Prozessen zugrunde liegen, zu verstehen In scRNA-Seq-Experimenten wird biologisches Rauschen mit technischem Rauschen vermischt. Mittels eines vereinfachten scRNA-Seq-Modells leiten wir eine analytische Verteilungsfunktion für die beobachtete Verteilung unter Kenntnis einer Ausgangsverteilung her. Charakteristiken und sogar ein allgemeines Moment der Ausgangsverteilung können aus der beobachteten Verteilung berechnet werden. Unsere Formeln stellen den Ausgangspunkt zur Quantifizierung von Zellvariabilität dar. Wir haben eine vollständig lineare Analyse von Transkriptomdaten entwickelt, die zeigt, dass sich Zellen während des Zellzyklus auf einer ebenen zirkulären Trajektorie im Transkriptomraum bewegen. In immortalisierten Zelllinien stellen wir fest, dass die Transkriptomdynamiken des Zellzyklus hauptsächlich unabhängig von den Dynamiken anderer Zellprozesse stattfinden. Unser Algorithmus (“Revelio”) bringt eine einfache Methode mit sich, um unsynchronisierte Zellen nach der Zeit zu ordnen und ermöglicht das exakte Entfernen von Zellzykluseffekten. Die Form der Zellzyklus-Trajektorie zeigt, dass der Zellzyklus sich dazu entwickelt hat, Änderungen der transkriptionellen Aktivitäten und der damit verbundenen regulativen Anstrengungen zu minimieren. Dieses Konstruktionsprinzip könnte auch für andere Prozesse relevant sein. Durch die Verwendung von metabolischer Molekülmarkierung erweitern wir Modelle zur mRNA-Kinetik, um dynamische mRNA-Ratenparameter für Transkription, Splicing und Degradation zu erhalten und die Lösungen auf den Zellzyklus anzuwenden. Wir zeigen, dass unser Modell zwischen Genen mit ähnlicher Genexpression aber unterschiedlicher Genregulation unterscheiden kann. Zwar enthalten scRNA-Seq-Daten aktuell noch zu viel technisches Rauschen, unser Modell wird jedoch für das zukünftige Errechnen von dynamischen mRNA-Ratenparametern von großem Nutzen sein., In this dissertation, we model and analyse scRNA-seq data to understand mechanisms underlying biological processes. In scRNA-seq experiments, biological noise gets convoluted with various sources of technical noise. With the help of a simplified scRNA-seq model, we derive an analytical probability distribution function for the observed output distribution given a true input distribution. We find that characteristics and even general moments of the input distribution can be calculated from the output distribution. Our formulas are a starting point for the quantification of cell-to-cell variability. We developed a fully linear analysis of transcriptome data which reveals that cells move along a planar circular trajectory in transcriptome space during the cell cycle. Additionally, we find in immortalized cell lines that cell cycle transcriptome dynamics occur largely independently from other cellular processes. Our algorithm (“Revelio”) offers a simple method to order unsynchronized cells in time and enables the precise removal of cell cycle effects from the data. The shape of the cell cycle trajectory indicates that the cell cycle has evolved to minimize changes of transcriptional activity and their related regulatory efforts. This design principle may be of relevance to other cellular processes. By considering metabolic labelling, we extend existing mRNA kinetic models to obtain dynamic mRNA rate parameters for transcription, splicing and degradation and apply our solutions to the cell cycle. We can distinguish genes with similar expression values but different gene regulation strategies. While current scRNA-seq data contains too much technical noise, the model will be of great value for inferring dynamic mRNA rate parameters in future research.

In dieser Dissertation haben wir das Verhalten menschlicher Zellen in Raum und Zeit untersucht. Hochwertige Datensätze subzellulärer Regionen in HEK293-Zellen wurden mit Hilfe der BirA* Proximity-Labelling-Aktivität erstellt, wobei die Lokalisierung auf zelluläre Regionen beschränkt wurde, die mit herkömmlichen Methoden nur schwer zu reinigen sind (d. h. die dem Zytosol zugewandten Seiten des ER, Mitochondrien und Plasma-membranen). Wir entwickelten daraufhin einen Ansatz zur Kartierung der Verteilung von Proteinen, die aktiv an RNA binden, und nannten ihn f-XRNAX. Wir stellten hintergrundkorrigierte Proteome für Zellkerne, Zytoplasma und Membranen von HEK293-Zellen her. Überraschenderweise wurden viele nicht-kanonische RBPs in der Membranfraktion identifiziert, und ihre Peptidprofile waren in Regionen mit hoher Dichte an intrinsisch ungeordneten Regionen angereichert, was auf eine möglicherweise schwache, durch diese nicht-strukturellen Motive vermittelte Interaktion mit RNA hinweist. Schließlich konnten wir die unterschiedliche Bindung desselben Proteins an RNA in verschiedenen HEK293-Kompartimenten nachweisen. Im zweiten Teil dieser Arbeit konzentrierten wir uns auf die Bestimmung und Quantifizierung von neu transkribierten RNAs auf Einzelzellebene. Die Kinetik der RNA-Transkription und -Degradation war bis vor kurzem auf Einzelzellebene nicht messbar. Daher haben wir einen neuen Ansatz (SLAM-Drop-seq genannt) entwickelt, indem wir die veröffentlichte SLAM-seq-Methode an Einzelzellen angepasst haben. Wir haben SLAM-Drop-seq verwendet, um die zeitabhängigen RNA-Kinetikraten der Transkription und des Umsatzes für Hunderte von oszillierenden Transkripten während des Zellzyklus von HEK293-Zellen zu schätzen. Wir fanden heraus, dass Gene ihre Expression mit unterschiedlichen Strategien regulieren und spezifische Modi zur Feinabstimmung ihrer kinetischen Raten entlang des Zellzyklus haben., In this PhD dissertation we investigated the behaviour of human cells through space and time. High quality datasets of subcellular regions in HEK293 cells were generated using BirA* proximity labelling activity and restricting its localization at cellular regions difficult to purified with traditional methods (i.e., the cytosol-facing sides of the endoplasmic reticulum, mitochondria, and plasma membranes). We then developed an approach to map the distribution of proteins actively binding to RNA, and named it f-XRNAX. We recovered background-corrected proteomes for nuclei, cytoplasm and membranes of HEK293 cells. Surprisingly, many non-canonical RBPs were identified in the membrane fraction, and their peptide profiles were enriched in regions with high density of intrinsically disordered regions, indicating a possibly weak interaction with RNA mediated by these non-structural motives. Lastly, we provided evidence of the differential binding to RNA of the same protein in different HEK293 compartments. In the second part of this thesis, we focused on the determination and quantification of newly transcribed RNAs at the single-cell level. The kinetics of RNA transcription, processing and degradation were until recently not measurable at the single-cell level. Thus, we have developed a novel approach (called SLAM-Drop-seq ) by adapting the published SLAM-seq method to single cells. We used SLAM Drop-seq to estimate time-dependent RNA kinetics rates of transcription and turnover for hundreds of oscillating transcripts during the cell cycle of HEK293 cells. We found that genes regulate their expression with different strategies and have specific modes to fine-tune their kinetic rates along the cell cycle.

