The liver is the central metabolic hub in organisms and a complex, intertwining regulatory network guarantees efficient liver processes. The morphogenic Hedgehog pathway was recently shown to play a role in regulating the underlying genetic program. Transgenic mouse models with hepatocyte-specific inactivation of Hedgehog signaling showed alterations in insulin-like growth factor homeostasis and in energy metabolism associated with increased lipid accumulation in the liver. In this thesis, it was possible to connect the observed infertility of female knockout mice with an unexpected activation of sex steroid synthesis in the liver. Associated with increased steroidogenic gene expression exclusively in hepatocytes, the plasma testosterone level was significantly elevated, which led to androgenization and an anovulatory phenotype. With these characteristics, the mouse model mimicked the human polycystic ovarian syndrome and suggested an influence of liver and hepatic Hedgehog signaling on reproduction under disease conditions. Further, murine liver metabolism was challenged with starvation starting at different times of day. The transcriptomic results were analyzed with a self-organizing map approach, allowing an intuitive interpretation of data and a thus far unknown diurnally different response of hepatic regulatory mechanisms due to starvation was revealed. In contrast to the manifoldly published and observed switch from energy-consuming to energy-providing processes due to starvation started in the morning, evening starvation led to a novel hepatic expression signature with decreased gluconeogenic gene expression and increased levels of lipid and steroid metabolism-related genes. These differences can be explained by the equally diurnally regulated expression of the corresponding regulatory transcription factors and hormones. Additionally, lipidome analysis confirmed the diurnal differences after starvation. Thus, this study emphasized the immense impact of circadian regulation on liver metabolism and suggests high accuracy when starvation is the focus of research to avoid varying results.:BIBLIOGRAPHISCHE DARSTELLUNG ................................................................................ II LIST OF ABBREVIATIONS .................................................................................................. III TABLE OF CONTENTS ....................................................................................................... IV SUMMARY ............................................................................................................................ 1 ZUSAMMENFASSUNG ......................................................................................................... 5 INTRODUCTION ................................................................................................................... 9 Liver architecture and metabolism ..................................................................................... 9 Diverse possibilities of liver metabolism regulation .......................................................... 10 Connection of Hedgehog signaling to hepatic metabolism ............................................... 10 Impact of feeding schemes on hepatic metabolism .......................................................... 13 Aims of the thesis ............................................................................................................ 14 References ...................................................................................................................... 15 CHAPTER 1 ........................................................................................................................ 18 CHAPTER 2 ........................................................................................................................ 39 PERSPECTIVE ................................................................................................................... 64 CURRICULUM VITAE ........................................................................................................... V PUBLICATIONS AND PRESENTATIONS ............................................................................ VI Publications ...................................................................................................................... VI Oral presentations ............................................................................................................ VI Poster presentations ........................................................................................................ VII AUTHOR CONTRIBUTION STATEMENT .......................................................................... VIII SELBSTSTÄNDIGKEITSERKLÄRUNG .............................................................................. XII DANKSAGUNG .................................................................................................................. XIII

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.

No description available.

