UNC-6/Netrin guides circumferential migrations along the dorso-ventral axis in C.elegans. Its receptors, UNC-40/DCC and UNC-5, mediate both attraction and repulsion of migrating cells or growth cones from sources of UNC-6. seu-2(ev523)and seu-3(ev555) (suppressor of ectopic unc-5) were identified as suppressors of ectopic UNC-5 in the touch receptor neurons(Colavita and Culotii, 1998). Like other components of UNC-6 signaling, seu-2 and seu-3 have roles not just in the migration of axon growth cones, but also in the repulsive migration of other cell types, specifically the distal tip cells (DTCs). Similar to observations in the touch receptor neurons, both seu-2 and seu-3 are able to suppress ectopic expression of the UNC-5 receptor in the DTCs. Genetic analysis of seu-2; seu-3 double mutants reveals that these genes function within the same signaling pathway for repulsive unc-6 guidance. seu-2 also appears to act in attractive unc-6 guidance. Mutations in seu-2 result in ventral to dorsal axon guidance defects in the HSN and ray 1 neurons. Double mutant analyses of seu-2 with either unc-40 or unc-6 null mutations exhibited HSN and ray 1 axon guidance defects at similar penetrance to either single mutant. These results suggest that seu-2 functions in the attractive unc-6-unc-40 dependent signaling pathway for HSN and ray 1 axon guidance. seu-2 was found to encode a G protein coupled receptor. Whole genome sequencing was used to identiy that seu-3 encodes the novel protein K09C6.9. K09C6.9 is predicted secreted protein that is expressed throughout development. Taken together, the phenotypes, method of isolation and genetic interactions of seu-2 and seu-3 make them interesting candidate mediators of UNC-6 signaling. I utilized genes, such as seu-2 and seu-3, to further elucidate other signaling components governing cell migration and axon guidance.

UNC-6/Netrin guides circumferential migrations along the dorso-ventral axis in C.elegans. Its receptors, UNC-40/DCC and UNC-5, mediate both attraction and repulsion of migrating cells or growth cones from sources of UNC-6. seu-2(ev523)and seu-3(ev555) (suppressor of ectopic unc-5) were identified as suppressors of ectopic UNC-5 in the touch receptor neurons(Colavita and Culotii, 1998). Like other components of UNC-6 signaling, seu-2 and seu-3 have roles not just in the migration of axon growth cones, but also in the repulsive migration of other cell types, specifically the distal tip cells (DTCs). Similar to observations in the touch receptor neurons, both seu-2 and seu-3 are able to suppress ectopic expression of the UNC-5 receptor in the DTCs. Genetic analysis of seu-2; seu-3 double mutants reveals that these genes function within the same signaling pathway for repulsive unc-6 guidance. seu-2 also appears to act in attractive unc-6 guidance. Mutations in seu-2 result in ventral to dorsal axon guidance defects in the HSN and ray 1 neurons. Double mutant analyses of seu-2 with either unc-40 or unc-6 null mutations exhibited HSN and ray 1 axon guidance defects at similar penetrance to either single mutant. These results suggest that seu-2 functions in the attractive unc-6-unc-40 dependent signaling pathway for HSN and ray 1 axon guidance. seu-2 was found to encode a G protein coupled receptor. Whole genome sequencing was used to identiy that seu-3 encodes the novel protein K09C6.9. K09C6.9 is predicted secreted protein that is expressed throughout development. Taken together, the phenotypes, method of isolation and genetic interactions of seu-2 and seu-3 make them interesting candidate mediators of UNC-6 signaling. I utilized genes, such as seu-2 and seu-3, to further elucidate other signaling components governing cell migration and axon guidance.

