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.

Many diseases, such as Parkinson's disease and heavy metal poisoning, are associated with impaired or aberrant locomotion. Because the underlying mechanisms are difficult to study in humans, simpler metazoans like Caenorhabditis elegans are commonly employed to model these diseases. C. elegans is especially useful in this respect because its innate electrotactic behaviour allows instantaneous manipulation of its locomotion using mild electric fields in a microfluidic environment, the results of which can be captured on video. However, extraction of locomotory data from these videos is a major bottleneck to the throughput of the microfluidic electrotaxis platform. In the present study, we describe the development of novel software to analyze electrotaxis videos in an automated fashion. The software, dubbed the Automated Nematode Tracking System (ANTS), uses efficient, parameterless computer vision techniques to simultaneously track and assess movement characteristics of ambulating animals. In combination with the previously described microfluidic electrotaxis platform, ANTS promises to accelerate research with C. elegans models of locomotory dysfunction. / Thesis / Master of Applied Science (MASc)

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.

The nexus of engineering and molecular biology has given birth to high-throughput technologies that allow biologists and medical scientists to produce previously unattainable amounts of data to better understand the molecular basis of many biological phenomena. Here, we describe the development of an enabling biotechnology, commonly known as microfluidics in the fabrication of high-throughput systems to study nerve degeneration and regeneration in the well-defined model nematode, Caenorhabditis elegans (C. elegans). Our lab previously demonstrated how femtosecond (fs) laser pulses could precisely cut nerve axons in C. elegans, and we observed axonal regeneration in vivo in single worms that were immobilized on anesthetic treated agar pads. We then developed a microfluidic device capable of immobilizing one worm at a time with a deformable membrane to perform these experiments without agar pads or anesthetics. Here, we describe the development of improved microfluidic devices that can trap and immobilize up to 24 individual worms in parallel chambers for high-throughput axotomy and subsequent imaging of nerve regeneration in a single platform. We tested different micro-channel designs and geometries to optimize specific parameters: (1) the initial trapping of a single worm in each immobilization chamber, simultaneously, (2) immobilization of single worms for imaging and fs-laser axotomy, and (3) long term storage of worms on-chip for imaging of regeneration at different time points after the initial axon cut. / text

Micromanipulation is considered a challenging task which requires high precision motion and measurement at the micro scale. When micromanipulation is concerned with living organisms important considerations need to be addressed. These include the physical or chemical properties of micro-organisms, living conditions, responses to the environment and achieving suitably delicate manipulation. Bio-micromanipulation can include micro surgery or cell injection operations, or to determine interaction forces as the basis to investigate behavior and properties of living micro-organisms. In order to achieve suitable bio-micromanipulation appropriate processes and/or sensory systems need to be investigated. This thesis aims to look into the force interaction and sensing addressing two distinctive challenges in the field of bio-micromanipulation. To this end, this thesis presents two major contributions to advancing bio-micromanipulation. Firstly, a novel Haptic Microrobotic Cell Injection System is introduced which is able to assist a bio-operator through haptic interaction. The system introduces a mapping framework which provides an intuitive method for the bio-operator to maneuver the micropipette in a manner similar to handheld needle insertion. To accurately control the microrobot, a neuro-fuzzy modeling and control scheme has been developed. Volumetric, axial and planar haptic virtual fixtures are introduced to guide the bio-operator during cell injection. Aside from improving real-time operator performance using the physical system, the system is novel in facilitating virtual offline operator training. Secondly, a first-of-its-kind micro-pillar based on-chip system for dynamic force measurement of C. elegans motion is introduced. The system comprises a microfabricated PDMS device to direct C. elegans into a matrix of micropillars within a channel mimicking its dwelling environment. An image processing algorithm is able to track the interaction of the C. elegans with the pillars and estimate contact forces based on micropillar deflections. The developed micropillar system is capable of measuring the force with sub-micron resolution while providing a continuous force output spectrum.

The nematode Caenorhabditis elegans is a unique resource for the study of axon guidance and cell migration. Genetic data collected from C. elegans research is integral to advancing our knowledge of genetic pathways that control axon and cell guidance, such as the UNC-6/netrin pathway, within a developing organism. In this thesis, I describe the characterization of ev821, an allele isolated from a screen for novel guidance mechanisms or components of the UNC-6 pathway. Furthermore, I show that ev821 is a maternal-effect and temperature-sensitive mutation with defects in phase 2 of distal tip cell migration in the C. elegans hermaphrodite, as well as ray-1 displacement and a blister phenotype in the C. elegans male. My research on ev821 could broaden our understanding of the mechanisms driving UNC-6 guidance, and has the potential to identify unknown factors that act within or in parallel to the UNC-6 signaling pathway.