La sclérose latérale amyotrophique (SLA) est une maladie neurodégénérative fatale caractérisée par une perte progressive et sélective des neurones moteurs. La SLA est incurable et il n’existe aucun traitement efficace pour les personnes atteintes de cette maladie. Environ 90% des cas sont sporadiques tandis que 10% sont familiaux, et les patients décèdent généralement deux à cinq ans après l'apparition des premiers symptômes. De nombreuses anomalies génétiques sont associées à la SLA, incluant des mutations dans les protéines FUS, C9orf72, SOD-1 et TDP-43. Le laboratoire a développé un modèle transgénique de Caenorhabditis elegans surexprimant la protéine humaine mutante TDP-43(Q331K) dans les neurones moteurs GABAergiques. Nous avons également obtenu par mutagénèse et CRISPR-Cas9 des modèles physiologiquement représentatifs du nématode basés sur des mutations dans tdp-1, l'orthologue de TARDBP chez le C. elegans. L'objectif est de caractériser ces modèles et de déterminer s'ils peuvent récapituler certains aspects phénotypiques clés de la SLA, tels que des déficits moteurs et une neurodégénérescence dépendante de l'âge générant une paralysie. L’hypothèse est que le modèle TDP-1 pourra refléter plus précisément l’expression physiologique du gène dans la maladie humaine grâce à la mutation dans un gène endogène, l’absence de surexpression et l’expression ubiquitaire de la protéine TDP-1. Les résultats montrent que les modèles TDP-43/TDP-1 ont des déficits moteurs, une transmission synaptique altérée et une neurodégénérescence liée à l’âge. Cependant, seule la mutation dans TDP-43 semble avoir un effet sur la durée de vie. Ces modèles procurent différentes expressions physiologiques des protéines mutantes et donc, des phénotypes de niveaux d'intensité variables. Ils constitueront des outils utiles pour élucider de nouveaux mécanismes pathogéniques de la SLA ainsi que de bons candidats pour le criblage de médicaments et le développement de stratégies thérapeutiques. / Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by a progressive and selective loss of motor neurons. ALS is incurable and there are no effective treatments available for people living with the disease. About 90% of the cases are sporadic whereas 10% are familial, and patients usually die two to five years after symptom onset. Many gene defects are associated with ALS, including mutations in genes encoding FUS, C9orf72, SOD-1 and TDP-43 proteins. We have developed a transgenic Caenorhabditis elegans model expressing human mutant TDP-43(Q331K) in GABAergic motor neurons. We have also obtained by mutagenesis and CRISPR-Cas9 physiologically accurate models based on mutations in tdp-1, the C. elegans ortholog of TARDBP. Our objective is to characterize these models and determine if they can recapitulate key aspects of the disease such as motor deficits and age-dependent neurodegeneration causing paralysis. We believe that the TDP-1 model will reflect more precisely the physiological expression of the gene in the human disease because of its mutation in an endogenous gene, the absence of overexpression and ubiquitous protein expression. Our results show that both TDP-43 and TDP-1 models have motor deficits, synaptic transmission impairments and age-dependent neurodegeneration. However, only the TDP-43 mutation seems to have an effect on lifespan. These models provide different physiological expression of mutant proteins and thus phenotypes of varying intensity levels. They will be useful tools to elucidate new pathogenic mechanisms of ALS as well as being good candidates for drug screening and developing therapeutic strategies.

CRISPR/Cas9 induced DNA breaks can be precisely repaired by cellular homology-directed repair (HDR) pathways using exogenously provided template DNA (donor). However, the full potential of precision editing is hindered in many model systems by low cutting efficiencies, low HDR efficiencies and, cytotoxicity related to Cas9 and donor DNA. In this thesis, I address these challenges and present methods that we developed to increase HDR efficiencies in multiple model organisms. In Caenorhabditis elegans, we show that by reducing toxicity high editing efficiencies can be achieved with single stranded oligonucleotide (ssODN) donors. We demonstrate that melting dsDNA donors dramatically improves the knock-in efficiencies of longer (1kb) edits. In addition, we describe 5′-terminal modifications to the donor molecules that further increase the frequency of precision editing. With our methodology a single optimally injected animal can yield more than 100 Green Fluorescent Protein (GFP) positive progeny, dramatically enhancing efficiency of genome editing. Next, we demonstrate the generality of 5′ modified donors by extending our studies to human cell cultures and mice zygotes. In mammalian models, 2′OMe-RNA modifications consistently increase HDR efficiencies by several fold over unmodified donors. Furthermore, end-modified donors exhibited a striking reduction in end-joining reactions including reduced concatemer formation and reduced direct ligation into the host genome. Our study demonstrates that HDR can be improved without inhibiting competing end-joining pathways and provides a platform to identify new chemical modifications that could further increase the potency and efficacy of precision genome editing.

No description available.