Obesity is a medical condition in which excess body fat has been accumulated. It is most commonly caused by imbalance between energy intake and energy expenditure (lack of physical activity and lower metabolic rate, etc.). The control of energy metabolism involves multiple tissues and signalling pathways and there is a great need for further understanding of these different interactions. In this study, I use Caenorhabditis elegans to study these complex pathways at the level of a whole organism. The downstream target of mTOR, p70 S6 kinase (S6K), has been implicated in the phosphorylation of multiple substrates and the regulation of growth and metabolism. In this study the worm homolog of S6K, rsks-1, found to be important for fat metabolism. Previous work in our lab found that RSKS-1::GFP is expressed at high levels in a set of sensory neurons and upregulated in ASJ, ASE and BAG sensory neurons in starved worms or mutants with low insulin activity. In this study, I found that the upregulation of rsks-1 expression was affected by serotonin, but not by the other neurotransmitters. Combined with the result that rsks-1 is required for the expression of TGFβ and insulin in ASI, rsks-1 may control dietary sensing by affecting insulin and TGFβ signalling within nervous system. Quantification of fat accumulation by TLC/GC revealed that in comparison to wild type worms, rsks-1 mutants have more than two-fold higher levels of triglycerides. This was confirmed by FT-IR microspectroscopy analysis. rsks-1 mutants also contain disproportionately high levels of C16:1n9 and C18:1n9 lipids compared with wild type worms. Genetic analysis has shown that rsks-1 acts either downstream of, or in parallel to the insulin and TGFβ pathways to affect fat levels. My studies showed that rsks-1 affects fat metabolism by influencing mRNA levels of genes encoding proteins in the β-oxidation pathway. Combined with defects in dietary sensing, fatty acid absorption, fertility and mitochondria function, the loss of rsks-1 activity induced much more energy storage than wild type by making a profound metabolic shift. These results are consistent with the metabolomics data analysis. Tissue specific RNAi showed that rsks-1 was required in many different tissues to regulate fat metabolism. Taken together, it can be concluded that RSKS-1 activity is needed for co-ordination of metabolic states in C. elegans. In order to understand more about the physiology behind fat accumulation, I analysed a mutant, aex-5, that has significantly lowered lipid levels. I found that this defect is associated with a significant reduction in the rate at which dietary fatty acids are taken up from the intestinal lumen. The aex-5 gene, which encodes a Kex2/subtilisin-family, Ca2+-sensitive proprotein convertase, is required for a discrete step in an ultraradian rhythmic phenomenon called the defecation motor program (DMP). Combined with other results, we conclude that aex-5 and other defecation genes may affect fat uptake by promoting the correct distribution of acidity within the intestinal lumen. This dissertation also described how to use Fourier transform infrared (FT-IR) microspectroscopy to detect lipids, proteins and carbohydrates directly in single worm. In conclusion, in this thesis I have uncovered several components that play roles in dietary sensing, fatty acid synthesis, adiposity regulation and fatty acid absorption in C. elegans.

The heterochromatin 1 (HP1) family of non-histone chromosomal proteins is evolutionarily conserved and is involved in numerous biological processes, including the stabilization of heterochromatin, a state of compacted DNA along a protein scaffold. HP1 proteins and trimethylated histone H3 on lysine 9 (H3K9me3) are major constituents of heterochromatin and have been characterized extensively in vitro. The binding of HP1 proteins to H3K9 methylation marks plays an essential role in mammalian development and chromatin organization. However, due to their critical function, dissecting the molecular mechanism by which HP1 proteins exert their function in vivo is difficult. C. elegans is a unique model because not only are deletion mutants of the two HP1 homologs, HPL-1 and HPL-2, viable, but also H3K9 methylation is not essential to worm development. Interestingly, HPL-2 is alternatively spliced to generate two HP1 proteins, but in vivo experimentation has vastly ignored the potential contributions of the alternative transcripts to hpl-2 function, thus obfuscating which phenotypes associated with hpl-2 knockdown are due to the loss of one or more of the splicing variants. In this dissertation, I characterized the HPL-2 splicing variants (A and B) on a biochemical level in relation to the canonical human HP1b protein and on a physiological level in splicing variant-specific knockout worms. I show that both recombinant HPL-2A and HPL-2B bind H3K9me3 through their chromodomain (CD). But while HPL-2A acts as a canonical HP1 protein, namely it dimerizes and phase-separates like hHP1b, HPL-2B does not. In contrast to recombinant protein, in extracts both proteins rely on other factors, such as the MBT domain-containing protein LIN-61, for their recruitment to H3K9me3. Although HPL-2A and HPL-2B display distinct characteristics in vitro, both hpl-2a and hpl-2b worms are phenotypically wildtype. In agreement, knockout of either splicing variant leads to upregulated expression of the other one, suggesting a certain level of functional redundancy. Nevertheless, I show that the C-terminal extension of HPL-2B, which is absent in HPL-2A, resembles that of the CEC-4 heterochromatin anchor. I therefore hypothesize that the main functions of HPL-2 are distinct: HPL-2A mediates chromatin compaction and HPL-2B facilitates heterochromatin anchoring to the nuclear periphery.