Viele biologische Prozesse im Verhalten von ganzen Organismen, aber auch in den Prozessen und der biochemischen Zusammensetzung von Zellen zeigen einen zirkadianen Rhythmus, also einen Rhythmus mit einer Periode von etwa 24 Stunden. Diese 24-Stunden-Rhythmen sind in der Genexpression auf allen Ebenen zu finden: von der Tran- skriptionsinitiation bis zur Proteindegradation. Auf Transkriptebene, zirkadiane mRNA-Produktion und mRNA-Abundanz ist umfassend gemessen. Auf der anderen Seite, zirkadiane posttranskriptionelle Regulation ist weit weniger verstanden. In dieser Arbeit untersuche ich, wie bisher ungemessene, posttranskriptionelle Prozesse die rhythmischen Eigenschaften der Genexpression beeinflussen. Dazu beschreibe ich die Lebensstadien eines Bio-Moleküls mit einem Modell-Motiv, einer einfachen Differentialgleichung mit zeitabhängigen, rhythmischen Raten. Als erstes diskutiere ich die Einschränkungen von Phase und Amplitude zirkadianer Transkripte, die nur von konstanter PTR beeinflusst werden. Bei vielen gemessenen Transkripten sind diese Einschränkungen verletzt. In diesen Fällen muss es eine rhythmische PTR geben. Ich untersuche, welche rhythmische PTR diese Fälle erklären können und führe einen statistischen Test ein, der auf unbeobachtete, rhythmische PTR testet. Durch die Analyse zweier Datensätze von Mausleber und -niere finde ich, dass 18% aller zirkadianen Gene in Niere und 34% in Leber rhythmisch posttranskriptionell reguliert sind. Im zweiten Teil analysiere ich weitere Aspekte von PTR in einem Hypothesen-getriebenen Ansatz. Ich zeige, dass Spleißen mit einem Rhythmus von 24 Stunden 12 Stunden-Rhythmen in der Abundanz von mRNA erzeugen kann. Als nächstes schlage ich ein Modell vor, das rhythmische Degradation von Mitgliedern der zirkadianen Uhr beschreibt. Schließlich erweitere ich das Modell-Grundmotiv zu einer partiellen Differentialgleichung (PDG), die das “Altern” von Molekülen beschreibt. / In almost all organisms on Earth, many behavioral, physiological, and biochemical activities oscillate with a circadian rhythm, a rhythm with a period of about 24 hours. In gene expression, the 24-hour-rhythm can be found on all stages: from transcription initiation to protein degradation. On the transcript level, circadian mRNA production and mRNA abundance are comprehensively charted through numerous genome-wide high throughput studies. Circadian post-transcriptional regulation, however, is less well understood. In this thesis, I will investigate how unobserved post-transcriptional processes influence rhythmic properties of gene expression. To this end, I quantify the life-stages of biomolecules using one modeling motif, a simple ordinary differential equation describing production and degradation with time-dependent rhythmic rates. This basic modeling motif is systematically varied to examine and discuss various influences of post-transcriptional regulation (PTR) on circadian mRNA expression. I first discuss the restrictions of rhythmic phase and amplitude of circadian transcripts influenced by non-rhythmic PTR. For many genes these restrictions are violated and we have to assume the existence of a rhythmic PTR. I discuss which rhythmic PTR can explain these findings and further introduce a statistical test to quantify the extent of unobserved rhythmic PTR. Analyzing two data sets on mouse liver and kidney, I find that 18% of circadian genes in kidney and 34% in liver are under rhythmic post-transcriptional control. In a second part, I analyze more specific aspects of PTR in a hypothesis-driven approach. Firstly, I find that splicing with a rhythm of 24 hours is able to generate 12-hour rhythms in abundance of mature mRNA. Secondly, I propose and analyze a model to investigate rhythmic degradation of core clock genes. And finally, I extend the core modeling motif to a partial differential equation (PDE) model that accounts for the “aging” process of molecules.

No description available.