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

To expand the understanding of aging in the model organism Caenorhabditis elegans, global quantification of metabolite and protein levels in young and aged nematodes was performed using mass spectrometry. With age, there was a decreased abundance of proteins functioning in transcription termination, mRNA degradation, mRNA stability, protein synthesis, and proteasomal function. Furthermore, there was altered S-adenosyl methionine metabolism as well as a decreased abundance of the S-adenosyl methionine synthetase (SAMS-1) protein. Other aging-related changes included alterations in free fatty acid levels and composition, decreased levels of ribosomal proteins, decreased levels of NADP-dependent isocitrate dehydrogenase (IDH1), a shift in the cellular redox state, an increase in sorbitol content, alterations in free amino acid levels, and indications of altered muscle function and sarcoplasmic reticulum Ca2+ homeostasis. There were also decreases in pyrimidine and purine metabolite levels, most markedly nitrogenous bases. Supplementing the culture medium with cytidine (a pyrimidine nucleoside) or hypoxanthine (a purine base) increased lifespan slightly, suggesting that aging-induced alterations in ribonucleotide metabolism affect lifespan. An age-related increase in body size, lipotoxicity from ectopic yolk lipoprotein accumulation, a decline in NAD+ levels, and mitochondrial electron transport chain dysfunction may explain many of these changes. In addition, dietary restriction in aged worms resulting from sarcopenia of the pharyngeal pump likely decreases the abundance of SAMS-1, possibly leading to decreased phosphatidylcholine levels, larger lipid droplets, and ER and mitochondrial stress. The complementary use of proteomics and metabolomics yielded unique insights into the molecular processes altered with age in C. elegans.

Organisms modulate their response to changing environmental conditions through changes in gene expression, and extensive variations in gene expression are prevalent among individuals even within a population. This widespread plasticity and variability of gene expression is thought to play roles in adaptation and drive novel phenotypes in species. Understanding the mechanisms that contribute to such variations requires the analysis of interactions between the genome and its environment and sequence variations within the genome. This work consists of two projects investigating the plasticity and variation of gene expression during post-embryonic development in the nematode C. elegans. In the first study, I examined the response to changes in population density in developmentally arrested L1 larvae. I systematically characterized arrested L1 larvae from low to high densities using single-worm RNA-seq and uncovered that the density of resuspended L1 larvae regulates the expression of hundreds of mRNAs. Further analysis revealed that the physiological response to changes in density is rapid and signaled by a non-canonical daf-22 ascaroside independent pathway. In the second study, I investigated the evolution of gene expression within species using two genetically divergent C. elegans strains (N2 and CB4856). I carried out RNA-seq and allele-specific analysis across six different conditions and four developmental stages, and we examined gene expression divergence using the homozygous parent and F1 hybrid system. This work provides a new experimental model for studying the evolution of gene expression and a comprehensive view of gene expression variation during development in C. elegans.

The intestinal epithelium represents one of the first lines of defense against pathogenic bacteria. Immune regulation at this critical barrier is necessary to maintain organismal fitness, and mis-regulation here has been linked to numerous debilitating diseases. Functional relationships between the nervous system and immune system have been found to be critical in the proper coordination of immune defenses at barrier surfaces, however the precise mechanisms underlying theses interactions remains unclear. Through conducting a forward genetic screen utilizing the model organism Caenorhabditis elegans, we uncovered a surprising requirement for the olfactory neuron gene olrn-1 in the regulation of intestinal epithelial immunity. During nematode development, olrn-1 is required to program the expression of odorant receptors in the AWC olfactory neuron pair. Here, we show that olrn-1 also functions in AWC neurons in the cell non-autonomous suppression of the canonical p38 MAPK PMK-1 immune pathway in the intestine. Low activity of OLRN-1, which activates the p38 MAPK signaling cassette in AWC neurons during larval development, also de-represses the p38 MAPK PMK-1 pathway in the intestine to promote immune effector transcription, increased clearance of an intestinal pathogen and resistance to bacterial infection. However, derepression of the p38 MAPK PMK-1 pathway also results in severe developmental and reproductive defects, demonstrating the critical function of OLRN-1 to both prime C. elegans intestinal epithelial cells for the induction of anti-pathogen responses, and to limit the deleterious effects of immune hyper-activation. These data reveal an unexpected connection between olfactory receptor development and innate immunity, as well as demonstrate how neuronal regulation of immune responses within the intestinal epithelium is critical for both reproductive and developmental fitness.