Nukleinske kiseline mogu poprimiti različite oblike sekundarne strukture. Jedna od takvih struktura nastaje u sljedovima bogatim dušičnom bazom gvanin, pri čemu se četiri gvanina vodikovim vezama povezuju u planarnu strukturu G-kvarteta. Nekoliko G-kvarteta naslaganih jedan na drugi i poduprijeti šećerno-fosfatnom okosnicom tvore sekundarnu strukturu G-kvadripleksa. G-kvadripleksi mogu nastati kao dio deoksiribonukleinskih kiselina i kao dio ribonukleinskih kiselina. Važan su dio genoma eukariota, prokariota i virusa. U eukariotskim genomima pronalaze se u telomerama, promotorima gena te u transkriptomu. Njihova funkcija određena je položajem u genomu. Važni su regulatori aktivnosti telomeraze, transkripcije gena i translacije proteina, ali i poliadenilacije i prekrajanja introna. Mogu se detektirati in vivo pomoću antitijela i imunofluorescencijom, a dijelovi genoma s potencijalom formiranja G-kvadripleksa određuju se računalnim algoritmima. Male molekule ili ligandi mogu se specifično vezivati i stabilizirati sekundarnu strukturu G-kvadripleksa. Stabilizacijom G-kvadripleksa u pojedinim regijama genoma moguće je potisnuti proliferaciju tumora, zbog čega se ovakvi ligandi intenzivno istražuju s ciljem pronalaska antitumorskih lijekova. Nucleic acids can adopt various types of secondary structures. One of such structures arises in guanine-rich sequences, where four guanines link with hydrogen bonds in the planar G-quartet structure. Few G-quartets stacked on each other and supported by the sugar-phosphate backbone create a G-quadruplex secondary structure. G-quadruplexes can arise as a part of the deoxyribonucleic acids, as well as ribonucleic acids. They are an important part of the eukaryote, prokaryote, and virus genomes. In eukaryotic genomes, they can be found in telomeres, gene promoters, and as part of the transcriptomes. Their function is determined by their position in the genome. They are important regulators of telomerase activity, gene transcription, protein translation, polyadenylation, and intron splicing. In vivo detection of G-quadruplexes is possible using antibodies and immunofluorescence, and part of the genome with the potential of forming G-quadruplex can be determined using computational algorithms. Small molecules or ligands can specifically bind and stabilize the secondary structure of Gquadruplexes. By stabilizing the G-quadruplex structures in certain regions of the genome, it is possible to suppress the proliferation of tumors, which is why such ligands are intensively researched with the aim of finding antitumor therapeutics.

Sekvenciranje RNA je molekularna tehnika z vrsto različnih aplikacij, za kar obstaja ogromno različnih pristopov in poti analize podatkov. V zadnjih nekaj desetletjih je RNA-seq znatno napredoval, kar ga je utemeljilo kot prvenstven pristop v transkriptomiki za določanje stopnje izraženosti genov oz. njihovih transkriptov. Le ta uporablja sposobnosti že obstoječih visoko pretočnih metod sekvenciranja naslednje generacije za pridobitev vpogleda v trenuten transkriptom tkiva. Posledično je tehnika zelo priljubljena v raziskavah interakcij bioloških sistemov in vplivov farmakoloških substanc, okolja in fiziološkega stanja na tkivo ali celice. Obstaja vrsto različnih pristopov k RNA-seq, tehnologija pa se tudi še vedno aktivno razvija. Običanjo sosledje korakov v analizi je filtriranje podatkov, kartiranje, sestavljanje transkriptov in kvantifikacija stopnje ekspresije. Vsak izmed teh korakov predstavlja velik nabor različnih možnosti in pristopov k analizi podatkov. Raziskujejo se tudi nove implementacije metode in inovacije na strani programske opreme in laboratorijskih metod pridobivanja podatkov. Vse to prispeva k boljšem razumevanju RNA biologije in odgovarja na vprašanja, kot so kje in kdaj se dejansko zgodi transkripcija ter kakšni so vplivi intramolekularnih interakcij, ki določajo funkcijo RNA molekulam. Moj namen je v naslednjih poglavjih strnjeno predstaviti ozadje, teorijo in praktično izvedbo vsakega od omenjenih korakov obdelave podatkov RNA-seq analize. RNA sequencing is a powerful technology with many applications, and consequently there are many data analysis paths available. Over the past few decades, RNA-seq has progressed significantly, becoming a predominant approach to the field of transcriptomics. It uses the capabilities of existing high-throughput next generation sequencing methods to provide insight into the current tissue transcriptome. This makes it very popular in studies of interaction and influence of different pharmacological substances, environment parameters or physiological states on tissue or cells. There are many different approaches to RNA-seq, as it is still a developing field of research. A typical path for the RNA-seq data analysis steps is filtering data, mapping, transcript construction and expression quantification. Each of those steps represent a massive array of different possibilities and approaches to data analysis. New implementations are being investigated together with innovations in software and wet lab methods, contributing to a better comprehension of RNA biology, giving answers to questions such as when and where transcription actually occurs to intermolecular interactions that govern the function of RNA. My intention in the following chapters is to plainly demonstrate the background, theory, and practical execution of each step involved in RNA-seq data analysis.

Die embryonale Neurogenese in Drosophila ist eine hochgradig koordinierte Abfolge von Zellschicksalsentscheidungen, die viele Ähnlichkeiten mit der Entwicklung des Nervensystems in Wirbeltieren aufweist. Diese Zellschicksalsentscheidungen sind räumlich und zeitlich koordiniert. Diese Zellen entstehen an stereotypen Positionen in jedem Segment und sind entlang zweier räumlicher Achsen angeordnet: der dorsoventralen und der anteroposterioren Achse. Neuroblasten teilen sich, um stereotype Zelllinien zu bilden, und die Zellen weisen charakteristische Zellmorphologien und -ziele auf, wobei die molekularen Mechanismen, die diese Merkmale bestimmen, noch weitgehend unbekannt sind. Jahrzehnte der Genetik haben einige Faktoren aufgedeckt, die für viele dieser Entscheidungen notwendig sind, aber ein Verständnis der einzelnen neurogenen Linien auf Genomebene war bis vor kurzem in vivo unmöglich. Ich habe mRNA aus Einzelzellen verwendet, um die Transkriptomdynamik von Schicksalsentscheidungen in der frühen Entwicklung des Nervensystems zu untersuchen. Mein Ziel ist es, zu entschlüsseln, wie sich Zellen unterscheiden, wenn Entscheidungen getroffen werden, die für die Entwicklung des Nervensystems wesentlich sind. Ich habe Transkriptomdaten von einzelnen Zellen aus Zehntausenden von Neuroblasten während der gesamten embryonalen Neurogenese erstellt. Es gelang mir, spezifische neurogene Populationen und ihre Genexpressionsprofile entlang ihrer Differenzierungswege zu identifizieren. Ich konnte die komplizierten zeitlichen Achsen, die das sich entwickelnde embryonale Nervensystem formen, teilweise entschlüsseln - ein Prozess, der von der Fliege bis zum Menschen konserviert ist. Diese Arbeit hat die Identifizierung lokalisierter Marker und sogar spezifischer Neuroblasten ermöglicht. Dieses Verständnis kann nun mit Informationen über die einzelnen Zellschicksale kombiniert werden, aus denen diese Neuroblasten hervorgehen, wie z. B. ihre spezifischen neuronalen und glialen Schicksale., Embryonic neurogenesis in Drosophila is a highly coordinated sequence of cell fate decisions that bears many similarities to the development of the nervous system in vertebrates. These cell fate decisions are spatially and temporally coordinated. These cells arise at stereotypic positions in each segment and are arranged along two spatial axes: the dorsoventral axis and the anteroposterior axis. Neuroblasts divide to give rise to stereotypic lineages and the cells exhibit characteristic cell morphologies, branching patterns, and targets, the molecular mechanisms that determine these characteristics are still largely unknown. Decades of genetics have uncovered some factors necessary for many of these decisions, but understanding individual neurogenic lineages at the genome level has been impossible in vivo until recently. I have used Single cell mRNA to study the transcriptome dynamics that accompany important fate decisions in early nervous system development. My goal is to decipher how cells differ when decisions are made that are essential for nervous system development. This knowledge is invaluable for developing models for the in vivo mechanisms that allow individual cells in the nervous system to specify and differentiate. I have generated transcriptome data of single cells from tens of thousands of neuroblasts throughout embryonic neurogenesis. I was able to identify specific neurogenic populations and their gene expression profiles along their differentiation pathways. I was able to partially decipher the intricate temporal axes that shape the developing embryonic nervous system, a process that is conserved from fly to human. Single-cell transcriptomics has enabled the identification of localized markers and even specific neuroblasts. This understanding can now be combined with information about the individual cell fates that give rise to these neuroblasts, such as their specific neuronal and glial fates.