Seit der Erfindung des Mikroskops haben mikroskopische Bilder zu neuen Erkenntnissen in der biomedizinischen Forschung geführt. Moderne Mikroskope sind in der Lage große Bilddatensätze von zunehmender Komplexität zu erzeugen, was eine manuelle Analyse ineffizient, wenn nicht gar undurchführbar macht. In dieser Arbeit stelle ich zwei neue Methoden für die automatische Bildanalyse von Mikroskopiedaten vor. 1. Die Fourier-Ringkorrelations-basierte Qualitätsschätzung (FRC-QE), ist eine neue Metrik für die automatisierte Bildqualitätsschätzung von 3D-Fluoreszenzmikroskopieaufnahmen, hier getestet am Beispiel von menschlichen Hirnorganoiden. FRC-QE automatisiert die Qualitätskontrolle, eine Aufgabe, die häufig manuell durchgeführt wird und somit einen Engpass bei der Skalierung bildbasierter Experimente auf tausend oder mehr Bilder darstellt. Die Methode kann die Clearing-Effizienz über experimentelle Replikate und Protokolle quantifizieren. Sie ist auf verschiedene Mikroskopiemodelle übertragbar und lässt sich effizient auf Tausende von Bildern skalieren. 2. Der von mir entwickelte "WormObserver" ermöglicht Langzeitaufnahmen, verarbeitet automatisch die aufgenommenen Videos und erleichtert die Datenintegration über Tausende von Individuen hinweg, um Verhaltensmuster zu entschlüsseln. Darauf aufbauend, habe ich mich auf ein Beispiel für die Plastizität des Nervensystems konzentriert: Die Verhaltenstrajektorie des "C. elegans Dauer Exits". Um den Entscheidungsmechanismus beim Verlassen des Dauer Larvenstadiums zu charakterisieren, habe ich Zeitrafferdaten von Larvenpopulationen in verschiedenen Umgebungen erfasst, analysiert und wichtige Entscheidungspunkte identifiziert. Indem ich die Verhaltensanpassung mit der Genexpression kontextualisiert habe, konnte ich neue Erkenntnisse gewinnen, wie ein sich entwickelndes Nervensystem externe Stimuli robust integrieren und das Verhalten des Organismus an neue Umgebungen anpassen kann. / Since the invention of the microscope, microscopy images have generated new insights in biomedical research. While in the past these images were used for illustrative purposes, state-of-the-art microscopy images provide quantitative measurements. Moreover, modern microscopes are capable of autonomously producing large image datasets of increasing complexity, rendering manual analysis inefficient if not infeasible. Thus, extracting biologically relevant information from these datasets requires computational analysis using appropriate algorithms and software. While some analysis methods generalize to different microscope set-ups and types of images, others need to be well tailored to a particular problem. In this work, I present two new methods for automated image analysis of microscopy data. First, Fourier ring correlation-based quality estimation (FRC-QE) is a new metric for automated image quality estimation of 3D fluorescence microscopy acquisitions. I benchmarked the method in the context of evaluating clearing efficiency in human brain organoids. FRC-QE automates image quality control, a task that is often performed manually and thereby represents a bottleneck when scaling image-based experiments to thousand or more images. The method can estimate clearing efficiency across experimental replicates and clearing protocols. It generalizes to different microscopy modalities and efficiently scales to thousands of images. Second, I have developed a new method for behavioral imaging of C. elegans larvae. The “WormObserver” enables long-term imaging (>12h, >80k images/experiment), automatically processes the acquired videos a

As technology improves, scientists are able to generate high-throughput data faster and cheaper. Consequently, the field of biological sciences is progressively becoming more reliant on data science tools like machine learning methods for analysis and sorting of big data. The Complex Object Parametric Analyzer and Sorter (COPAS) is a large particle flow cytometer that can perform high-throughput fluorescence screens on small animals, like Caenorhabditis elegans. The outputs of the COPAS are extinction coefficient (EXT), Time of Flight (TOF, arbitrary length unit) and fluorescence. However, the COPAS outputs include unwanted objects like bubbles or bacteria and some animals pass