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Tumor necrosis factor-alpha (TNF-α) is a cytokine produced primarily by macrophages during acute inflammation. In this study we examined the differential effect of retinoic acid (RA) and phorbol 12-myristate 13-acetate (PMA) on the induction of TNF-α secretion from U937 monocytic cell populations by using the reverse hemolytic plaque assay (RHPA). The RHPA will allow us to investigate both changes in TNF-α secreting populations as well as monitor the relative amount of TNF-α released from individual cells. Our results indicate that treatment of U937 cells with RA (10-6M) moderately increases the secreting cell populations, and dramatically enhances the amount of TNF-α secreted from cells already committed to secretion. In contrast, treatment with PMA (250ng/ml) drastically increased the secreting population, but only slightly increasing the amount of TNF-α released. These results suggest that induction of TNF-α secretion from U937 cells occurs by different pathways.

Lysosomal storage disorders are a group of rare neurodegenerative diseases that are collectively common, sharing many aspects with other neurodegenerative disorders, including substrate build-up and neuroinflammation. The GM2 Gangliosidoses, Tay-Sachs disease and Sandhoff disease, are pathologically overlapping lysosomal storage disorders, with high prevalence within specific ethnicities. Their effects are neurologically devastating and often fatal at young ages. Current treatments only slow or stall an inevitable decline in health. Novel treatment targets are needed for these disorders, and others with similar pathologies. In these works we demonstrate the negative effect the inflammatory cytokine tumour necrosis factor-alpha has on survival of a model of Sandhoff disease. We demonstrate its role in the upregulation of astrogliosis, and apoptosis, and we present evidence that this effect on astrogliosis occurs through an upregulation of the JAK-2/STAT3 pathway. Though fruitful, a singular focus on inflammation/gliosis in these diseases has left a vacuum in the research into neuron specific molecular processes. We observe the development of inflammation, astrogliosis and neuronal processes in our model, and demonstrate a bi-phasic disease progression, in which early onset microgliosis precedes terminal astrogliosis, apoptosis, and a decline in excitatory glutamate receptors, suggesting neuron-specific malfunction. Furthermore, we show that knockout of the synaptic protein neuronal pentraxin 1 retards neurodegeneration and extends the lifespan of Sandhoff disease mice, independent of inflammation or astrogliosis. Through electrophysiology, we provide evidence of dysregulation of glutamate receptors in Sandhoff disease, and show that knockout of neuronal pentraxin 1 provides rescue from this dysregulation. This work expands on research into gliosis in GM2 gangliosidoses, presents the finding of a novel protein isoform, and presents a new focus on non-glial disease mechanisms and treatments for these and other neurodegenerative disorders. / Thesis / Doctor of Philosophy (PhD) / Lysosomal storage disorders are a group of neurological diseases that are debilitating, and often fatal at a young age. Two diseases of this group- Tay-Sachs disease and Sandhoff disease – are similar in their causes and symptoms. Current treatments for these diseases only slow or stall an inevitable decline in health. New targets for treatment are required, and we provide data suggesting several proteins that may fit this criterion. We also provide evidence of the discovery of a new form of one of these proteins, which is found in high levels in the disease, indicating it may be important in these and other neurodegenerative diseases. Finally, we provide findings indicating that a certain cell type, which is largely ignored in current research for these diseases, may be important in the disease progress. These findings increase our knowledge of Tay-Sachs disease and Sandhoff disease, and open new avenues for medicinal intervention.

No description available.