Links between ontogeny and phylogeny in animals have been discussed for more than two centuries. With the uprising of molecular biology and bioinformatics, several studies have revealed the presence of the phylogeny-ontogeny correlation on molecular level in developmental transcriptomes of eukaryotic clades with complex multicellularity. These findings open a possibility to test the phylogeny-ontogeny correlation in more basal organisms, with more obscure development and multicellularity characteristics. Using time-resolved transcriptome and proteome profiles, this study showed that Bacillus subtilis biofilm ontogeny correlates with the evolutionary measures through recapitulation pattern, in a way that evolutionary younger and more diverged genes were increasingly expressed towards the later timepoints of the biofilm growth. Molecular and morphological signatures also revealed that biofilm growth is highly regulated and organized into discrete ontogenetic stages. Together, this suggests that the biofilm formation in Bacillus subtilis is a true developmental process comparable to organismal development in animals, plants and fungi Korelacija između filogenije i ontogenije životinja je predmet rasprave među znanstvenicima od početka 19. stoljeća. Razvojem molekularne biologije i bioinformatike te analizama transkriptoma razvojnih stadija, uočena je prisutnost korelacije filogenije i ontogenije na molekularnoj razini u eukariotskim evolucijskim granama sa složenom višestaničnosti. Ova saznanja potaknula su nas da testiramo korelaciju filogenije i ontogenije u bazalnim organizmima s nejasnim razvojnim i višestaničnim karakteristikama. Analizom transkriptomskih i proteomskih podataka, pokazali smo da ontogenija Bacillus subtilis biofilmova korelira s evolucijskim mjerama u obliku rekapitulacijskog profila, odnosno evolucijski mlađi i divergentniji geni su sve više eksprimirani prema kasnijim stadijima rasta biofilma. Dodatne molekularne analize pokazale su da je rast biofilma reguliran i organiziran u jasno odvojene stadije ontogenije. Zaključno, rezultati pokazuju da je rast Bacillus subtilis biofilmova istinski razvojni proces usporediv s razvojem životinja, biljaka i gljiva.

Some species of parasites, including helminths, can inhibit carcinogenesis in their hosts. The antitumoral effect has been discovered in the tapeworms Taenia crassiceps and Echinococcus granulosus, which genes associated with cancer regression have been identified. The effect of melanoma suppression has also been observed in tapeworm Mesocestoides corti by the Laboratory of Helminthology, Department of Parasitology, Faculty of Science, Charles University, however the mechanism-of-action, remains unknown. For the upcoming research it was essential to have the complex molecular data such as transcriptome of the developmental stage s of M. corti. This work is focused on the transcriptomic profiling of the tapeworm M. corti and the differential gene expression in two different strains of murine hosts (inbred and outbred) using the RNA-Seq. The main goal was to identify upregulated transcripts in the tapeworms from the murine hosts that could have a potential effect on cancer regression. Differential gene expression analysis was performed, and the results showed that tapeworms in murine hosts (regardless of strain) had more upregulated transcripts than tapeworms cultured in vitro. Analysis of highly upregulated transcripts in the tapeworms that were grown in the murine hosts identified several...

Leukemia is a cancer of hematopoietic cells affecting the whole organism. Currently, there are many treatment options for all disease types, but it is still not always possible to fully cure the patient. The single-cell RNA sequencing method offers a new insight into the heterogeneity of both cancerous and non-cancerous cells in the leukemic environment. This thesis aims to briefly present the method and its history and to highlight current findings about leukemia obtained with the help of it. Keywords leukemia (AML, CML, ALL, CLL), sequencing, scRNA-seq, cells, transcriptome, treatment Okomentoval(a): [o1]: To the reader se v odborné literatuře moc nepoužívá

Kampilobakterioza je ena najpogostejših bakterijsko povzročenih črevesnih bolezni. Povzročajo jo predvsem bakterije Campylobacter jejuni. Število okužb v zadnjih letih narašča, zato je pomembno vpeljati nove strategije nadzora teh bakterij. Velik problem predstavlja tvorba biofilma, saj so v tej obliki bakterije veliko bolj odporne. Namen našega dela je bil zato pregledati študije o mehanizmih tvorbe biofilma bakterij Campylobacter jejuni na nivoju transkriptoma in izpostaviti potencialne tarče za delovanje protimikrobnih snovi za nadzor nad temi bakterijami. Raziskovali smo mehanizme gibljivosti, pritrjevanja, medcelične komunikacije, stresnega odziva in tvorbe zunajceličnega matriksa pri nastanku biofilma. Za ključna mehanizma za začetek tvorbe biofilma sta se izkazala mehanizma gibljivosti in pritrjevanja. Prav tako je bil za uspešno tvorbo biofilma pomemben sistem medcelične komunikacije. Z mutacijami genov (flaA, flaB, flgE, cadF, flpA, jlpA, luxS) vključenih v te tri mehanizme se je tvorba biofilma zmanjšala ali bila onemogočena. Bakterijske celice so v biofilmu zaščitene z zunajceličnim matriksom, ki omogoča vezavo adhezinov oz. ireverzibilno pritrditev bakterij na podlago. Ob pojavu neugodnih pogojev so se povečano izražali geni vključeni v splošen stresni odgovor (groEL, groES). Campylobacteriosis is one of the most common bacterial intestinal infections. The main cause are the bacteria Campylobacter jejuni. In recent years, the number of these infections has increased. A major problem is the formation of a biofilm. In a biofilm the bacteria are more resistant. The aim of our work was to investigate the mechanisms of biofilm formation of bacteria Campylobacter jejuni at the transcriptome level and to highlight potential targets to control these bacteria. We investigated motility, attachment to surfaces, cell communication, stress response and formation of the extracellular matrix. Motility and attachment were shown to be the main mechanisms for the onset of biofilm formation. The mechanisms of cell communication were also very important for successful formation. Mutations of genes (flaA, flaB, flgE, cadF, flpA, jlpA, luxS) involved in these three mechanisms inhibited or prevented biofilm formation. Bacterial cells are protected in the biofilm by the extracellular matrix, which also allows adhesins to bind and bacteria to irreversibly attach to the surface. When stress conditions occurred, genes involved in the general stress response (groEL, groES) were up-regulated.