the flow cell in a non-straight manner producing abnormal profiles leading to inaccurate developmental staging. In this thesis, I have created an R package, named COPASProfiler, that generates experiment-specific supervised machine learning (ML) classification models which can detect and remove abnormal profiles enabling standardized fluorescence quantification and analysis. I used COPASProfiler to develop a pipeline to automate fluorescence analysis of high-throughput COPAS data sets. Using R shiny, I created a web program with a graphical user interface that allows users to view, annotate, quantify fluorescence, and classify COPAS-generated datasets. The COPASProfiler is available on GitHub and can be installed using one single R command. Lastly, the COPASProfiler comes with multiple tutorials and examples, and was designed to accommodate users with minimal programming experience. COPASProfiler should enable robust high-throughput fluorescence studies of regulatory elements (e.g., enhancers, promoters, and 3’UTRs) and long-term epigenetic silencing in C. elegans.

Pour comprendre les mécanismes de l'immunité innée, nous utilisons Caenorhabditis elegans comme un organism model host et coniospora Drechmeria comme un pathogène. D. coniospora adhère à la cuticule de C. elegans pour infecter son épiderme. Le ver répond par une régulation de gènes de défense multiples, y compris des gènes codant pour des peptides antimicrobiens (AMP) comme nlp-29. En utilisant des vers transgéniques portant des constructions rapporteurs fluorescents comme nlp-29p :: gfp, nous pouvons suivre l'expression des gènes in vivo AMP et de chercher des gènes nécessaires à l'induction de gènes AMP à travers les écrans génétiques. Le but de mon projet était de caractériser des mutants qui ont été identifiés dans un nouvel écran génétique saturée, où 57 nouveaux allèles Nipi qui manquent nlp-29 d'induction après l'infection ont été isolés. Adaptation d'une nouvelle cartographie combinée SNP et toute stratégie de séquençage du génome, nous avons pu isoler 15 allèles de gènes nouveaux Nipi déjà connus et 12 allèles de 6 «nouveaux» gènes. Notre travail a confirmé le rôle principal de la MAPK PKCδ/p38 dans la régulation de l'expression nlp-29 AMP après l'infection, ainsi que le facteur de transcription STA-2/STAT et le transporteur SNF-12/SLC6. Nous faire progresser nos connaissances en identifiant NIPI-4 comme un régulateur positif de l'expression des gènes nlp peptide antimicrobien après l'infection. NIPI-4 est un membre de la famille des kinases nématode spécifique et est prévu pour être un pseudokinase. Nous avons montré qu'il agit dans l'épiderme partie aval de la MAPK p38. / To understand the mechanisms of innate immunity, we use Caenorhabditis elegans as a model host and Drechmeria coniospora as a fungal pathogen. D. coniospora adheres to the cuticle of C. elegans to infect its epidermis. The worm responds by an up-regulation of multiple defence genes, including genes encoding anti-microbial peptides (AMP) like nlp-29. Using transgenic worms carrying fluorescent reporter constructs like nlp-29p::gfp, we can follow AMP gene expression in vivo and look for genes required for the induction of AMP genes through genetic screens. The aim of my project was to characterize mutants that have been identified in a new saturated genetic screen, where 57 new Nipi alleles that lack nlp-29 induction after infection were isolated. Adapting a new combined SNP mapping and whole genome sequencing strategy we were able to isolate 15 new alleles of previously known Nipi genes and 12 alleles of 6 “new” genes. Our work confirmed the primary role of the PKCδ/p38 MAPK in the regulation of nlp-29 AMP expression after infection, as well as the STA-2/STAT transcription factor and the SNF-12/SLC6 transporter. We further advance our knowledge by identifying NIPI-4 as a positive regulator of nlp antimicrobial peptide genes expression after infection. NIPI-4 is a member of a nematode-specific kinase family and is predicted to be a pseudokinase. We showed that it acts in the epidermis partially downstream of the p38 MAPK. It also controls the constitutive expression of antimicrobial peptide genes of the cnc family that are targets of TGFß regulation. Together these suggested that NIPI-4 acts with STA-2 and SNF-12 to regulate AMP gene expression in the epidermis.