Cocaine abuse poses a substantial health and economic burden for which no effective treatment currently exists. Exposure to cocaine results in altered signaling in a number of central nervous system (CNS) pathways. Previous studies have primarily focused on neurotransmitter systems, such as the dopaminergic and glutamatergic systems, as well as on drug-induced neuroplasticity within the mesolimbic system, which is believed to contribute to reward, addiction, and relapse following withdrawal. Furthermore, cocaine exerts a number of effects on gene regulation that contribute to many pathological conditions commonly afflicting users such as mood disturbances, psychotic symptoms, and long-term cognitive dysfunction. While some mechanisms by which cocaine regulates gene expression have been well-characterized, a large gap in our understanding regarding its downstream actions still exists and must be elucidated in order to develop effective treatment strategies. One pathway we have discovered to be disrupted in an animal model of chronic cocaine abuse is the retinoid X receptor (RXR) signaling pathway. Retinoid X receptors serve as obligate heterodimer partners for a number of nuclear receptor transcription factors, including the thyroid hormone receptor (TR), retinoic acid receptor, vitamin D receptor, and peroxisome proliferator activated receptor. Heterodimeric complexes bind to specific recognition sequences in or around the promoter of target genes to activate, or in some cases, repress, transcriptional activity. Therefore, alterations in the levels and function of RXRs can potentially disrupt numerous signaling cascades. In this context, we observed a significant down-regulation in mRNA and protein levels of RXR-y, an isoform predominantly expressed in the CNS that is involved in dopaminergic signaling, in brains of cocaine-administered mice. Additionally, we observed significantly decreased levels of the neuroplasticity protein, neurogranin, which is regulated transcriptionally by TR/RXR heterodimers. Mechanisms underlying regulation of RXR levels in cells of the CNS are vastly unexplored. Studies in other organ systems, including liver and cardiac systems, demonstrate pro-inflammatory cytokines and cellular stress pathways exert repressive effects on RXR signaling, although these studies solely investigated regulation of the RXR-a isoform. Recently, studies have highlighted the role of the immune system during chronic drug abuse, and demonstrate that significant amounts of proinflammatory factors are produced in the brains of chronic cocaine abusers. Therefore, we hypothesized that cocaine-mediated induction of inflammatory cytokines, such as tumor necrosis factor (TNF)-a may contribute to decreased RXR-y expression within the CNS. Utilizing in vitro neuronal systems, we have demonstrated that cocaine exposure induces neuronal expression of TNF-a and that this contributes to decreased levels of RXR-y, as inhibition of TNF-a or its downstream effector c-Jun-NH2-terminal kinase (JNK) prevents cocaine-mediated reductions in RXR-y protein levels. Furthermore, treatment of neurons with TNF-a alone mimics the effects on RXR-y levels observed in cocaine-treated cells. Additionally, we show that proteasome-dependent protein degradation likely plays a role, as inhibition of the 26 S proteasome with Bortezomib during cocaine or TNF-a exposure blocks the down-regulation of RXR-y levels. Degradation of RXR-y in response to cocaine and TNF-a may involve nuclear export, as our results show an increased level of RXR-y in the cytoplasmic compartment shortly after treatment, and inhibiting nuclear export during treatment with Leptomycin B prevents decreases in whole cell protein levels of RXR-y. In addition to the effects of chronic cocaine abuse on neurons, other CNS cell types such as oligodendrocytes may be negatively impacted by exposure to cocaine. Imaging studies and post-mortem microarray data from human cocaine abuse patients reveal loss of myelin and down-regulated expression of myelin-related genes in the nucleus accumbens and frontal cortex. Altered myelin integrity likely contributes to cognitive deficits that present in many chronic cocaine abuse patients and may also exacerbate damage to neurons. However, limited investigation has been performed to evaluate the effects of cocaine on oligodendrocyte health and function. We have employed an in vivo murine model of chronic cocaine administration to evaluate the impact of cocaine on white matter protein levels. Our data reveal that cocaine induces a significant decrease in white matter protein levels, even following an extended period of withdrawal, in the nucleus accumbens. One potential mechanism for cocaine-mediated