Candida species are constituents of the healthy human microbiota on the mucosal epithelial surfaces, such as the gut, oral cavity and vaginal tract. Although normally commensals, these yeasts can cause a wide variety of diseases, inlcuding vulvovaginal candidiasis (VVC). This is the second most common vaginal infection, affecting millions of women worldwide. In this thesis, the main focus was to dissect the interaction of vaginal epithelial cells with the four most prevalent human pathogenic Candida species. Using a time-course in vitro vaginal infection model, pathogenicity mechanisms of these Candida species were characterized. Comparative transcriptomics approach based on dual RNA-Sequencing indicated that these species exhibited species-specific transcriptional patterns, supporting the view that their virulence strategies have evolved independently. On the host side, an early response to all four species was characterized by a protective type-I interferon signalling induced via via transient host mitochondrial dysfunction. At later stages of infection, epithelial responses were driven by Candida species-specific capacities to inflict host cell damage. Moreover, the influence of host factors and a variety of newly synthesized compounds on Candida virulence properties was tested in the in vitro epithelial infection model and indicated their potential role during infection. Taken together, the results of this thesis provide new knowledge on Candida-epithelial interactions in the context of vaginal infections, both on the pathogen and the host side. The results are strengthened by extending the focus on the four most common Candida species, thus considering the evolutionary perspective of host-pathogen interaction. The described mechanisms may contribute to the development of novel diagnostic tools and immunotherapeutic applications.

The rise of second generation sequencing enabled the study of non-model organisms. Without the requirement of having a reference genome, de novo transcriptomics allows the study of functional elements of their genomes. That way, the great complexity of non-model organisms can be explored. This thesis gives a comprehensive overview of the de novo transcriptomics experiment workflow from a bioinformatics perspective. The emphasis was placed on both theoretical background and practical approaches. This work also highlights new methods in de novo transcriptomics which may start to dominate in the near future. The practical part of the work presents transXpress - a de novo transcriptome assembly and annotation pipeline. Its use is demonstrated on a non-model plant long pepper (Piper longum) with medicinal potential. Keywords: transcriptomics, de novo transcriptomics, transcriptome, RNA-Seq, non-model organism, assembly

Thermothelomyces thermophilus is a thermophilic, filamentous fungus, and a very efficient natural producer of hydrolases, like lignocellulolytic enzymes. To optimize this fungus as a production host for hydrolases and heterologous proteins, the understanding of hydrolase expression and regulation is of great interest. With that the expression of specific hydrolases could either be enhanced for applications in, e.g. biorefineries or lowered to increase yield and purity of heterologously expressed proteins. Therefore, this dissertation aimed to 1) establish and apply molecular biological methods to delete regulators of cellulase expression, 2) establish and perform bioreactor cultivations with subsequent biochemical analyses of the above-mentioned strains, and 3) analyze the transcriptome with a focus on hydrolase expression and regulation. Molecular biological methods were established and successfully used to delete known regulators of cellulase expression (Clr1, Clr2, Clr4, and Vib1) and a potentially new one (Vib2). The split marker approach was applied to delete the genes clr2 and vib2, whereas clr4 and vib1 were deleted via the newly established CRISPR/Cas12a system. Deletion of clr1 required the simultaneous use of the split marker approach and the CRISPR/Cas12a system. Bioreactor cultivations in batch or chemostat operation were successfully established. This allows studies on T. thermophilus under controlled, industrially relevant conditions. The transcriptomic analysis revealed a strong expression of genes encoding for predicted lignocellulolytic enzymes including cellulases, hemicellulases, pectinases, and esterases in response to cellulose. Almost all predicted LPMO genes were strongly expressed as well, supporting the application of T. thermophilus in biorefineries. In addition, the analysis uncovered highly expressed predicted protease genes and so far uncharacterized potential regulators of lignocellulolytic enzyme expression. These can be deleted or overexpressed to optimize T. thermophilus for industrial applications. Furthermore, the role of the T. thermophilus orthologs of known regulators of plant biomass degradation was examined in detail, revealing the essential function of the regulators Clr1 and Clr2 for cellulase expression. The expression of clr2 and therefore the complete lignocellulolytic response to cellulose was observed to be Clr1 dependent. Moreover, the data indicate that the regulator Clr4, which was recently published to be an activator of cellulase expression, functions as a repressor, instead. Here, the deletion was leading to a higher gene expression of predicted lignocellulolytic enzymes and corresponding regulators (e.g. Clr2). Furthermore, the function of the potentially new regulator Vib2 was investigated and revealed a repressing function in the early lignocellulolytic response to cellulose. The deletion mutant of vib1 showed growth defects on many carbon sources including cellulose on solid medium, whereas cellulose was successfully utilized during submerged cultivation. The transcriptome of that mutant was not analyzed in this study. Finally, the data suggest a strong involvement of genes predicted to be orthologs of other known regulators like Stk12 and McmA/1. Thus, more than 20 targets for future overexpression or deletion experiments were identified within this work., Der thermophile, filament��se Pilz Thermothelomyces thermophilus ist ein sehr effizienter nat��rlicher Produzent von Hydrolasen, wie z.B. lignocellulolytischen Enzymen. Um die Produktion von Hydrolasen und heterologen Proteinen weiter zu optimieren, ist das Verstehen der Expression und Regulation von Hydrolasen von gro��em Interesse. Dadurch kann die Expression von spezifischen Hydrolasen f��r Anwendungen in z.B. Bioraffinerien gesteigert, oder mit dem Ziel einer erh��hten Reinheit und Ausbeute von heterolog exprimierten Proteinen, verringert werden. Die Dissertation hatte deshalb zum Ziel: 1) molekularbiologische Methoden zu etablieren und anzuwenden, um Regulatoren der Cellulaseexpression zu deletieren, 2) Bioreaktorkultivierungen zu etablieren, mit den erstellten St��mmen durchzuf��hren und anschlie��end biochemisch zu analysieren und 3) das Transkriptom im Hinblick auf Expression und Regulation von Hydrolasen zu analysieren. Molekularbiologische Methoden wurden etabliert und erfolgreich angewendet, um bekannte Regulatoren der Cellulaseexpression (Clr1, Clr2, Clr4 und Vib1) und einen potenziell neuen (Vib2) zu deletieren. Die Gene clr2 und vib2 wurden mittels Split Marker Methode deletiert und clr4 und vib1 durch das neu etablierte CRISPR/Cas12a System. Die Deletion von clr1 erforderte die gleichzeitige Nutzung der Split Marker Methode und des CRISPR/Cas12a Systems. Protokolle f��r Bioreaktorkultivierungen mit T. thermophilus (Batch und Chemostat) wurden erfolgreich etabliert und erm��glichen zuk��nftige Studien unter kontrollierten, industriell relevanten Bedingungen. Die Transkriptomanalyse zeigte eine starke Expression vorhergesagter lignocellulolytischer Enzyme, insbesondere von Cellulasen, Hemicellulasen, Pektinasen und Esterasen als Antwort auf Cellulose. Fast alle vorhergesagten LPMOs wurden ebenfalls stark exprimiert. Demnach ist T. thermophilus f��r die Anwendung in Bioraffinerien geeignet. Zus��tzlich offenbarte die Transkriptomanalyse stark exprimierte vorhergesagte Proteasen und bisher unbekannte Regulatoren der Expression von lignocellulolytischen Enzymen, welche deletiert oder ��berexprimiert werden k��nnten, um T. thermophilus f��r industrielle Anwendungen zu optimieren. Des Weiteren konnte die Rolle von Orthologen zu bekannten Regulatoren des Abbaus von pflanzlicher Biomasse detailliert untersucht werden, wodurch die essenzielle Funktion von Clr1 und Clr2 f��r die Cellulaseexpression gezeigt werden konnte. Hierbei ist die Expression von clr2 und damit die komplette lignocellulolytische Antwort auf Cellulose abh��ngig von Clr1. Die vorliegenden Daten f��r Clr4, bekannt als Aktivator der Cellulaseexpression, weisen stattdessen auf eine reprimierende Funktion hin, da eine Deletion zu einer h��heren Expression putativer lignocellulolytischer Enzyme und den zugeh��rigen Regulatoren (z.B. Clr2) als Antwort auf Cellulose f��hrte. Weiterhin konnte die Funktion des potenziell neuen Regulators Vib2 untersucht werden, welcher f��r eine Repression der fr��hen lignocellulolytischen Antwort auf Cellulose sorgt. Die Deletionsmutante von vib1 zeigte Wachstumsdefekte auf vielen Kohlenstoffquellen (einschlie��lich Cellulose) auf festem Medium, wogegen Cellulose bei fl��ssiger Kultivierung erfolgreich genutzt werden konnte. Die Transkriptomanalyse dieser Mutante wurde innerhalb dieser Arbeit nicht durchgef��hrt. Letztlich suggerieren die Daten eine starke Beteiligung von Genen die als Orthologe von anderen bekannten Regulatoren, wie z.B. Stk12 und McmA/1, identifiziert wurden. Somit konnten ��ber 20 Gene f��r zuk��nftige ��berexpressions- und Deletionsexperimente innerhalb dieser Arbeit identifiziert werden.

