La transmission de l’information au niveau des synapses chimiques requiert la concentration de récepteurs appropriés face aux sites de libération des neurotransmetteurs. Afin d'identifier de nouveaux gènes impliqués dans la localisation synaptique des récepteurs de l’acétylcholine (RAChs), un crible génétique basé sur l'observation in vivo de ces récepteurs à la jonction neuromusculaire (JNM) de C. elegans a été conduit et a permis d’isoler 29 mutants. J’ai identifié des gènes candidats dans cinq lignées et la mutation causale dans trois autres en caractérisant 9 de ces mutants par séquençage de leur génome. Par ailleurs, l’analyse fonctionnelle de 2 autres des mutants nous a permis de mettre en évidence deux activités mobilisatrices des récepteurs très différentes. D’une part, nous avons mis en évidence le rôle déterminant d’isoformes de Ce-Punctine/MADD-4, sécrétées spécifiquement par les motoneurones cholinergiques et GABAergiques, dans le recrutement local des récepteurs appropriés à la membrane de la cellule musculaire. Chef d’orchestre de la transmission neuronale, la Punctine définit ainsi l’identité excitatrice ou inhibitrice des JNMs. D’autre part, nous avons montré que les RAChs extrasynaptiques normalement diffus à la membrane de la cellule musculaire s’agrègent en amas ectopiques dans un mutant du gène suppresseur de tumeurs rsu-1 (ras suppresseur 1). L’absence de RSU-1, protéine localisée aux sites d’adhésion focale en aval des intégrines, engendre une diminution des récepteurs synaptiques, ce qui met à jour un mécanisme actif prévenant l’agrégation illégitime des récepteurs extrasynaptiques et permettant d’assurer leur recrutement effectif à la synapse. / Chemical neurotransmission relies on the concentration of receptors in front of neurotransmitter release sites. To identify new genes involved in the synaptic localization of receptors, a genetic screen based on the in vivo visualization of acetylcholine receptors (AChRs) at the C. elegans neuromuscular junction (NMJ) was performed and twenty-nine mutants were retrieved. By whole genome sequencing of nine mutated lines, I identified candidate genes for five mutants and the causal mutation for three others. The functional analysis performed in parallel of two other mutants from the same screen revealed two novel and distinct activities that are required for the synaptic localization of AChRs. We evidenced the fundamental role of Ce Punctin/MADD-4 isoforms, which are specifically secreted by cholinergic and GABAergic motoneurons, in recruiting locally appropriate receptors at the muscle cell membrane. This showed that Punctin is a key factor specifying the excitatory or the inhibitory identity of NMJs and defined a new paradigm with isoforms from the same gene that promote specific post-synaptic domains assembly. The tumor suppressor gene rsu-1 (ras suppressor 1) is evolutionary conserved and encodes a protein localized at focal adhesion sites. In the absence of RSU-1, normally diffused extrasynaptic AChRs are found aggregated and the synaptic receptor content is diminished. This reveals that an RSU-1 dependent mechanism is required to maintain receptors dispersed in the extrasynaptic membrane, allowing for their proper recruitment at the synapse.

