After ejaculation, spermatozoa undergo a series of transformation in the female genital tract, named capacitation, that enables them to bind to the zona pellucida, initiate the acrosome reaction and fertilize the egg. Mammalian sperm motility, capacitation and acrosome reaction are regulated by signal transduction systems involving cAMP and cAMP-dependent protein kinase A (PKA). Intracellular levels of cAMP are regulated by adenylyl cyclase (AC) and phosphodiesterases (PDEs), responsible for its production and degradation, respectively. Of the 11 PDE families that exist, calmodulin (CaM)-dependent PDE (PDE1) and cAMP-specific PDE (PDE4) activities were previously identified in human spermatozoa. The aims of the present study were to evaluate the role played by some PDEs in human sperm function, to determine which PDEs are present and to evaluate the changes in cAMP, PDE and PKA activities during sperm capacitation and acrosome reaction. PDE activity did not change during either capacitation or acrosome reaction while PKA activity was increased in both phenomena. Moreover, sperm PDEs were associated with the membrane (50--60%) as well as with the particulate fraction (30--50%) and had much more affinity for cAMP than cGMP as substrate. Type-specific PDE inhibitors such as EHNA for cGMP-stimulated PDE (PDE2), milrinone for cGMP-inhibited PDE (PDE3) and rolipram for PDE4 but not sildenafil for cGMP-specific PDE (PDE5), decreased sperm PDE activity suggesting that PDE2, PDE3 and PDE4 but not PDE5 were present both in membrane and particulate fractions. Moreover, EHNA and rolipram increased sperm capacitation while milrinone increased sperm cAMP. Anti-PDE1A and anti-PDE3A antibodies recognized proteins of 67 kDa (PDE1A) and 97 kDa (PDE3A). Immunolocalization indicated that PDE1A was present on the equatorial segment of the sperm head as well as on the mid- and principal pieces of the flagellum and that PDE3A was present on the post-acrosomal segment of the head. Furth

Protein disulfide isomerase (PDI) is the most abundant and best characterized member of the extensive PDI family of proteins found in the endoplasmic reticulum. PDI plays an important role in protein folding. It is composed of four thioredoxin-like domains denoted as a-b-b'-a'. Recent studies have identified a hydrophobic surface on the b' domain as the main binding site for the unfolded protein substrates of PDI. Here, the main hydrophobic residues of the b' domain were mutated in a bb' construct and tested for their ability to bind to the 14-residue mastoparan peptide. Most of the mutants had a significant effect on the binding affinity. In reverse experiments the mastoparan peptide was mutated and NMR titrations used to determine the orientation of the peptide on the bb' binding surface. Finally, the catalytic a and a' domains were individually tested for binding to unfolded RNaseA by NMR. Neither catalytic domain exhibited any direct interaction with the unfolded substrate. / La protéine disulfure isomérase (PDI) est le membre le mieux connu et caractérisé de la nombreuse famille de PDI qui se trouve dans le réticulum endoplasmique. PDI joue un rôle important dans le repliement des chaînes protéiques. Elle est composée de quatre domaines désignés a-b-b'-a' de type thiorédoxine. Des études récentes ont identifié une surface hydrophobe dans le domaine b' comme étant le lieu principal d'interaction avec les substrats déroulés de la PDI. Ici, les résidus hydrophobes principaux du domaine b' ont été mutés et testés pour leur interaction avec un peptide de quatorze résidus nommé mastoparan. La plupart des mutants ont eu un impact important sur l'affinité de la PDI pour son substrat. Dans des expériences contraires où le peptide mastoparan a été muté, des titrations par RMN ont révélé l'orientation du peptide sur la surface d'interaction du domaine b'. Finalement, les domaines catalytiques a et a' ont été individuellement testés par RMN pour leur affinité envers l'ARNase A déroulée. Les deux domaines n'ont montré aucune interaction avec le substrat déroulé.