Antibody-mediated rejection (AMR) is the main cause of the kidney graft dysfunction and its failure after transplantation. Antibodies lead to vascular damage that is either acute or chronic and manifests as sudden or progressive graft dysfunction. Risk factors for development of AMR are time spent on haemodialysis, retransplantation, previous sensitisation against HLA antigens, and persistence of panel-reactive antibodies. Diagnosis is based on detection of deposits of C4d component of complement in peritubular capilaries and presence of donor-specific antibodies (DSA). We can also observe injury caused by antibodies against non-HLA antigens without detection of anti-HLA DSA. Use of "molecular microscope" can be beneficial in diagnosis and stratification of the risk of graft failure. High expression of ENDAT (endothelial activation and injury transcript) improves prediction of kidney graft failure more than C4d staining. Based on gene expression, the AMR scoring system correlates with the diagnosis of AMR and predicts graft loss in the future. The main goal of our work was to recognize patients at risk of AMR. In our study, we confirmed the efficacy and safety of acute AMR therapy with plasmaphereses and administration of intravenous immunoglobulins for improving outcomes of kidney transplantation....

Single cell multi omics techniques have shown an advancement in unrevealing complex diseases like cancer heterogeneity by providing multi-faceted insight into their individual cellular regulations. In this study, a machine learning approach, Bayesian network (BN), has been applied to integrate genomic, epigenomic, and transcriptomic data in hepatocellular carcinoma at single cell resolution. Hepatocellular carcinoma (HCC) is the most common type of liver cancer with a high metastatic rate and reckoned for poor prognosis. Heterogeneity of tumor cells is concerned with cancer progression, metastasis, therapeutic resistance, and mortality. For this purpose, a dataset from a published study of 25 single cell sequencing of hepatocellular carcinoma were used. First, DNA methylome and transcriptome data were analyzed on their own. Copy number variations were estimated from DNA methylome data by using the Hidden Markov Model method. To reveal the causal relationship between the omics, three BN models were constructed. The models were fitted to their parameters by using maximum likelihood estimation. For model evaluation, score-based criteria, Akaike information criterion and Bayesian information criterion, were used. 207 genes with significant models have been detected. The heterogeneity of the omics and their regulation mechanisms with each other have been shown, by pointing to genes that follow different BN models that take place in major pathways in HCC. Tek hücreli çoklu omik teknikleri, kendi bireysel hücresel düzenlemelerine çok yönlü bir bakış açısı sağlayarak kanser heterojenliği gibi kompleks hastalıkların ortaya çıkarmada bir ilerleme göstermiştir. Bu çalışmada, tek hücre temelli hepatoselüler karsinomda genomik, epigenomik ve transkriptomik verileri entegre etmek için bir makine öğrenimi yaklaşımı olan Bayesian ağları (BN) uygulanmıştır. Bu amaçla, yayınlanmış bir çalışmadan hepatoselüler karsinomun 25 tekil hücre dizileme veri seti kullanılmıştır. Hepatosellüler karsinom (HSK), yüksek metastatik oranla en yaygın karaciğer kanseri türüdür ve kötü prognoza sahip olduğu düşünülmektedir. Tümör hücrelerinin heterojenliği, kanserin ilerlemesi, metastaz, terapötik direnç ve mortalite ile ilgilidir. Önce, DNA metilom ve transkriptom verileri tek başlarına analiz edilmiştir. Kopya sayısı varyasyonu, Gizli Markov Modeli yöntemi kullanılarak DNA metilom verilerinden tahmin edilmiştir. Omikler arasındaki nedensel ilişkiyi incelemek için üç BN modeli oluşturulmuştur. Modeller, en çok olabilirlik kestirimi (MLE) kullanılarak parametrelerine uydurulmuştur. Model değerlendirme için puana dayalı kriterler, Akaike bilgi kriteri (AIC) ve Bayes bilgi kriteri (BIC) kullanılmıştır. Anlamlı modele sahip 207 gen tespit edilmiştir. Farklı BN model izleyen genlerin HCC’de aynı yolakta yer aldığına işaret ederek, omiklerin ve birbirleriyle regülasyon mekanizmalarının heterojenliği gösterilmiştir.

Pojavom metoda sekvenciranja druge generacije, duge nekodirajuće RNA postale su vrlo zanimljiv predmet bioloških istraživanja. Njihove uloge dokazane su u velikom broju bioloških procesa, od kojih je najvažnije spomenuti regulaciju ekspresije brojnih gena. Ipak, ova skupina RNA još uvijek nije istražena u brojnim koljenima životinja, uključujući i spužve. Radi bolje karakterizacije navedene skupine RNA spužvi, u ovom istraživanju korištene su dvije sekvencirane knjižnice RNA primarnih kultura ogulinske špiljske spužvice. Serijom bioinformatičkih metoda u sastavljenim transkriptomima pronađen je velik broj dugih nekodirajućih RNA. Utvrđeno je postojanje brojnih sličnih svojstava dugih nekodirajućih RNA spužvi i drugih životinjskih koljena, uključujući izrezivanje introna, mogućnost alternativnog prekrajanja i pristranost u sastavu nukleotida. Također, ustanovljena je potencijalna uloga transpozona u nastanku dugih nekodirajućih RNA, kao i moguća uloga navedene skupine RNA u regulaciji ekspresije brojnih protein-kodirajućih gena. Isto tako, opisan je raznolik skup stabilnih sekundarnih struktura dugih nekodirajućih RNA. Na kraju, pronađena je relativno slaba očuvanost dugih nekodirajućih RNA u koljenu spužvi, pri čemu iznimku čini izrazito velika sličnost navedene skupine RNA ogulinske špiljske spužvice i srodne spužve Ephydatia mulleri, što baca novo svjetlo na raspravu o mogućnosti pogrešne klasifikacije ogulinske špiljske spužvice. With the advances in next-generation sequencing technologies, long non-coding RNA have become one of the focal points of RNA research. Their roles have been demonstrated in a plethora of biological processes, most important being the regulation of gene expression. However, key information surrounding this type of RNA in many phyla is missing, which is why this research focused on characterizing long non-coding RNAs in sponges (Porifera). In this study, I used two RNA libraries isolated from the endemic cave sponge to identify a comprehensive set of highly-confident long non-coding RNAs. This study showed many similarities between long non-coding RNAs of sponges and other animals, including the phenomena of intron excision, alternative splicing, and nucleotide content bias. Furthermore, this research demonstrated the potential role of transposable elements in the origin of sponges' long non-coding RNAs, as well as a possible role of these RNAs in the regulation of expression of many important genes. Finally, this study found a low level of conservation of sponges' long non-coding RNAs, with the exception of the similarity found between endemic cave sponge and a closely related sponge, Ephydatia mulleri, which sheds new light on the potential misclassification of the endemic cave sponge.