La réplication de l'ADN chez les eucaryotes commence lorsque le complexe de reconnaissance de l'origine (ORC) se lie à l'ADN puis recrute les facteurs nécessaires à la duplication du génome. Bien que les mécanismes biochimiques et les facteurs impliqués dans l'initiation de la réplication semblent être conservés, les séquences d'ADN (les origines de réplication) sur lesquelles ces événements ont lieu ne le sont pas. L'ensemble des données connues suggèrent fortement un rôle prépondérant de la mise en place des origines de réplication dans la structuration du génome et l'organisation des autres processus cellulaires lors de la différenciation. Comprendre la coordination de ces processus in vivo et au cours du développement est primordial pour déchiffrer la régulation cellulaire dans son contexte réel. Le modèle du développement embryonnaire du nématode C.elegans constitue un outil génétique de premier choix pour l'étude de la mise en place des origines de réplication au cours du développement. Au cours de ma thèse, j'ai dû premièrement développer une technique de culture de C.elegans synchronisée en grosse échelle, afin d'obtenir le matériel nécessaire pour identifier les origines de réplication. Cette technique nous a aussi permis de caractériser, pour la première fois, la croissance synchronisée en bioréacteur d'un métazoaire. D'autre part, l'étude des origines de réplication a révélé une distribution hétérogène des origines de réplication dans les chromosomes qui corrèle avec des domaines de certaines marques épigénétiques, une corrélation avec des séquences d'ADN capables de former des structures cruciformes de l'ADN, ainsi comme une confirmation de la corrélation avec la transcription. Nous avons aussi vu que la corrélation de origines de réplication avec des CpG, est fortement établie après le début de la gastrulation, et que l'association avec des éléments fonctionnels spécifiques du génome, comme les operons, est perdu une fois la transcription embryonnaire deviens nécessaire après la gastrulation. L'ensemble de résultats suggèrent fortement un changement dans l'organisation des origines de réplication après gastrulation, qui corrèle avec des éléments fonctionnels du génome. / Eukaryotic DNA replication begins when the origin recognition complexes (ORC) binds to DNA and recruits the necessary factors for genome duplication. Even though, biochemical mechanisms as well as the factors involved seem to be well conserved, the DNA sequences (replication origins) where these events take place are not. The known data strongly suggest that replication origins establishment may play an important role in genome structuring as well as in the organization of other cellular processes during cell differentiation. To understand how these processes are coordinated in vivo and during development, is essential for deciphering cellular regulation in its real context. C.elegans embryonic development is a genetic tool of first choice for studying replication origins in vivo and their correlation with other genome features and processes during development.During my thesis and with the aim of obtaining enough material for the replication origins identification method, I've had to develop a new technique of synchronized high-scale liquid culture of the nematode C.elegans. This technique has allowed the characterization for the first time of the synchronize growth of a metazoan in bioreactor. Furthermore, the study of replication origins has revealed a heterogenic distribution of replication origins along chromosomes that correlates with specific epigenetic marks. Moreover, replication origins are strongly associated with specific DNA structures able to form cruciforms, and we have confirmed the correlation of replication origins and transcription. This study also show that the association of replication origins with CpGs is greatly increased after gastrulation, and that the association with some genetic elements, such as operons, is reduced after gastrulation begins. Taken together these results show a change of replication origins before and after cell differentiation during embryonic development that correlate with functional genome elements.