This thesis describes the alignment of cloned DNA with the genetic map, and the identification of coding elements within the aligned DNA. The region of study was the dpy-5 unc-29 interval from chromosome I of the nematode Caenorhabditis elegans, with an emphasis on the region surrounding the gene dpy-14. The objectives of this thesis were: 1) to align the physical and genetic maps .of the region; 2) to identify and characterize the coding elements in the vicinity of dpy-14; and 3) to cross-hybridize the identified C. elegans coding elements to mammalian DNA in an attempt to identify evolutionarily conserved genes. Six polymorphisms from the dpy-5 unc-29 interval were mapped with respect to the free duplication sDp2. The polymorphisms hP5, sPl, and hP9 were found to.be inside the region spanned by sDp2 while the polymorphisms hP4, hP6, and hP7 were found to be outside this interval. In addition, these six polymorphisms were mapped with respect to visible markers from the dpy-5 unc-29 interval. These analyses demonstrated the genetic order to be dpy-5, hP5, unc-37, (dpy-14, sPl), hP9, unc-13, hP7, (hP4, hP6), unc-29. Lambda phage containing the hP5, sPl, and hP6 sites identified and anchored cosmid contigs to the genetic map. The interval from the left of hP5 to the right of unc-13 is contained in a single contig of approximately 1400 Kb. The amount of DNA in Kb across the hP5 and unc-13 interval was compared to the genetic distance in map units. The DNA per map unit value was found to vary in this interval with the greatest value found between hP9 and unc-13. Seven cosmids representing 173 Kb of N2 genomic DNA near the gene dpy-14 were isolated. Using cross-species hybridization to C. briggsae DNA ten conserved regions were identified within these seven cosmids. The ten conserved fragments were used to identify seven cDNAs, six of which also identified RNAs on Northern blots. The relative abundance of the isolated cDNAs varied 250 fold with the most abundant having a level similar to that found for actin. The first comprehensive survey of mammalian homologies in a contiguous set of ten coding regions found three coding elements to be. conserved. One was demonstrated to be the small nuclear RNA gene U1. Another shared sequence similarities with the gene S-adenosyl-L-homocysteine hydrolase. No detectable homologies were identified with the third. A formaldehyde-induced mutation that failed to complement the genes unc-37, unc-87, dpy-14, let-83 and let-86 was isolated. This mutation appeared to be the result of a DNA rearrangement which had one breakpoint within the cosmid C14A12. Using the conserved elements identified in this thesis together with the rearrangements and mapped genes from the region, a detailed physical and genetic map in the vicinity of dpy-14 was constructed. / Medicine, Faculty of / Medical Genetics, Department of / Graduate

Caenorhabditis elegans fait partie des modèles animaux les plus étudiés en laboratoire. La découverte des premiers virus infectant naturellement des Caenorhabditis apporte de nouveaux modèles animal-virus très prometteurs.Le virus d'Orsay, infectant spécifiquement C. elegans, et le virus de Santeuil, infectant spécifiquement C. briggsae, sont des virus à ARN positif simple brins (Hépatites A, C et E, Chikungunya, Coronavirus etc¿) qui provoquent une désorganisation de la structure des cellules intestinales de leur hôte. Par ailleurs, une variation dans la sensibilité à ce virus au sein des espèces est observée. Grâce à une étude statistique d'association pangénomique portant sur la sensibilité au virus d'Orsay de 97 isolats naturels de C. elegans, nous avons pu mettre en évidence une région chromosomique qui contient un polymorphisme responsable de cette sensibilité. Il s'agit une délétion dans le gène drh-1 qui code une protéine similaire à la protéine RIG-I, connue pour initier une réponse antivirale chez les vertébrés. Cependant, C. elegans est incapable de produire la même réponse antivirale que ces derniers. Ainsi, DRH-1 est impliquée dans la reconnaissance des ARN viraux et dans la production d'une réponse spécifique à l'infection virale par les petits ARN. Les gènes impliqués dans l'immunité sont soumis à une forte pression de sélection. Pourtant, de manière surprenante, la délétion du gène drh-1 est présente dans 23% des isolats étudiés. Cette délétion est génétiquement liée à une région plus large de 3 Mb. Cependant, au laboratoire, cette région n’apporte pas d’avantage sélectif qui expliquerait comment cette délétion peut se propager dans les populations. / Caenorhabditis elegans is a commonly studied animal model in laboratories. The discovery ofthe first natural viral infections of Caenorhabditis brings new models to study animal-virusinteractions.The Orsay virus, specifically infecting C. elegans, and the Santeuil virus, specificallyinfecting C. briggsae, are positive single strand RNA viruses (Hepatites, Chikungunya,Coronavirus etc…) disrupting the structure of intestinal cells of their host. However, weobserved a strong variability in the sensitivity to those viruses at the intraspecific level.To identify the genetic basis of the sensitivity, we performed a genome wide association studyon 97 wild isolates of C. elegans. We were able to identify the center of chromosome IV as aregion containing the locus responsible for this sensitivity. A deletion in the drh-1 gene,coding for a RIG-I-Like protein, confers sensitivity to their carrier. RIG-I is known torecognize viral RNA and to trigger an antiviral response through the production of interferonsin vertebrates. However, C. elegans is not able to produce interferons but it appears thatDRH-1 initiates a viral specific siRNA pathway.Immunity genes are under strong selective pressure. Thus, it is surprising that such animportant protein for the antiviral pathway appears to be disrupted in 23% of the wild isolates.This deletion shows high linkage disequilibrium with a broader region of 3Mb, suggestingthat the deletion propagates with this region. However, this region does not seem to provideany advantage to their owner under laboratory conditions.