During mammalian skeletal development, endochondral ossification is preceded by the establishment of a cartilage template through a process termed chondrogenesis. Disruptions in chondrogenesis result in chondrodysplasia, a disorder of the appendicular skeleton which is characterized by disproportionately shortened limbs. A severe chondrodysplasia phenotype is observed when site-1 protease (S1P) is disrupted in both the zebrafish and the mouse, indicating that S1P is essential for normal skeletal development. To investigate the role of S1P during chondrogenesis, ATDC5 cells were used as an in vitro model. Inhibition of S1P activity in these cells decreases the expression of collagens type II and X, while type I collagen and aggrecan remain unaffected. The substrates of S1P are membrane-bound proteins which, when cleaved, release transcription factors promoting cholesterol biosynthesis and the unfolded protein response. Real time-PCR analysis determined that the expression of two substrates, ATF6 and CREB3/Luman, were significantly up-regulated during chondrogenesis. However, silencing of CREB3/Luman expression revealed no cartilage-related phenotype. / Pendant le dévelopment du squelette des mammifères, l'ossification endochondrale est précédée par l'établissement d'une matrice cartilagineuse par un processus appelé chondrogenesis. Toute altération dans la chondrogenèse produisant une telle matrice résulte en une chondrodysplasie, maladie affectant le squelette appendiculaire et caractérisée par un raccourcissement disproportionnel des membres. Chez le poisson zèbre et la souris, l'ablation du gène encodant S1P (site-1-protease) engendre une chondrodysplasie sévère, démontrant ainsi que S1P est une protéine essentielle au développement normal du squelette. Ainsi, afin d'étudier le rôle de S1P dans l'ossification endochondrale, les cellules ATDC5 on été utilisées comme modèle in vitro de chondrogenèse. L'inhibition de l'activité de S1P dans ces cellules a eu pour effet de diminuer l'expression des collagènes de type II et X, alors que l'expression du collagène de type I et de l'aggrecane est restée inchangée. Les substrats de S1P sont des protéines membranaires qui, une fois clivées, libèrent des facteurs de transcription actifs lesquels, après translocation nucléaire, vont promouvoir la synthèse du cholestérol, ainsi que la voie UPR (unfolded protein response). L'analyse par PCR en temps réel a permis de démontrer que l'expression génique de deux substrats de S1P, ATF6 et CREB3/Luman, est augmentée de façon significative lors de la chondrogenèse. Cependant, les expériences de suppression génique de CREB3/Luman n'ont pas permis de révéler de phénotype spécifique du cartilage.