white matter damage involves perturbations of glutamate homeostasis, as glutamatergic signaling can induce excitotoxicity in CNS cells, including oligodendrocytes. In this context, we found that administration of the B-lactam antibiotic, ceftriaxone, during cocaine withdrawal ameliorates loss of white matter proteins. Ceftriaxone has previously been shown to upregulate expression and activity of the glial glutamate transporter GLT-1, lending support to the theory that cocaine-mediated myelin loss may be due, in part, to disruption of glutamatergic signaling. Ceftriaxone treatment also decreased expression of cleaved caspase-3, a pro-apoptotic signaling molecule activated during excitotoxic cell death, in cocaine-administered mice. Taken together, our studies characterize two novel consequences of cocaine exposure: (1) decreased neuronal RXR-y expression and down-regulation of RXR-target genes, such as neurogranin, and (2) loss of myelin proteins in the nucleus accumbens which can be attenuated by administration of ceftriaxone. These findings yield insight into mechanisms underlying cocaine-mediated CNS cell death, and highlight potential treatment avenues for restoring brain health. Additionally, as inflammatory processes were identified as key mediators in some of these observations, our findings likely extend to a number of neurodegenerative diseases which are characterized by a neuroinflammatory component. / Biomedical Neuroscience

Immunoassays are widely used for detection of antigens in a wide range of applications including assays in pharmaceutical development. Immunoassays are continuously improved in many aspects including automatization, miniaturization and extending the dynamic range. The need to measure low abundance molecules are challenging and the need to improve the sensitivity is desired. The Gyrolab technology is a miniaturized immunoassay performed in an automated system covering a broad concentration range. In order to  extend the sensitivity, the technology is combined with Binding Oligo Ladder Detection (BOLD) amplification. The technology behind BOLD or Exazym ® utilizes a DNA primer, a polymerase, and a template (RNA) to generate a ladder-like modified DNA strand. Antibodies with affinity for the polymerized DNA:RNA hybrid strand (duplex) conjugated with reporter molecules are introduced to the system, resulting in an increased number of signal-generating molecules associated with each bound analyte molecule. In this thesis, the development of an ultra-sensitive immunoassay is pursued by applying Exazym ® add-on reagents to the Gyrolab platform, comparing performance with the standard Gyrolab sandwich assay and other commercially available high-performing TNF-α assays. The work includes characterization of a wide range of reaction variables involved in the BOLD signal amplification process including hybridization, polymerization, and detection of a synthetic oligonucleotide. The breakthrough involves the introduction of Allophycocyanin (APC) as a fluorescent conjugate, significantly improving sensitivity and signal-to-noise ratios. The BOLD amplified sensitivity for the TNF-α assay approaches levels seen in ultra-sensitive biomarker assays like Erenna ® and Simoa®. Exazym® technology on the Gyrolab platform allows highly sensitive biomarker assays with minimal sample volume and a 1–2-hour run-time. The study marks substantial progress in achieving ultra-sensitive biomarker assays on the Gyrolab platform through BOLD signal amplification. The use of APC-conjugated detection reagents holds promise for future optimization studies.

L’autophagie est une voie hautement conservée de dégradation lysosomale des constituants cellulaires qui est essentiel à l’homéostasie cellulaire et contribue à l’apprêtement et à la présentation des antigènes. Les rôles relativement récents de l'autophagie dans l'immunité innée et acquise sous-tendent de nouveaux paradigmes immunologiques pouvant faciliter le développement de nouvelles thérapies où la dérégulation de l’autophagie est associée à des maladies auto-immunes. Cependant, l'étude in vivo de la réponse autophagique est difficile en raison du nombre limité de méthodes d'analyse pouvant fournir une définition dynamique des protéines clés impliquées dans cette voie. En conséquence, nous avons développé un programme de recherche en protéomique intégrée afin d’identifier et de quantifier les proteines associées à l'autophagie et de déterminer les mécanismes moléculaires régissant les fonctions de l’autophagosome dans la présentation antigénique en utilisant une approche de biologie des systèmes. Pour étudier comment l'autophagie et la présentation antigénique sont activement régulés dans les macrophages, nous