Prezgodnji porod (PP) predstavlja vodilni vzrok neonatalne obolevnosti in umrljivosti. Kljub poročani povezavi med sestavo vaginalnega mikrobioma in PP, so bile predhodne raziskave v sklepih neskladne, sistemski mehanizmi preko katerih vaginalni mikrobiom vpliva na dovzetnost za PP, pa v veliki meri nepojasnjeni. Postavili smo naslednji hipotezi: (1) z analizo vaginalnega mikrobioma bomo ugotovili razlike v sestavi med slovenskimi nosečnicami, ki so rodile pred 37. tednom nosečnosti in nosečnicami, ki so rodile ob predvidenem terminu poroda, (2) potencialne razlike v sestavi vaginalnega mikrobioma so povezane s specifičnim globalnim ekspresijskim profilom v materini periferni krvi. Z namenom oceniti vlogo vaginalnega mikrobioma pri PP smo izvedli neodvisno raziskavo 16S sekvenciranja. Pri preiskovankah s PP smo ugotovili večjo mikrobno bogatost in raznolikost, zmanjšano zastopanost rodu Lactobacillus ter povečano zastopanost rodu Gardnerella in drugih z bakterijsko vaginozo in aerobnim vaginitisom povezanih rodov. Nadalje smo izvedli globalno transkriptomsko analizo RNA-sekvenciranja materine polne periferne krvi, z namenom ugotoviti ali je tvegani vaginalni mikrobiomski profil (CST IV/CST III) povezan s sistemskimi posledicami. Zaznali smo številne biološke poti, značilno povezane s PP (CST IV/CST III) med njimi signalno pot fosfatidilinositola, JAK-STAT, Fc gama-R fagocitozo in adherentne stike ter obogatene bolezenske in funkcijske procese, vključno z vnetnim odzivom, celičnim gibanjem, aktivacijo granulocitov, fagocitozo, kemotakso, organizacijo citoskeleta in adhezijo. V opravljeni raziskavi predstavljamo nadaljnje dokaze, da je sestava vaginalnega mikrobioma povezana s PP ter morebitno povezavo tveganega profila vaginalnega mikrobioma pri PP s hkratnimi transkriptomskimi spremembami periferne krvi. Z analizo prispevamo nova znanja potencialnih dejavnikov, ki privedejo do PP. Preterm birth (PTB) represents a leading cause of neonatal morbidity and mortality. Despite the reported association between vaginal microbiome and PTB, previous research has been inconsistent in its conclusions, and the systemic mechanisms through which the vaginal microbiome influences susceptibility to PTB remain unexplained. We set the following hypotheses: (1) analysis of the vaginal microbiome will reveal differences in the composition between Slovenian pregnant women who delivered before completed 37th week of gestation and pregnant women who delivered at term, (2) potential differences in the composition of the vaginal microbiome are associated with a specific global expression profile in maternal peripheral blood. To re-evaluate the role of the vaginal microbiome in PTB, we conducted an independent 16S sequencing study. In PTB women, we report higher microbial richness and diversity, decreased abundance of genus Lactobacillus, increased abundance of Gardnerella, and other bacterial vaginosis and aerobic vaginitis-related genera. We performed a global transcriptomic analysis of maternal whole peripheral blood to determine whether a high-risk vaginal microbiome profile (CST IV/CST III) is associated with systemic consequences. We identified several biological pathways associated with PTB (CST IV/CST III), including phosphatidylinositol signaling pathway, JAK-STAT, Fc gamma-R phagocytosis, and adherent junctions, as well as enriched diseases and functions, including inflammatory response, cell movement, granulocyte activation, phagocytosis, chemotaxis, cytoskeletal organization, and adhesion. The present study represents further evidence that the vaginal microbiome composition is associated with PTB and the possible association of risk vaginal microbiome profile with concomitant transcriptomic changes in peripheral blood. We contribute new knowledge about the potential factors leading to PTB.

Knochenmarks-Stromazellen sind in den letzten Jahren in den Fokus der Forschung gerückt. Es konnte gezeigt werden, dass sie durch Bereitstellung von Überlebenssignalen essenziell für die Erhaltung hämatopoetischer Nischen sind. Stromales Interleukin-7 (IL-7) konnte dabei für T Zellen als Überlebenssignal identifiziert werden. Gemeinsam ist allen Stromazellen die Expression des Oberflächenmarkers CD106/VCAM-1. Ein effizientes Protokoll erlaubte die qualitative wie quantitative Isolation von Stromazellen aus dem murinen Knochenmark mit anschließender ex vivo Microarray-Analyse. Die auf diese Weise ermittelten Kandidaten-Marker wurden auf Proteinebene via Histologie und (Hochdurchsatz-) Durchflusszytometrie validier. Dazu gehören z.B. die Marker CD1d, gas6 oder ANXA2R. CD1d wurde als guter Interimsmarker für VCAM-1+PECAM-1- Stromazellen identifiziert, wohingegen die IL-7-Produzenten in der Population von CD200int/BP 1+/CD73+/CD105- angereichert sind. Gleiches gilt für den Transkriptionsfaktor Prrx1. CD55, BP-1 and Cadherin-11 zeigten eine Expressionsmuster in Abhängigkeit des verwendeten IL-7-Reportermaus-Haplotyps. Für BP-1 und Cadherin 11 konnte die Abwesenheit von reifen Lymphozyten als Ursache des Feedbacks ausgeschlossen werden. Die Haplotypen der Reportermaus legten auch eine monoallele Expression des IL-7 nahe. Die Ergebnisse dieser Arbeit zeigen VCAM-1+ (IL-7+/-) Stromazellen als heterogene Population, wenn es nach der Vielzahl der möglichen exprimierten Marker geht. Zwischen vielen dieser Marker gibt es aber wiederum auf Zelloberflächenebene einen großen Überlapp. Die funktionelle Relevanz dieser Oberflächenmarker-Diversität wird in weiteren Arbeiten zu klären sein, gibt aber den Stromazellen ein breites Repertoire vor, um Interaktionen mit Lymphozyten zu initiieren, modulieren und inhibieren. Abschließend ist zu erwarten, dass diese Erkenntnisse in die klinische Behandlung der Stroma-Nischen in Autoimmun-Fragestellungen einfließen. Bone marrow stromal cells receive increasing amounts of attention lately. They have been shown to support survival of hematopoietic stem cells as well as memory lymphocytes which is of great importance when targeting the perseverance of autoimmune diseases. CD4+ memory T lymphocytes reside in the proximity of VCAM-1 expressing stromal cells which provide them with survival signals such as Interleukin-7. Herein, a protocol was developed to quantitatively obtain VCAM-1+ and VCAM-1+ IL-7+/- stromal cells via enzymatic/mechanic digestion and cytoskeleton-inhibition. Ex vivo gene expression analysis was performed from sorted, pure cells with good recovery. Candidate genes/markers were validated in (high-throughput) flow cytometry and histological analysis including subsequent semi-automated colocalization was performed. CD1d was found to be good surrogate marker for VCAM-1+PECAM-1- non-endothelial stroma while the population of CD200int/BP-1+/CD73+/CD105- stromal cells is greatly enriched in IL-7 producers which was equally true for the stromal transcription factor Prrx1. CD55, BP-1 and Cadherin-11 were found to be differentially expressed in differing IL-7 reporter mice haplotypes. The reporter mice haplotypes revealed monoallelic expression features of IL-7. All methodologies suggest that VCAM-1+ as well as IL-7+/- stromal cells are heterogeneous by marker expression yet don’t cluster extensively in flow cytometry co-stains. The functional relevance of the marker diversity described in this thesis remains to be tested but insinuates a broad repertoire for bone marrow stroma cells for new interaction pathways with lymphocyte subsets. Ultimately, this knowledge will hopefully feedback to clinical questions of autoimmunity for targeted treatment of stromal niches.