Abstract The serine/threonine kinase mTOR plays a critical role in the regulation of cellular proliferation, growth, differentiation, autophagy, and cytoskeletal organization. Two mTOR containing complexes exist in mammalian cells: mTORC1 and mTORC2. mTORC1 mediates its effects through the control of mRNA translation initiation via phosphorylation of its two major downstream targets: the 4E-BPs and S6Ks. In order to study the individual roles of S6Ks and 4E-BPs downstream of mTORC1, we utilized new active-site mTOR inhibitors and cells deficient for either 4E-BPs or S6Ks. Surprisingly, we found that 4E-BPs and S6Ks play distinct roles in mediating the activity of mTORC1, with the 4E-BPs mediating cell proliferation and the S6Ks regulating cell growth. The effects of active-site mTOR inhibitors on cell proliferation, cell cycle progression, and protein synthesis were highly attenuated in cells lacking expression of 4E-BPs (4E-BP DKO), while these processes were significantly inhibited in wild-type cells. However, the growth of 4E-BP DKO cells was equally sensitive to active site mTOR inhibitors, as compared to their wild-type counterparts. In contrast, the growth of S6K1/2 null cells was refractory to the effects of active site mTOR inhibitors, whereas these cells were fully sensitive to the effects of the inhibitors on proliferation and cell cycle progression. These data support a model whereby the mTORC1 proliferative and growth signals diverge in mammalian cells to provide a system that allows for increased flexibility in the maintenance of full body homeostasis. mTOR is often over-activated in human cancers and as a result, has emerged as a target for anti-cancer therapies. The anti-diabetic drug metformin was recently identified as a potential anti-cancer agent. To elucidate its mechanism of action, we studied its effects on breast cancer cell proliferation, mTOR signalling and mRNA translation. In b / RésumémTOR est une sérine/thréonine kinase qui joue un rôle primordial dans lecontrôle de plusieurs fonctions cellulaires, incluant la prolifération, la croissance, ladifférentiation, l'autophagie et la réorganisation du cytosquelette. Les cellulesmammifères possèdent deux complexes protéiques contenant mTOR, soit mTORC1 andmTORC2. mTORC1 exerce son action en contrôlant l'initiation de la traduction desARN messagers via la phosphorylation de ses principaux effecteurs : les 4E-BP et lesS6K. Dans le but de mieux comprendre les fonctions spécifiques des 4E-BP et des S6Ken aval de la signalisation cellulaire initiée par mTORC1, nous avons utilisé de nouveauxinhibiteurs spécifiques au site actif de mTOR, ainsi que des cellules n'exprimant pas les4E-BP ou les S6K. Nous avons découvert que les 4E-BP et les S6K jouent des rôlesdistinct en aval de mTORC1, les 4E-BP étant principalement impliquées dans laprolifération cellulaire, alors que les S6K jouent un rôle dans le contrôle de la croissancecellulaire. Les effets des inhibiteurs spécifiques au site actif de mTOR sur la proliférationcellulaire, la progression à travers le cycle cellulaire et la synthèse protéique sontfortement atténués dans les cellules nulles pour les 4E-BP, alors qu'ils sont inhibés demanière importante dans les cellules de type sauvage. Cependant, la croissance descellules 4E-BP nulles, en présence des inhibiteurs spécifiques au site actif de mTOR, estla même que celle observée pour les cellules de type sauvage. À l'opposé, la croissancedes cellules S6K nulles n'est pas affectée par ces inhibiteurs, tandis que les inhibiteursconservent leurs effets négatifs sur la prolifération et la progression au travers du cyclecellulaire dans ces mêmes cellules. Ces résultats sont en accord avec un modèle danslequel la signalisation en aval de mTORC1, pour promouvoir la prolifération et lacroissance cellulair

Tumor suppressor p53 is a mediator of stress-induced cell cycle arrest and apoptosis. The kinase inhibitor 2-aminopurine (2-AP) is an adenine analog shown to cause cells to bypass DNA damage-induced cellular arrest through unknown mechanisms, and may potentially target p53. Although p53 plays vital roles in adaptation to many stresses, its role in cellular response to endoplasmic reticulum (ER) stress is unclear. Here, stress-induced p53 stabilization and checkpoint control in the presence of 2-AP are examined, as well as p53 regulation upon ER stress induction. I show that 2-aminopurine suppresses p53 stabilization in response to different forms of DNA damage. Biologically, 2-AP exposure enables cells to bypass adriamycin-induced G2/M arrest in a p53-dependent manner, but rescues the clonogenic survival of cells exposed to adriamycin in a p53-independent manner. Next, I show that pharmacological and physiological inducers of ER stress can inhibit stress-induced p53 function by promoting p53 cytoplasmic retention.

The HIV-1 protein Nef is a critical factor in the viral pathogenesis and decline of CD4 T-cells during HIV infection. Nef has been implicated in modulating several cellular pathways, including apoptosis. This thesis herein describes our attempt to elucidate the mechanism by which Nef modulates apoptosis in T-cells. Using Jurkat cells inducibly expressing wild-type Nef, we observe that Nef renders cells more sensitive to apoptosis upon cross-linking with anti-Fas or TRAIL. Enhancement of Fas-mediated apoptosis required the presence of Lck, as apoptosis was abrogated in Nef expressing Lck-/- cells as compared to wild type Jurkat cells. Nef does not modulate expression of pro or anti-apoptotic proteins, or cell surface Fas receptor. Furthermore, Nef differentially mediates activation signaling pathways upon anti-CD3 stimulation. Enhancement of apoptosis by Nef may represent one of the mechanisms by which HIV depletes CD4 T-cells during infection.