No description available.

C. elegans est infecté par divers agents pathogènes ; bactéries, champignons et virus. Lors d'une infection fongique, C. elegans surexprime de nombreux gènes codant pour des peptides antimicrobiens (AMP). Le principal objectif de ma thèse était de construire un réseau de régulation génique intégré représentant l'induction de ces gènes AMP pendant l'infection. Pour trouver les principales composantes du réseau de régulation, via un criblage ARNi du génome entier (Zugasti et al 2016), nous avons identifié 278 clones Nipi (pour "Absence d'induction de peptides antimicrobiens après infection") qui abrogent l’induction d’AMP. En utilisant "CloneMapper" (Thakur et al. 2014), nous avons identifié 338 gènes cibles pour ces clones. Nous avons montré que les voies de MAPK sont au cœur de l'induction des AMP. Parmi les 50 gènes arbitrairement sélectionnés et surexprimant les AMP, nous en avons validé 48 en utilisant Fluidigm. Pour attribuer des fonctions aux gènes identifiés dans ces études à haut débit, nous avons développé un outil d'enrichissement fonctionnel pour la communauté C.elegans (MS en préparation). Nous avons utilisé cet outil pour analyser les cibles du criblage ARNi sur le génome entier et sur d'autres bases de données concernant divers agents pathogènes. Nous avons fait une analyse de l'enrichissement fonctionnel des cibles ChIPseq de CEBP-1, un facteur de transcription lié à la régulation de la réponse immunitaire innée (Kim et al, Soumis). Enfin, pour mieux comprendre l'interaction entre l'hôte et le pathogène, nous avons séquencé, assemblé, annoté et analysé le génome de D. coniospora. Nous avons identifié plusieurs facteurs de virulence potentiels dans ce génome. / C. elegans is infected by diverse pathogens, including bacteria, fungi and viruses. Upon fungal infection, C. elegans up-regulates the expression of many antimicrobial peptide (AMP) genes. The main aim of my thesis was to build an integrated gene regulatory network representing the induction of these AMP genes upon infection. To find the main/backbone components of the regulatory network, through a genome-wide RNAi screen (Zugasti et al. 2016), we identified 278 Nipi (for “no induction of antimicrobial peptides after infection”) clones that abrogate AMP induction. Using “CloneMapper” (Thakur et al. 2014), we identified 338 target genes for these clones. We showed that MAPK pathways are central to the induction of AMPs. We also characterized the transcriptional changes provoked by infection using RNA-sequencing and identified more than 300 genes that are dynamically up-regulated after infection, including 13 AMPs. We validated 48 (96%) of 50 arbitrary selected up-regulated genes using Fluidigm. To assign functions to genes identified in these high-throughput studies, we developed a functional enrichment tool for C.elegans community (MS in preparation). We used this tool to analyse the genome-wide RNAi screen targets and other pathogen-related datasets. We did functional enrichment analysis of ChIPseq targets of CEBP-1, TF linked to the regulation of the innate immune response (Kim et al., submitted). Finally, to understand better the interaction between host and pathogen, we sequenced, assembled, annotated and analysed the D. coniospora genome (Lebrigand et al. 2016). We identified various potential virulence factors in the fungal genome.