The nutrient exchange between plant and fungus is the key element of the arbuscular mycorrhizal (AM) symbiosis. The fungus improves the plant’s uptake of mineral nutrients, mainly phosphate, and water, while the plant provides the fungus with photosynthetically assimilated carbohydrates. Still, the knowledge about the mechanisms of the nutrient exchange between the symbiotic partners is very limited. Therefore, transport processes of both, the plant and the fungal partner, are investigated in this study. In order to enhance the understanding of the molecular basis underlying this tight interaction between the roots of Medicago truncatula and the AM fungus Rhizophagus irregularis, genes involved in transport processes of both symbiotic partners are analysed here. The AM-specific regulation and cell-specific expression of potential transporter genes of M. truncatula that were found to be specifically regulated in arbuscule-containing cells and in non-arbusculated cells of mycorrhizal roots was confirmed. A model for the carbon allocation in mycorrhizal roots is suggested, in which carbohydrates are mobilized in non-arbusculated cells and symplastically provided to the arbuscule-containing cells. New insights into the mechanisms of the carbohydrate allocation were gained by the analysis of hexose/H+ symporter MtHxt1 which is regulated in distinct cells of mycorrhizal roots. Metabolite profiling of leaves and roots of a knock-out mutant, hxt1, showed that it indeed does have an impact on the carbohydrate balance in the course of the symbiosis throughout the whole plant, and on the interaction with the fungal partner. The primary metabolite profile of M. truncatula was shown to be altered significantly in response to mycorrhizal colonization. Additionally, molecular mechanisms determining the progress of the interaction in the fungal partner of the AM symbiosis were investigated. The R. irregularis transcriptome in planta and in extraradical tissues gave new insight into genes that are differentially expressed in these two fungal tissues. Over 3200 fungal transcripts with a significantly altered expression level in laser capture microdissection-collected arbuscules compared to extraradical tissues were identified. Among them, six previously unknown specifically regulated potential transporter genes were found. These are likely to play a role in the nutrient exchange between plant and fungus. While the substrates of three potential MFS transporters are as yet unknown, two potential sugar transporters are might play a role in the carbohydrate flow towards the fungal partner. In summary, this study provides new insights into transport processes between plant and fungus in the course of the AM symbiosis, analysing M. truncatula on the transcript and metabolite level, and provides a dataset of the R. irregularis transcriptome in planta, providing a high amount of new information for future works. / In der arbuskulären Mykorrhiza (AM) Symbiose werden die Wurzeln fast aller Landpflanzen von Pilzen der Abteilung Glomeromycota besiedelt. Der Pilz erleichtert der Pflanze die Aufnahme von Mineralien, hauptsächlich Phosphat, und Wasser. Im Gegenzug versorgt die Pflanze ihn mit Photoassimilaten. Trotz der zentralen Bedeutung der Austauschmechanismen zwischen Pilz und Pflanze ist nur wenig darüber bekannt. Um die molekularen Grundlagen der Interaktion zwischen den Wurzeln der Leguminose Medicago truncatula und dem arbuskulären Mykorrhizapilz Rhizophagus irregularis besser zu verstehen, werden hier die Transportprozesse, die zwischen den Symbiosepartnern ablaufen, näher untersucht. Die zellspezifische Regulation der Transkription potentieller M. truncatula Transporter Gene in arbuskelhaltigen und nicht-arbuskelhaltigen Zellen mykorrhizierter Wurzeln wird bestätigt. Ein Modell zur möglichen Verteilung von Kohlenhydraten in mykorrhizierten Wurzeln, nach dem Zucker in nicht-arbuskelhaltigen Zellen mobilisiert und symplastisch an arbuskelhaltige Zellen abgegeben werden, wird vorgestellt. Die Analyse eines Mykorrhiza-induzierten Hexose/H+ Symporter Gens, MtHxt1, liefert neue Einsichten in die Mechanismen der Kohlenhydratverteilung in mykorrhizierten Pflanzen. Metabolitanalysen von Wurzeln und Blättern einer knock-out Mutante dieses Gens zeigen dessen Einfluss auf den Kohlenhydrathaushalt der ganzen Pflanze und auf die Interaktion mit dem Pilz. Die Metabolitzusammensetzung von M. truncatula wird durch die Mykorrhiza Symbiose signifikant beeinflusst. Darüber hinaus werden durch Transkriptomanalysen die molekularen Grundlagen der AM Symbiose auf der Seite des Pilzes analysiert. Arbuskeln wurden mittels Laser Capture Mikrodissektion direkt aus mykorrhizierten Wurzeln isoliert. Über 3200 pilzliche Transkripte weisen in diesen Arbuskeln im Vergleich zu extraradikalen Geweben ein deutlich verändertes Expressionslevel auf. Unter diesen Transkripten sind auch sechs zuvor unbekannte Gene, die für potentielle Transporter codieren und mit großer Wahrscheinlichkeit eine Rolle im Nährstoffaustausch zwischen Pilz und Pflanze spielen. Während die Substrate von drei potentiellen MFS Transportern noch unbekannt sind, spielen zwei potentiellen Zuckertransporter möglicherweise eine Rolle im Transport von Kohlenhydraten in Richtung des Pilzes. Zusammengefasst bietet diese Arbeit neue Einsichten in Transportprozesse zwischen Pilz und Pflanze im Laufe der AM Symbiose. M. truncatula Transkript- und Metabolitlevel werden analysiert und die Transkriptomanalyse von R. irregularis liefert einen umfassenden Datensatz mit einer großen Menge an Informationen zu der noch unzureichend erforschten pilzlichen Seite der Symbiose für folgende Arbeiten.