avons d'abord procédé à une étude protéomique à grande échelle sous différentes conditions connues pour stimuler l'autophagie, tels l’activation par les cytokines et l’infection virale. La cytokine tumor necrosis factor-alpha (TNF-alpha) est l'une des principales cytokines pro-inflammatoires qui intervient dans les réactions locales et systémiques afin de développer une réponse immune adaptative. La protéomique quantitative d'extraits membranaires de macrophages contrôles et stimulés avec le TNF-alpha a révélé que l'activation des macrophages a entrainé la dégradation de protéines mitochondriales et des changements d’abondance de plusieurs protéines impliquées dans le trafic vésiculaire et la réponse immunitaire. Nous avons constaté que la dégradation des protéines mitochondriales était sous le contrôle de la voie ATG5, et était spécifique au TNF-alpha. En outre, l’utilisation d’un nouveau système de présentation antigènique, nous a permi de constater que l'induction de la mitophagie par le TNF-alpha a entrainée l’apprêtement et la présentation d’antigènes mitochondriaux par des molécules du CMH de classe I, contribuant ainsi la variation du répertoire immunopeptidomique à la surface cellulaire. Ces résultats mettent en évidence un rôle insoupçonné du TNF-alpha dans la mitophagie et permet une meilleure compréhension des mécanismes responsables de la présentation d’auto-antigènes par les molécules du CMH de classe I. Une interaction complexe existe également entre infection virale et l'autophagie. Récemment, notre laboratoire a fourni une première preuve suggérant que la macroautophagie peut contribuer à la présentation de protéines virales par les molécules du CMH de classe I lors de l’infection virale par l'herpès simplex virus de type 1 (HSV-1). Le virus HSV1 fait parti des virus humains les plus complexes et les plus répandues. Bien que la composition des particules virales a été étudiée précédemment, on connaît moins bien l'expression de l'ensemble du protéome viral lors de l’infection des cellules hôtes. Afin de caractériser les changements dynamiques de l’expression des protéines virales lors de l’infection, nous avons analysé par LC-MS/MS le protéome du HSV1 dans les macrophages infectés. Ces analyses nous ont permis d’identifier un total de 67 protéines virales structurales et non structurales (82% du protéome HSV1) en utilisant le spectromètre de masse LTQ-Orbitrap. Nous avons également identifié 90 nouveaux sites de phosphorylation et de dix nouveaux sites d’ubiquitylation sur différentes protéines virales. Suite à l’ubiquitylation, les protéines virales peuvent se localiser au noyau ou participer à des événements de fusion avec la membrane nucléaire, suggérant ainsi que cette modification pourrait influer le trafic vésiculaire des protéines virales. Le traitement avec des inhibiteurs de la réplication de l'ADN induit des changements sur l'abondance et la modification des protéines virales, mettant en évidence l'interdépendance des protéines virales au cours du cycle de vie du virus. Compte tenu de l'importance de la dynamique d'expression, de l’ubiquitylation et la phosphorylation sur la fonction des proteines virales, ces résultats ouvriront la voie vers de nouvelles études sur la biologie des virus de l'herpès. Fait intéressant, l'infection HSV1 dans les macrophages déclenche une nouvelle forme d'autophagie qui diffère remarquablement de la macroautophagie. Ce processus, appelé autophagie associée à l’enveloppe nucléaire (nuclear envelope derived autophagy, NEDA), conduit à la formation de vésicules membranaires contenant 4 couches lipidiques provenant de l'enveloppe nucléaire où on retrouve une grande proportion de certaines protéines virales, telle la glycoprotéine B. Les mécanismes régissant NEDA et leur importance lors de l’infection virale sont encore méconnus. En utilisant un essai de présentation antigénique, nous avons pu montrer que la voie NEDA est indépendante d’ATG5 et participe à l’apprêtement et la présentation d’antigènes viraux par le CMH de classe I. Pour comprendre l'implication de NEDA dans la présentation des antigènes, il est essentiel de caractériser le protéome des autophagosomes isolés à partir de macrophages infectés par HSV1. Aussi, nous avons développé une nouvelle approche de fractionnement basé sur l’isolation de lysosomes chargés de billes de latex, nous permettant ainsi d’obtenir des extraits cellulaires enrichis en autophagosomes. Le transfert des antigènes HSV1 dans les autophagosomes a été determine par protéomique quantitative. Les protéines provenant de l’enveloppe nucléaire ont été préférentiellement transférées dans les autophagosome lors de l'infection des macrophages par le HSV1. Les analyses protéomiques