Oxygen deprivation can be pathological for many organisms, including humans. Consequently, there are several biologically and economically relevant negative impacts associated with oxygen deprivation. Developing an understanding of which genes can influence survival of oxygen deprivation will enable the formulation of more effective policies and practices. In this dissertation, genes that influence adult anoxia survival in the model metazoan system, C. elegans, are identified and characterized. Insulin-like signaling, gonad function and gender have been shown to influence longevity and stress resistance in the soil nematode, C. elegans. Thus, either of these two processes or gender may influence anoxia survival. The hypothesis that insulin-like signaling alters anoxia survival in C. elegans is tested in Aim I. The hypotheses that gonad function or gender modulates anoxia survival are tested in Aim II. Insulin-like signaling affects anoxia survival in C. elegans. Reduction of insulin-like signaling through mutation of the insulin-like receptor, DAF-2, increases anoxia survival rates in a gpd-2/3 dependent manner. The glycolytic genes gpd-2/3 are necessary for wild-type response to anoxia, and sufficient for increasing anoxia survival through overexpression. Gonad function and gender both affect anoxia survival in C. elegans. A reduction of ovulation and oocyte maturation, as measured by oocyte flux, is associated with enhanced anoxia survival in all cases examined to date. Reduction of function of several genes involved in germline development and RTK/Ras/MAPK signaling reduce ovulation and oocyte maturation while concurrently increasing anoxia survival. The act of mating does not influence anoxia survival, but altering ovulation through breeding or chemical treatment does. The male phenotype also increases anoxia survival rates independent of genotype. These studies have identified and characterized over ten different genotypes that affect adult survival of anoxia in C. elegans. Before these studies were conducted, there were no genes known to influence adult anoxia survival in C. elegans. Furthermore, these studies have begun to uncouple mechanisms of longevity and stress resistance.

La macroautophagie est un processus cellulaire qui permet la dégradation et le recyclage de constituants cytoplasmiques par formation de vésicules à double membrane, les autophagosomes qui fusionnent ensuite avec les lysosomes. Ce processus intervient dans divers processus physiologiques tels que le développement, la longévité, la mort cellulaire et dans des pathologies humaines comme des cancers ou maladies neurodégénératives. Mes travaux de thèse ont révélé l’existence de rôles séquentiels et spécifiques des protéines autophagiques, LGG-1 et LGG-2, homologues d’Atg8/LC3 chez le nématode Caenorhabditis elegans. Cette étude a été réalisée dans l’embryon précoce sur une population particulière d’autophagosomes responsables d’un processus physiologique stéréotypé : la dégradation des mitochondries paternelles au moment de la fécondation. Nous avons montré que LGG-1 est recruté au niveau des autophagosomes précoces et permet le recrutement de LGG-2 qui intervient plus tardivement dans le processus autophagique pour permettre la fusion des autophagosomes avec les lysosomes. De plus, la fonction de LGG-1 peut être complémentée par son homologue humain témoignant de l’intérêt du système modèle C. elegans pour l’analyse des homologues d’Atg8.Par ailleurs, des études récentes ont démontré que la protéine autophagique LC3 était recrutée au cours de la phagocytose des corps apoptotiques. Ce processus a été appelé LAP pour LC3-associated phagocytosis. Par des approches génétiques et cellulaires, utilisant la microscopie optique et électronique, j’ai montré qu’il existait une implication différente de protéines autophagiques LGG-1 et LGG-2 dans la dégradation des