Improving the short-term results of kidney transplantation did not result in improving the long-term function and survival of kidney allograft. Organ shortage and increasing number of marginal donors remains the key problem in transplant today. The quality of donor organ is critical for graft function development and survival. The aim is to improve understanding to ischemia/reperfusion injury and its consequences, predict delayed graft function and rejection, improve organ allocation strategy and identify patients suitable for safe drug minimization or complete withdrawal of immunosuppressive therapy. Analysis of donor kidneys identified poor tubular cell quality and low survival factor, Netrin-1 expression levels, to be associated with delayed graft function. We confirmed that reperfusion phase of ischemia/reperfusion injury leads to minimal morphological but significant molecular abnormalities. Dissociation observed in histology and molecular pathology finding calls for an integrated approach in donor quality organ evaluation and allocation for transplantation. Significant heterogeneity within donors with expanded criteria was shown and subgroup of organs at low risk of delayed graft function was identified. We suggested donor biopsies to be performed as a routine praxis in all kidneys...

Screening of a deeply sequenced transcriptome using Illumina sequencing as well as the genome of the tardigrade Hypsibius dujardini revealed a set of five opsin genes. To clarify the phylogenetic position of these genes and to elucidate the evolutionary history of opsins in Panarthropoda (Onychophora + Tardigrada + Arthropoda), we reconstructed the phylogeny of broadly sampled metazoan opsin genes using maximum likelihood and Bayesian inference methods in conjunction with carefully selected substitution models. According to our findings, the opsin repertoire of H. dujardini comprises representatives of all three major bilaterian opsin clades, including one r-opsin, three c-opsins, and a Group 4 opsin (neuropsin/opsin-5). The identification of the tardigrade ortholog of neuropsin/opsin-5 is the first record of this opsin type in a protostome, but our screening of available metazoan genomes revealed that it is also present in other protostomes. Our opsin phylogeny further suggests that two r-opsins, including an "arthropsin", were present in the last common ancestor of Panarthropoda. While both r-opsin lineages were retained in Onychophora and Arthropoda, the "arthropsin" was lost in Tardigrada. The single (most likely visual) r-opsin found in H. dujardini supports the hypothesis of monochromatic vision in the panarthropod ancestor, whereas two duplications of the ancestral panarthropod c-opsin have led to three c-opsins in tardigrades. Although the early-branching nodes are unstable within the metazoans, our findings suggest that the last common ancestor of Bilateria possessed six opsins: two r-opsins, one c-opsin, and three Group 4 opsins, one of which (Go opsin) was lost in the ecdysozoan lineage.

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...

Mammalian oocyte is a highly differentiated cell which gives rise to an embryo after fertilization. Importantly, fully-grown oocytes become transcriptionally inactive at the end of the growth phase. During following stages of development, i. e. meiotic maturation of the oocyte and early embryonic development, only transcripts previously synthesized and stored are used. The tight correlation between mRNA distribution and subsequent protein localization and function provides a mechanism of spatial and temporal regulation of gene expression used by various cell types. However, not much is known about mRNA localization and translation in the mammalian oocyte and early embryo. The aim of my thesis was to determine the localization of transcripts and components of translational machinery in the mammalian oocyte and embryo and to uncover the mechanisms of spatiotemporal regulation of translation as a prerequisite for correct oocyte and embryo development. We have shown that nuclei of both mouse and human oocytes contain RNA molecules and RNA binding proteins. Following the nuclear envelope breakdown (NEBD), translational hot-spots occur in the area surrounding the nuclear region. We suppose that mRNAs previously retained in the nucleus are released to the cytoplasm during NEBD and their subsequent...