d’autophagosomes impliquant NEDA ou la macroautophagie ont permis de decouvrir des mécanismes jouant un rôle clé dans l’immunodominance de la glycoprotéine B lors de l'infection HSV1. Ces analyses ont également révélées que diverses voies autophagiques peuvent être induites pour favoriser la capture sélective de protéines virales, façonnant de façon dynamique la nature de la réponse immunitaire lors d'une infection. En conclusion, l'application des méthodes de protéomique quantitative a joué un rôle clé dans l'identification et la quantification des protéines ayant des rôles importants dans la régulation de l'autophagie chez les macrophages, et nous a permis d'identifier les changements qui se produisent lors de la formation des autophagosomes lors de maladies inflammatoires ou d’infection virale. En outre, notre approche de biologie des systèmes, qui combine la protéomique quantitative basée sur la spectrométrie de masse avec des essais fonctionnels tels la présentation antigénique, nous a permis d’acquérir de nouvelles connaissances sur les mécanismes moléculaires régissant les fonctions de l'autophagie lors de la présentation antigénique. Une meilleure compréhension de ces mécanismes permettra de réduire les effets nuisibles de l'immunodominance suite à l'infection virale ou lors du développement du cancer en mettant en place une réponse immunitaire appropriée. / Autophagy is a highly conserved lysosomal-mediated protein degradation pathway that plays a crucial role in maintaining cellular homeostasis and contributes to antigen processing and presentation. The emerging roles of autophagy in both innate and adaptive immunity underpin novel immunological paradigms that may provide opportunities for the development of new therapies where impaired autophagy is associated with autoimmune diseases. However, the in vivo study of autophagic response is challenging in view of the limited number of analytical approaches that can provide a dynamic definition of the key proteins involved in this pathway. Accordingly, we developed an integrated proteomics research program to unravel the molecular machines associated with autophagy and to decipher the fine details of the molecular mechanisms governing the functions of the autophagosome in antigen presentation using a systems biology approach. To study how autophagy and antigen presentation are actively modulated in macrophages, we first conducted comprehensive, global proteomics studies under different conditions known to stimulate autophagy. Autophagy is modulated by cytokines as well as by viral infection in various ways. TNF-alpha is one of the major proinflammatory cytokines that mediate local and systemic responses and direct the development of adaptive immunity. Label-free quantitative proteomics analysis of membrane extracts from TNF-alpha activated and resting macrophages revealed that TNF-alpha activation led to the downregulation of mitochondrial proteins and the differential regulation of several proteins involved in vesicle trafficking and immune response. Importantly, we found that the downregulation of mitochondria proteins occurred through Atg5-dependent mitophagy, and was specific to TNF-alpha. Furthermore, using a novel antigen presentation system, we observed that the induction of mitophagy by TNF-alpha enabled the processing and presentation of mitochondrial antigens at the cell surface by MHC class I molecules, suggesting that TNF-alpha induced mitophagy contributes to the modification of the MHC class I peptide repertoire. These findings highlight an unsuspected role of TNF-alpha in mitophagy and expanded our understanding of the mechanisms responsible for MHC class I presentation of self-antigens. A complex interplay also exists between viral infection and autophagy. Recently, our lab provided the first evidence that macroautophagy can contribute to the presentation of viral proteins on MHC class I molecules during Herpes Simplex Virus type 1 (HSV1) infection. HSV1 are among the most complex and widespread human viruses. While the composition of viral particles has been studied, less is known about the expression of the whole viral proteome in infected cells. To comprehensively characterize the system, we analyzed the proteome of the prototypical HSV1 in infected macrophages by LC-MS/MS. We achieved a very high level of protein coverage and identified a total of 67 structural and non-structural viral proteins (82% of the HSV1 proteome) using LC-MS/MS on a LTQ-Orbitrap instrument. We also obtained a comprehensive map of 90 novel phosphorylation sites and ten novel ubiquitylation sites on different viral proteins. Interestingly all ubiquitylated proteins could either localize to the nucleus or