Ciliates are the most diversified protists in suboxic and anoxic habitats where they often form symbioses with prokaryotes. Although the diversity of anaerobic ciliates has been overlooked for a long time, anaerobic representatives can be found in most ciliate classes. This study focuses on anaerobic ciliates from the subclass Scuticociliatia, a neglected lineage, which belongs to the species-rich class Oligohymenophorea. One of the main outcomes resulting from this study is the discovery of a novel anaerobic clade of ciliates, from which only one species has been described molecularly to date. We have shown that the clade represents a diversified lineage, likely a new order. Thanks to the sampling of many freshwater and marine anoxic sediments, we have established the largest culture collection of anaerobic scuticociliates in the world. This has enabled us to determine the 18S rRNA gene sequences of 55 cultured anaerobic scuticociliates and to study their morphology both in-vivo and using various silver- impregnation methods. Besides, we applied transmission and scanning electron microscopy techniques to study the ultrastructure of both ciliates and symbionts. To identify the symbionts, we also employed other methods including microbiome sequencing and fluorescence in-situ hybridization. Since all...

Bei enterohämorrhagischen Escherichia coli (EHEC) handelt es sich um humanpathogene Bakterien, welche beim Menschen schwerwiegende gastrointestinale Erkrankungen auslösen können. Der Gastrointestinaltrakt von Rindern wird als Hauptreservoir von EHEC angesehen und die primäre Infektionsquelle des Menschen stellt kontaminiertes, rohes Fleisch dar. Jedoch kam es in den letzten Jahrzehnten vermehrt zu EHEC-Infektionen, welche mit dem Verzehr von rohem Gemüse und Salatpflanzen assoziiert wurden. Heute ist es anerkannt, dass diese Infektionen dadurch begünstigt werden, dass EHEC-Bakterien in der Lage sind, Pflanzen als Sekundärwirte zu nutzen und damit eine Übertragung auf den Menschen erleichtern. Um diese Interaktion zwischen Pathogen und Pflanze besser zu verstehen, sind grundlegende Kenntnisse in der molekularen Anpassung an pflanzliche Bestandteile nötig. Im Rahmen dieser Arbeit wurde die Anpassung verschiedener EHEC Stämme an pflanzliche Bestandteile untersucht, dazu wurden O157:H7 Stamm Sakai, O104:H4 Stamm C227-11phicu und O157:H- Stamm 3072/96 als Prototypen ausgewählt. Zunächst konnte in Wachstumsexperimenten in einem artifiziellen Salatmedium gezeigt werden, dass Inhaltsstoffe des Salatmediums für ein erfolgreiches Wachstum aller drei Stämme ausreichend waren. In der RNA-Sequenzierung wurde die differentielle Genexpression der drei Stämme nach Wachstum in Salatmedium gegenüber dem Wachstum in M9-Minimalmedium bestimmt. Um Gene zwischen den Stämmen anhand einer einheitlichen Genbezeichnung miteinander zu vergleichen, wurde die differentielle Genexpression mit der Grundlage eines geteilten Genoms der drei pathogenen Stämme inklusive Referenzstamm Escherichia coli Stamm K-12 Substamm MG1655 durchgeführt. Analog zum erfolgreichen Wachstum in Anwesenheit pflanzlicher Bestandteile zeichnete sich eine erhöhte Transkription von zahlreichen Genen des Kohlenhydrat- und Peptidmetabolismus in allen drei Stämmen ab. Besonders die Gene des Lactose- (lacZ), Ribose- (rbsAC) und Xylosemetabolismus (xylF) waren in allen Stämmen deutlich hochreguliert. Die deutlichsten transkriptionellen Unterschiede zwischen den Stämmen zeigten sich in der Regulation von Motilitäts- und Chemotaxis-Genen. O104:H4 Stamm C227-11phicu zeigte in Anwesenheit pflanzlicher Bestandteile eine starke Expression aller Gene der drei Flagellenklassen (Klasse I, II und III). Dazu gehören die Gene, welche für den Aufbau der Flagellengrundstruktur (fli, flg), der Ausbildung des Filaments (fliC) sowie des Chemotaxis-Systems (che, tar, tap) verantwortlich sind. Dem entgegen konnte bei O157:H7 Stamm Sakai ausschließlich eine erhöhte Expression der Klasse I und II Flagellengene beobachtet werden. In Übereinstimmung mit den Transkriptomdaten zeigten diese beiden Stämme ein gesteigertes Schwimm- und Schwärmverhalten auf Motilitätsplatten in Anwesenheit von Salatextrakt. Ausschließlich bei dem, ausgelöst durch eine Deletion im Gen flhC, unbeweglichen O157:H- Stamm 3072/96 konnte eine gesteigerte Expression von Virulenzfaktoren der LEE-Pathogenitätsinsel beobachtet werden. Dazu gehörten Gene für den Aufbau des T3SS (esc) sowie T3SS-vermittelten Effektoren (esp). Interessanterweise äußerte sich dieser Stamm als leistungsfähiger Biofilmbildner in M9-Minimalmedium. Des Weiteren konnte gezeigt werden, dass die Komplementierung des intakten flhC-Gens die Beweglichkeit von O157:H- Stamm 3072/96 wieder herstellen konnte. In diesem Zusammenhang konnte ebenfalls festgestellt werden, dass die Deletion im Gen flhC nicht der alleinige Grund für die erhöhte Biofilmbildung dieses Stammes war. Zusätzlich zur Bestimmung der Biofilmbildung wurden die Stämme auf ihre Adhärenz an HT-29 Zellen untersucht. Hierbei konnte für O157:H- Stamm 3072/96 gegenüber den motilen Stämmen eine signifikant erhöhte Adhärenz beobachtet werden, das geringste Adhärenzpotential konnte bei O157:H7 Stamm Sakai festgestellt werden. Die Ergebnisse dieser Studie liefern klare Hinweise, dass die verschiedenen EHEC-Stämme fähig sind, sich an die Nährstoffverfügbarkeiten in einem pflanzlichen Wirt anzupassen. Es kann davon ausgegangen werden, dass Flagellen und das Chemotaxis-System eine entscheidende Rolle in der Auffindung von Pflanzen und deren Erschließung spielen. Außerdem könnten Curli-Strukturen eine elementare Funktion bei der initialen Anheftung und der anschließenden Biofilmbildung auf pflanzlichem Gewebe spielen. Vermutlich sind, neben dem typischen Pflanzen-assoziierten Ausbruchstamm O157:H7 Stamm Sakai, auch weitere EHEC-Stämme in der Lage ihr genetisches Repertoire auszunutzen, um eine Antwort auf die atypische Bedingungen dieser Nische zu gewährleisten. Die in dieser Arbeit gewonnenen Ergebnisse legen nahe, dass die Stämme aufgrund ihrer unterschiedlichen genetischen Ausstattung, neben zahlreichen übereinstimmenden Mechanismen, zusätzlich auch stammspezifische Strategien bei einer Interaktion mit Pflanzen als Sekundärwirte zeigen. Enterohemorrhagic Escherichia coli (EHEC) are human pathogens which are able to cause severe gastrointestinal diseases in humans. The gastrointestinal tract of cattle is considered as the main reservoir for EHEC and contaminated raw meat represents the primary source of infection. Yet there have been increasing reports over the last few decades of EHEC infections that were linked to the consumption of raw vegetables. Today it is generally accepted that EHEC bacteria are able to use plants as their secondary hosts, thus favouring the transmission to humans. To improve the understanding of this pathogen-plant interaction fundamental knowledge about the pathogens’ molecular adaptions towards plant material is urgently required. In the cope of this study the adaption of different EHEC strains towards components of the plant was examined. Therefore O157:H7 strain Sakai, O104:H4 strain C227-11phicu and O157:H strain 3072/96 were chosen as surrogates. In growth experiments performed with an artificial lettuce medium it could be shown that components of the lettuce were sufficient for the proliferation of the three strains. RNA-sequencing was performed to study the differential gene expression of the three strains after the growth in lettuce medium compared to the growth in M9 minimal medium. In order to compare genes according to standardized gene denotations, the differential gene expression analysis was performed on the basis of a shared genome including the genomes of the three pathogenic strains as well as the genome of Escherichia coli strain K-12 substrain MG1655. Analogous to the successful growth in presence of components of the plant an upregulation of genes involved in carbohydrate and peptide metabolism throughout all three strains was observed. Especially genes involved in the catabolism of lactose (lacZ), ribose (rbsAC) and xylose (xylF) were found to be uniformly upregulated. The greatest differences among the strains accounted for the regulation of motility and chemotaxis genes. O104:H4 strain C227-11phicu showed a strong upregulation of all three classes of the flagellar hierarchy (class I, II and III) in presence of plant derived compounds. These included genes involved in the establishment of the basal body hook structure (fli, flg), the synthesis of the flagellar filament (fliC), and the chemotaxis-system (che, tap, tar). In contrast, O157:H7 strain Sakai only featured upregulation of class I and class II genes. According to the transcriptional data both of these strains also showed increased swimming and swarming behaviour on motility plates in presence of lettuce extract. Solely O157:H- strain 3072/96, which is non-motile due to a deletion in the flhC gene, showed an upregulation of virulence factors encoded on the LEE pathogenicity island, including genes involved in the establishment of the T3SS (esc) and T3SS secreted effectors (esp). Interestingly, it was shown for O157:H- strain 3072/96 to have a powerful capacity to form biofilms in M9 minimal medium. Furthermore it was proven that the complementation of an intact flhC gene restored motility in O157:H- strain 3072/96. In this regard it could be shown that the deletion in flhC was not the mere reason for the augmented biofilm formation capacity. In addition to the biofilm formation, the strains’ potential to adhere to HT-29 cells was examined. Here a significantly increased adherence potential for O157:H- strain 3072/96 with respect to the motile strains could be observed, the lowest adherence potential was determined for O157:H7 strain Sakai. The results presented in this study clearly indicate that the different EHEC strains are capable to adapt towards the nutrient availability provided by their plantal host. It can be assumed that flagella and the chemotaxis system play a fundamental role in the finding and exploitation of the plant. Furthermore curli structures might play a crucial role in the initial adherence and the subsequent establishment of a biofilm on plant tissues. Presumably, besides the typical plant associated outbreak strain O157:H7 strain Sakai, there are further strains capable of utilizing their genetic repertoire in order to adapt towards the atypical environmental conditions within this niche. The findings of this study suggest that the strains, besides sharing multiple coinciding mechanisms, are able to adapt in a strain specific manner and use different strategies in coping with plants as their secondary hosts.