Progression of metazoans through different developmental programs requires temporal control that is achieved by molecules originating from endocrine tissues that diffuse throughout the whole body of the animal to coordinate program execution by activating cell specific gene expression patterns. These programs then define cascades of successive, distinct developmental stages or the choice between alternative fates for the same stage. A model for this developmental control is found in the nematode C. elegans, where environmental cues signal through insulin and TGF-beta cascades to regulate the daf-12/nuclear hormone receptor (NHR) ligand synthesis that then coordinates organism wide developmental timing and fate choice. For cell intrinsic aspects of C. elegans temporal control of development, microRNAs play an important role but their connection to organism wide endocrine control is unknown. This work shows how the DAF-12/NHR directly activates let-7 family microRNAs during the L3 stage to repress L2 stage activator hbl-1 to prevent L2 stage programs from reoccurring. The interaction of upstream transcription factors with the downstream cis-regulatory elements in promoters of the let-7 family microRNAs are further analyzed in detail and identify potential DAF-12 coregulators that might connect daf-12 endocrine signaling also to later stage developmental control. These observations are the first to integrate microRNAs into establishedendocrine signaling cascades. In addition they reveal specific details about how organism wide upstream, endocrine signaling pathways induce downstream cell intrinsic changes of gene expression and developmental progression. This work postulates a "molecular switch" that actively drives stage transitions, consisting of a NHR that directly activates microRNAs to actively repress mediators of old stages while directly activating translation of protein coding genes mediating the new stage.

La maladie d’Alzheimer (MA) se présente souvent comme un déclin progressif des fonctions cognitives et comportementales. Au niveau biologique, on observe une accumulation de la protéine bêta-amyloïde (Aß) et de la protéine tau, une altération de la fonction synaptique ainsi qu’une mort neuronale. Des travaux récents ont démontré qu’une souche spécifique de probiotiques, Lacticaseibacillus rhamnosus HA-114 (HA-114), avait des propriétés neuroprotectrices dans plusieurs modèles de maladies neurodégénératives. Le projet de recherche présent visait à caractériser les aspects neurophysiologiques d’une souche de nématodes transgéniques exprimant l’Aß et valider la neuroprotection de HA-114 dans ce modèle ainsi que dans un modèle murin de la MA (APP/PS1). Les résultats présentés dans ce manuscrit montrent que les vers transgéniques exprimant le fragment 1-42 de l’Aß récapitulent des caractéristiques clés de la MA, soit une altération de la santé neuronale et de la mémoire liée à l’âge. Il s’agit donc d’un nouvel outil pour l’investigation de la physiopathologie de la MA et pour l’essai de molécules ayant un potentiel thérapeutique. Les données expérimentales montrent aussi que l’administration de HA-114 permet d’atténuer les phénotypes de neurodégénérescence et de paralysie liée à l’âge. Cela témoigne d’un potentiel en vue de l’amélioration de la qualité de vie des patients qui souffrent de la MA. L’étude des effets de HA-114 dans un modèle murin pourrait révéler de nouvelles avenues d’investigations pour la physiopathologie de la MA et les mécanismes par lesquels on pourrait un jour la traiter. / Alzheimer’s disease (AD) often presents itself as a progressive decline of behavioral and cognitive functions such as memory impairment. On a biological level, this disease presents extracellular accumulation of amyloid-beta (Aß), intracellular accumulation of tau, alteration of synaptic function and neuronal loss. Recent data showed that a supplementation with a specific probiotic strain of bacteria, Lacticaseibacillus rhamnosus HA-114 (HA-114), had a neuroprotective effect in several neurodegenerative disease models. This project aimed to characterize the neurophysiological aspects of a specific nematode strain expressing the Aß1-42 fragment and to validate the neuroprotective effect of HA-114 in this nematode strain as well as in the APP/PS1 murin model of AD. Following biochemical and behavioral experiments, data showed that this strain recapitulates key traits of AD: an age-dependent alteration of neuronal health and memory impairment. It was also observed that administrating HA-114 probiotic strain could attenuate age-related phenotypes of neurodegeneration and paralysis. This transgenic strain is therefore a new tool for drug screening and investigating AD’s pathogenesis. The neuroprotective effect of HA-114 observed in this strain shows potential towards an amelioration of the patient’s quality of life. Studying the effects of HA-114 in a murin model could reveal new investigation opportunities for AD’s pathogenesis and mechanisms that could one day cure this disease.

Amyloidosis are diseases caused by misfolded proteins that have transformed into extracellular insoluble amyloid fibrils. One type of amyloidosis is AA amyloidosis caused by AA amyloid and diseases connected are Rheumatoid arthritis and Tuberculosis. C. Elegans is a nematode used as a model organism and in this experiment. They are transgenic and express GFP (green fluorescent protein), a probe that mark the body-wall muscle cells in order to be visible in fluorescence microscopes.  Worms expressing AA45 and AA76 as well as a GFP control were fed with E. Coli OP50 and amyloid AA896 or Lin100. One control group was only fed with OP50. Three different aspects were researched: the size of the worms, their movements and a confocal microscope was used to detect amyloid. Neither the size of the worms nor the movements seemed to be linked to AA amyloid formation. However, amyloid were detected in worms expressing AA45 and AA76 but not in the GFP control when studied through a confocal microscope.  In this research it is shown that worms fed with amyloid fibrils and expressing AA45 or AA76 start forming amyloid in the body-wall muscle. This can be of much help in future research in order to make simpler diagnostical methods which can reduce the time for patients to start treatment and improve chances of survival or living with the disease with less complications.