participate in membrane fusion events, suggesting that ubiquitylation of viral proteins might affect their trafficking. Treatment with inhibitors of DNA replication induced changes of both viral protein abundance and modifications, highlighting the interdependence of viral proteins during the life cycle of the virus. Given the importance of expression dynamics, ubiquitylation and phosphorylation for protein function, these findings will serve as important tools for future studies on herpes virus biology. Interestingly, HSV1 infection in macrophages triggers a novel form of autophagy which remarkably differs in many ways from macroautophagy. This process, referred to as nuclear envelope-derived autophagy (NEDA), leads to the formation of 4-membrane layered vesicles originating from the nuclear envelope where some viral protein such as glycoprotein B are highly enriched. To which extent this process differs from macroautophagy and participates in the pathogenesis of HSV infection is still largely unknown. Using a novel antigen presentation assay we could show that NEDA is an Atg5-independent pathway that participates in the capture of viral proteins, and their processing and presentation on MHC class I molecules. To understand the involvement of NEDA in antigen presentation it is crucial to characterize the autophagosomal proteome in HSV1 infected macrophages. We developed a novel isolation method based on the loading of the lysosomal compartment with latex beads, a unique tool to obtain very pure cell extracts, upon autophagy induction. The transfer of HSV1 antigens into autophagosomes was monitored using quantitative proteomics. Nuclear enveloped-derived proteins were preferentially transferred to the autophagosome during HSV1 infection. Detailed proteomics characterization of autophagosomes formed during NEDA and macroautophagy led to the discovery of mechanisms that play a key role in glycoprotein B immunodominance during HSV1 infection. These analyses also revealed that various autophagic pathways can be induced to promote the capture of selective sets of viral proteins, thus actively shaping the nature of the immune response during infection. In conclusion, the application of quantitative proteomics methods played a key role in identifying and quantifying important regulators of autophagy in macrophages and allowed us to identify changes occurring during the remodeling of autophagosomes in response to disease and inflammatory conditions such as viral infections. Furthermore, our systems biology approach that combined mass spectrometry-based quantitative proteomics with functional screens such as antigen presentation assays revealed novel biological insights on the molecular mechanisms governing the functions of autophagy in antigen presentation. Harnessing the contribution of autophagy in antigen presentation has the potential to minimize the deleterious effects of immunodominance in viral infection and cancer by shaping an appropriate immune response.

L’élimination des cellules infectées par apoptose constitue un mécanisme de défense antivirale. Les virus de l’herpès simplex (HSV) de type 1 et 2 encodent des facteurs qui inhibent l’apoptose induite par la réponse antivirale. La sous-unité R1 de la ribonucléotide réductase d’HSV-2 (ICP10) possède une fonction anti-apoptotique qui protège les cellules épithéliales de l’apoptose induite par les récepteurs de mort en agissant en amont ou au niveau de l’activation de la procaspase-8. Puisqu’une infection avec un mutant HSV-1 déficient pour la R1 diminue la résistance des cellules infectées vis à vis du TNFα, il a été suggéré que la R1 d’HSV-1 (ICP6) pourrait posséder une fonction anti-apoptotique. Le but principal de cette thèse est d’étudier le mécanisme et le potentiel de la fonction anti-apoptotique de la R1 d’HSV-1 et de la R1 d'HSV-2. Dans une première étude, nous avons investigué le mécanisme de la fonction anti-apoptotique de la R1 d’HSV en utilisant le TNFα et le FasL, deux inducteurs des récepteurs de mort impliqués dans la réponse immune anti-HSV. Cette étude a permis d’obtenir trois principaux résultats concernant la fonction anti-apoptotique de la R1 d’HSV. Premièrement, la R1 d’HSV-1 inhibe l’apoptose induite par le TNFα et par le FasL aussi efficacement que la R1 d’HSV-2. Deuxièmement, la R1 d’HSV-1 est essentielle à l’inhibition de l’apoptose induite par le FasL. Troisièmement, la R1 d’HSV interagit constitutivement avec la procaspase-8 d’une manière qui inhibe la dimérisation et donc l’activation de la caspase-8. Ces résultats suggèrent qu’en plus d’inhiber l’apoptose induite par les récepteurs de mort la R1 d’HSV peut prévenir l’activation de la caspase-8 induite par d’autres stimuli