corps apoptotiques chez C. elegans. La protéine LGG-2, spécifiquement, joue un rôle dans la cellule phagocytaire afin de dégrader le corps apoptotique. Ces travaux suggèrent également une implication de l’autophagie dans le corps apoptotique pour permettre la phagocytose. / Macroautophagy is a major ubiquitous catabolic process which allows the bulk degradation and recycling of cytoplasmic constituents by formation of double membrane vesicles called autophagosomes which then fuse with lysosomes. This process is involved in a large variety of physiological processes such as development, anti-aging, cell death and in human pathologies like cancers or neurodegenerative diseases. My thesis work revealed the existence of sequential and specific roles of autophagic proteins LGG-1 and LGG-2, homologs of Atg8/LC3 in Caenorhabditis elegans. In this study, we focused on a particular population of autophagosomes involved in a physiological process in early embryos: the degradation of paternal mitochondria during fertilization. We showed that LGG-1 is recruited at the early autophagosomes and allows LGG -2 recruitment which acts later in the autophagic process to allow the fusion of autophagosomes with lysosomes. Moreover, the function of LGG -1 can be complemented with its human homologs revealing the interest of the C. elegans model system for analyzing Atg8 homologs.Furthermore, recent studies have identified the recruitment of autophagic proteins during phagocytosis of apoptotic cells in the so called LC3-associated phagocytosis (LAP). By genetic and cellular approaches, using optical and electron microscopy, I showed that there is a different involvement of autophagic proteins, LGG-1 and LGG-2 in the degradation of apoptotic cells in C. elegans. LGG-2 protein, specifically, plays a role in phagocytic cell to degrade apoptotic corpses. Moreover, this work suggest a function of autophagy in the apoptotic corpses to allow phagocytosis.

Under unfavorable environmental conditions Caenorhabditis elegans larvae enter a dauer stage which is a specialized non-feeding larval stage. In the dauer stage, worms display astonishingly low metabolism, which allows them to adapt themselves to environmental stress and to dwell without food for several months. Dauer larvae can enter into the reproductive larval stage, when environmental conditions become favorable. In this study, the metabolic transition of dauers into the reproductive larval stage is analyzed in detail: a. During the exit of dauers, several metabolic traits were examined. Primarily, dauer larva initiates the metabolic transition by activating feeding, which is followed by upregulated oxygen consumption and mitochondrial remodeling, as well as enhanced protein synthesis. b. To better understand the metabolic transition, inhibitors of the dauer exit were introduced. Lithium ions were shown to inhibit the transition of dauers to reproductive larvae and prevent the upregulation of metabolic activities required for this process. c. In liquid culture, the transition from the dauer to the reproductive larva is also inhibited, presumably because of the hypoxic character of the liquid culture. Thus, hypoxia has a negative effect on the metabolic transition. d. In the course of our investigation we discovered that the dauer larva is not a closed system but indeed, it can dwell on the externally available ethanol as a carbon source by incorporating it into the energy metabolism. This allows dauers to survive for longer periods in the absence of bacteria, the preferred food of worms. These findings clarify the nature of dauers, how they utilize distinct pathways during the metabolic transition and how they take advantage of the externally available carbon source. These results may in the future enable us to elucidate the complex pathways of metabolism, as well as the ways in which it can be regulated.