Sand flies (Diptera, Phlebotominae) are small biting insects and vectors of Leishmania spp. which cause medically and veterinary important disease - leishmaniasis. During the piercing of the host skin, sand fly females inject saliva to facilitate the blood feeding. The sand fly saliva is composed of many bioactive molecules which were shown to possess anti-inflammatory and anti-haemostatic functions. The saliva affects host's immunity in the bite site and consequently enhances the survival and development of transmitted pathogens. Most of the studies focus on salivary proteins and enzymes of sand flies belonging to Phlebotomus and Lutzomyia genera, while salivary proteins from sand flies of the third genus Sergentomyia were neglected so far. In this thesis we focused on comparison of salivary proteins from two Phlebotomus species, namely Phlebotomus perniciosus and Phlebotomus orientalis, and Sergentomyia schwetzi. These sand fly species differ not only by the ecology and geographical distribution but also by host preferences. Both Phlebotomus species prefer large or medium-size mammals as the bloodmeal source, particularly rabbits, hares and dogs for P. perniciosus and cattle, goats, sheep and humans for P. orientalis. Contrarily, Sergentomyia sand flies are known for preferred feeding on reptiles...

The proposed thesis describes stage-specific transcription of Ixodes ricinus tick. Based on the transcriptome assemblies of I. ricinus tick life stages this work describes processes typical for each I. ricinus life stage and infers the significance of methylation and glycosylation for ticks.

Ebolaviruses are negative strand RNA viruses which are known to cause Ebola virus disease (EVD) with a fatal outcome in primates. All five species of Ebolavirus can infect humans, but only four lead to EVD. The Ebolavirus with the most provoked outbreaks and highest fatality rate (above 80%) is Zaire ebolavirus (EBOV), while the one without any provoke symptoms in humans is Reston ebolavirus (RESTV). In order to determine the features which lead to the different outcomes from EBOV and RESTV the cellular response against these viruses, and the divergence between RESTV and EBOV life cycle inside human cells was investigated. To study the cellular response RNA of two human cell lines (HuH7 and THP1) infected with RESTV, EBOV and uninfected (Mock) at two different time points was analyzed. Using whole transcriptome screening with smallRNAseq, Microarray, de novo annotation and expression profiles it was possible to elucidate that the cellular response against RESTV and EBOV infection differs the most at 3 h p.i., this was consistent in HuH7 and THP1 cell lines. The transcriptomic study showed RESTV and EBOV stimulate a distinct set of genes related to cellular entry. Also, the transcriptomic data suggests EBOV transcribes and replicates faster than RESTV, supported by cellular components like snoRNAs, while RESTV is similar to Mock in this aspect. This finding was backed with an entry assay which showed EBOV releases its content into the cytosol faster than RESTV, pointing to differences in entry pathway or a better time controlled response from the cell against RESTV. To understand the life cycle of RESTV and EBOV in human cells transcription/replication, inclusion bodies, nucleocapsid (NC) transport, viral particle formation, and infection was studied. Selected genes which were differentially expressed between RESTV and EBOV infected cells were further analyzed on the virus life cycle context.