pro-apoptotiques. Les ARN double-brins (ARNdb) constituant un intermédiaire de la transcription du génome des HSV et activant l’apoptose par une voie dépendante de la caspase-8, nous avons testé dans une seconde étude l’impact de la R1 d’HSV sur l’apoptose induite par l’acide polyriboinosinique : polyribocytidylique (poly(I:C)), un analogue synthétique des ARNdb. Ces travaux ont montré qu’une infection avec les HSV protège les cellules épithéliales de l’apoptose induite par le poly(I:C). La R1 d’HSV-1 joue un rôle majeur dans l’inhibition de l’activation de la caspase-8 induite par le poly(I:C). La R1 d’HSV interagit non seulement avec la procaspase-8 mais aussi avec RIP1 (receptor interacting protein 1). En interagissant avec RIP1, la R1 d’HSV-2 inhibe l’interaction entre RIP1 et TRIF (Toll/interleukine-1 receptor-domain-containing adapter-inducing interferon β), l’adaptateur du Toll-like receptor 3 qui est un détecteur d’ARNdb , laquelle est essentielle pour signaler l’apoptose induite par le poly(I:C) extracellulaire et la surexpression de TRIF. Ces travaux démontrent la capacité de la R1 d’HSV à inhiber l’apoptose induite par divers stimuli et ils ont permis de déterminer le mécanisme de l’activité anti-apoptotique de la R1 d’HSV. Très tôt durant l’infection, cFLIP, un inhibiteur cellulaire de la caspase-8, est dégradé alors que la R1 d’HSV s’accumule de manière concomitante. En interagissant avec la procapsase-8 et RIP1, la R1 d’HSV se comporte comme un inhibiteur viral de l’activation de la procaspase-8 inhibant l’apoptose induite par les récepteurs de mort et les détecteurs aux ARNdb. / Elimination of infected cells by apoptosis constitutes an ancestral mechanism of host defense against viral infection. Herpes simplex viruses (HSVs) encode several viral factors to counteract the apoptotic antiviral response. Among them, the R1 subunit of HSV type-2 ribonucleotide reductase (HSV-2 R1, also named ICP10), protects cells by interrupting death receptor-mediated signaling at, or upstream of, caspase-8 activation. Since protection against tumor necrosis factor alpha (TNFα)-induced apoptosis is decreased un cells infected with an HSV type-1 R1 null mutant, it has been proposed that HSV-1 R1 (ICP6) could also possess an antiapoptotic activity. The fundamental goal of this thesis is to better understand the mechanism and the potential of the HSV R1s antiapoptotic activity. In a first study, we investigated the mechanism of the antiapoptotic activity of HSV R1s by using TNFα and Fas ligand (FasL), two death-receptor inducers involved in anti-HSVs immune response. From this work, we report three main findings on the antiapoptotic activity of HSV R1s. First, HSV-1 R1 like HSV-2 R1 has the ability to protect cells against TNFα- and FasL-induced apoptosis. Second, HSV-1 R1 contributes in protecting infected cells against FasL. Third, HSV R1s and procaspase-8 interact in a way that inhibits the dimerization/activation of caspase-8. These results suggest that in addition to counteracting death receptor-induced apoptosis, HSV R1s could inhibit apoptosis induced by other signals that trigger caspase-8 activation during HSV infection. Double-stranded RNA (dsRNA) are viral intermediates notably produced by HSVs and have been shown to induce apoptosis via caspase-8 activation. We tested in a second study whether HSV R1s have the ability to counteract apoptosis triggered by polyriboinosinic : polyribocytidylic acid (poly(I:C)), a synthetic analog of dsRNA that triggers caspase-8 activation. We showed that HSVs infection protect epithelial cells from apoptosis induced by poly(I:C). We established that HSV-1 R1 is essential for the protection of HSV-1-infected cells against poly(I:C)-induced caspase-8 activation. HSV R1s interact not only with procaspase-8 but also with the receptor interacting protein 1 (RIP1). The interaction between RIP1 and HSV-2 R1 inhibits the binding of RIP1 to the Toll/interleukine-1 receptor-domain-containing adapter-inducing interferon β (TRIF), the adaptor of Toll-like receptor 3 that is an extracellular dsRNA sensor, which is required to activate caspase-8 following extracellular poly(I:C) stimulation and TRIF overexpression. Thus, HSV R1s have the ability to inhibit poly(I:C)-induced apoptosis at several levels by preventing caspase-8 dimerization/activation and TRIF RIP1 interaction. This work sheds light on the ability of HSV R1s to manipulate apoptosis. Early during the lytic cycle, protein levels of the cellular inhibitors of caspase-8 as cFLIP drop but HSV R1s accumulate concomitantly and act as a viral inhibitor of apoptosis by binding to procaspase-8 and RIP1 in a way that impairs caspase-8 activation by death-receptors and dsRNA detectors stimulation.