<p>  </p> <p>Edible insects, a novel source of protein, are gaining interest for their health promoting attributes. In this study, the <em>in vivo and in vitro</em> antioxidant effect of tropical banded cricket (<em>Gryllodes sigillatus</em>) peptides was evaluated. Antioxidant activity by 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) radical scavenging activity, oxygen radical antioxidant capacity (ORAC) and caco-2 cellular antioxidant activity, were measured in hydrolyzed followed by simulated gastrointestinal digested (SGD) cricket peptides. <em>In vivo</em> analysis was conducted using <em>Caenorhabditis elegans</em> as a model. <em>In vitro</em> analysis showed cricket peptides had greater (p< 0.05) antioxidant activity than the unhydrolyzed protein (control). In <em>C. elegans,</em> the lifespan of nematodes fed SGD peptides increased under chronic and acute oxidative stress conditions. Reactive oxygen species (ROS) levels of nematodes fed SGD peptides under paraquat induced oxidative stress were lower (p<0.05) than that of the control group. Further studies using polymerase chain reaction (PCR) indicated that the increased resistance to oxidative stress in <em>C. elegans</em> fed SGD peptides could be due to the increased expression of the stress-related gene gst-4. Taken together, these results indicate that tropical banded cricket peptides could be used as a functional food and nutraceutical to combat oxidative stress. </p>

Le stress thermique résulte d'une exposition à une température située au-delà de la plage optimale pour un organisme. L’impact du stress thermique est variable selon son intensité, allant d’un effet bénéfique à la mort de l’organisme. Mon travail de thèse a établi un modèle de stress thermique aigu (aHS pour acute Heat Stress) chez C. elegans et a étudié ses effets sur l'homéostasie cellulaire, le développement des vers et la réponse autophagique. Un aHS au cours du 4ème stade larvaire induit un retard de développement, mais aucune létalité ni stérilité. Ce stress de développement entraîne la fragmentation massive mais transitoire des mitochondries, la formation d'agrégats dans la matrice et la diminution de la respiration mitochondriale. En outre, l’aHS déclenche un flux autophagique associé à des événements de mitophagie dans de nombreux tissus et en particulier dans l'épiderme. Nous avons montré que la réponse autophagique à l’aHS était protectrice pour les animaux. De plus, nous avons découvert que dans l’épiderme, les mitochondries sont les principaux sites de biogenèse des autophagosomes, en conditions physiologique et en aHS. Nous avons également constaté que la protéine DRP-1 (dynamin related protein 1) est impliquée dans le processus de mitophagie induite par l'aHS. Chez les animaux mutants drp-1 soumis au aHS, la fission mitochondriale est impossible, l’autophagie est induite mais les autophagosomes sont anormaux et agrégés sur la mitochondrie. À partir de ces données, nous proposons que DRP-1 participe au contrôle de la qualité des mitochondries stressées en coordonnant la fission mitochondriale et la biogenèse des autophagosomes. J'ai également étudié plusieurs protéines pouvant être impliquées dans les zones de contact entre le réticulum endoplasmique et les mitochondries, ainsi que leurs rôles sur la morphologie mitochondriale et l'autophagie, dans des conditions physiologiques ou d’aHS. De plus, nous avons développé de nouveaux outils pour analyser les sites de contact ER-mitochondries. / Heat stress results from an exposure to a temperature beyond the optimum range of an organism. The impact of heat stress can range from beneficial to lethal due to the severity of stress. My thesis work established an acute heat stress (aHS) model in C. elegans and studied its effects on cell homeostasis, worm development and autophagy response. aHS during the 4th larval stage induces a developmental delay but no lethality or sterility. This developmental stress results in the massive but transitory fragmentation of mitochondria, the formation of aggregates in the matrix and the decrease of mitochondrial respiration. In addition, aHS triggers an active autophagy flux associated to mitophagy events in many tissues and particularly in epidermis. We showed that the autophagy response upon aHS is protective for the animals. Moreover, we discovered that in the epidermis, the mitochondria are the major sites for autophagosome biogenesis in both standard and aHS. We also found that the dynamin related protein DRP-1 is involved in aHS-induced mitophagy process. In drp-1 animals submitted to aHS, mitochondrial fission is unable to achieve, and despite autophagy induction the autophagosomes cluster and elongate abnormally on mitochondria. From these data, we propose that DRP-1 is involved in the quality control of stressed mitochondria by coordinating mitochondrial fission and autophagosomes biogenesis. I also studied several proteins which may be involved in contact zones between endoplasmic reticulum and mitochondria, and their roles on mitochondrial morphology and autophagy, in physiological or aHS conditions. Furthermore, we have developed new tools for further studying the ER-mitochondria contact sites.

The specification and development of nervous system diversity is a driving question in the field of Neurobiology. The overarching goals of the projects described in this thesis are to describe tools to aid in the description of nervous system development and to show the use of the described tools to study nervous system development in the nematode Caenorhabditis elegans. The first chapter of this thesis describes a complete map of the male C. elegans nervous system using a tool developed in the lab to uniquely label all neurons in the C. elegans nervous system, NeuroPAL. The second chapter of this thesis largely focuses on a well-studied homeobox gene, unc-86, and its role in fate transformations in dopaminergic and GABAergic neuron types. These two seemingly disparate projects are united in their effort to investigate nervous system development and neuronal fate determination. NeuroPAL is a multicolor transgene that uniquely labels all neurons of the C. elegans hermaphrodite nervous system and here I show it can be used to disambiguate all 93 neurons of the male-specific nervous system. I demonstrate the wide utility of NeuroPAL to visualize and characterize numerous features of the male-specific nervous system, including mapping the expression of gfp-tagged reporter genes and neuron fate analysis. NeuroPAL can be used in combination with any gfp-tagged reporters to unambiguously map the expression of any gene of interest in the male, or hermaphrodite, nervous system. Furthermore, NeuroPAL is used in mutants of several developmental patterning genes to confirm previously described defects in neuronal identity acquisition. Additionally, I show that NeuroPAL can be used to uncover novel neuronal fate losses and identity transformations in these mutants because of the unique labeling of every neuron. Lastly, we show that even though the male-specific neurons are generated throughout all four larval stages, the neurons only terminally differentiate in the fourth and final larval stage, termed ‘just-in-time’ differentiation. In the second part of this thesis, I describe a few examples of mutant analysis of homeobox gene family members and describe their function in the C. elegans nervous system. I focus largely on a couple potential examples of homeotic fate transformations in mutants of the POU homeobox gene, unc-86. In unc-86 mutants, I describe the ectopic expression of multiple GABAergic terminal identity features in one cell in the head of C. elegans. I raise the hypothesis that this cell may be a transformation of a non-GABAergic ring interneuron, RIH, into that of its GABAergic sister cell, AVL, in unc-86 mutants. While ectopic dopaminergic neurons were previously described in unc-86 mutants, I expand the study to show the ectopic expression of all dopaminergic synthesis and packaging genes. I show support that all non-dopaminergic anterior deirid neurons, ADA, AIZ, FLP, and RMG, lose the expression of some of their wild type terminal fate genes and transform to a fate like that of their dopaminergic sister cell, ADE, as assessed by NeuroPAL expression. Taken together, these studies describe tools and methods for studying nervous system development as well as describe many examples of cell fate transformations.

BACKGROUND:The SPE-8 group gene products transduce the signal for spermatid activation initiated by extracellular zinc in C. elegans. Mutations in the spe-8 group genes result in hermaphrodite-derived spermatids that cannot activate to crawling spermatozoa, although spermatids from mutant males activate through a pathway induced by extracellular TRY-5 protease present in male seminal fluid.RESULTS:Here, we identify SPE-8 as a member of a large family of sperm-expressed non-receptor-like protein-tyrosine kinases. A rescuing SPE-8::GFP translational fusion reporter localizes to the plasma membrane in all spermatogenic cells from the primary spermatocyte stage through spermatids. Once spermatids become activated to spermatozoa, the reporter moves from the plasma membrane to the cytoplasm. Mutations in the spe-8 group genes spe-12, spe-19, and spe-27 disrupt localization of the reporter to the plasma membrane, while localization appears near normal in a spe-29 mutant background.CONCLUSIONS:These results suggest that the SPE-8 group proteins form a functional complex localized at the plasma membrane, and that SPE-8 is correctly positioned only when all members of the SPE-8 group are present, with the possible exception of SPE-29. Further, SPE-8 is released from the membrane when the activation signal is transduced into the spermatid.