Netrins are a small family of secreted proteins that function as chemotropic cues directing cell and axon migration during neural development. They are bifunctional molecules attracting and repelling different classes of axons. DCC (deleted in colorectal cancer) is a transmembrane receptor for netrin-1 implicated in mediating both responses. The intracellular mechanisms mediating the response of an axon to netrin-1 are currently unclear. Previous studies indicated that extracellular guidance cues induce the neuronal growth cone to advance, retract, or turn by regulating the actin cytoskeleton within the growth cone. The Rho family GTPases, in particular, RhoA, Rac1 and Cdc42, are well-described regulators of actin reorganization in non-neuronal cells, and there is now compelling evidence implicating a role for them as signaling components within the neuronal growth cone. / In the first part of this thesis, we have demonstrated that the Rho GTPases are required for embryonic spinal commissural axon outgrowth induced by netrin-1. Using NIE-115 neuroblastoma cells we found that both Rac1 and Cdc42 activities are required for DCC-induced neurite outgrowth. In Swiss 3T3 fibroblasts, DCC was found to trigger actin reorganization through activation of Rac1. These results implicate the small GTPases as important signaling components in the molecular mechanisms underlying DCC. / In the second part, we found that DCC interacts constitutively with the adaptor protein Nck in commissural neurons. Moreover, dominant negative Nck-1 inhibits the ability of DCC to induce neurite outgrowth in NIE-115 cells and to activate Rac1 in fibroblasts in response to netrin-1. These studies provide evidence for an important role of Nck-1 in a novel signaling pathway from an extracellular guidance cue to changes in the actin-based cytoskeleton responsible for axonal guidance. / In the last part, we found that disruption of each of the PxxP motifs in the cytoplasmic domain of DCC is not able to block the interaction of DCC with Nck-1, suggesting that more than one PxxP motifs or non-PxxP sequences may mediate the interaction of DCC with Nck-1. / Taken together, the data in this thesis contribute to our understanding of the intracellular mechanisms mediating the response of an axon to netrin-1 during neural development.

The tyrosine kinase receptor HER2/neu (ErbB2) plays fundamental roles in the development, proliferation and differentiation processes. Overexpression of ErbB2 is present in 25%--30% of breast cancers and is associated with poor prognosis. Trastuzumab (Herceptin), a humanized anti-ErbB2 antibody used to treat ErbB2-overexpressing breast cancer, is often cited as a prototype for rationally-designed anti-neoplastic drugs targeting critical signal transduction pathways. However, development of resistance to trastuzumab is common, and the duration of the clinical response rarely exceeds 12 months. The insulin-like growth-I receptor (IGF-IR) is a transmembrane tyrosine kinase receptor activated by binding of the IGF ligands. The IGF-IR signaling provokes mitogenic and antiapoptotic effects in a number of normal and transformed cell types. IGF-I receptors and ErbB2 receptors share several common signaling transduction pathways, which raises the possibility of interaction between two signaling pathways on modulation of cell proliferation, survival and differentiation. This thesis provides evidence that cross-talk exists between the IGF-IR and ErbB2 signaling pathways and this is associated with trastuzumab resistance. We show here that MCF7/HER2--18 cells which overexpress ErbB2 receptors and express IGF-IR are responsive to trastuzumab treatment only when IGF-IR signaling is minimized. SKBR3 cells genetically altered to overexpress IGF-IR lose sensitivity to trastuzumab treatment in the presence of IGF-I. In the second part of this thesis, we examine the mechanisms by which IGF-IR activation antagonizes the effects of trastuzumab on cell cycle regulation. We show that IGF-I reproducibly reduces the trastuzumab-induced increase of p27 Kip1, decreases association of p27Kip1 with CDK2, and dramatically increases CDK2 activity in IGF-IR-overexpressing SKBR3 cells. In the last part of this thesis, a trastuzumab-resistant SKBR3 cell line was establishe

The morphogenetic plasticity of adult pancreatic islets of Langerhans has been implicated in the development of pancreatic adenocarcinomas and in islet transplant failure. The objective of this doctoral work was to investigate the extent of this plasticity and to characterize the unknown cell types and intracellular mechanisms involved in changes of adult islet phenotype. / In the first published study, isolated adult canine islets were induced to undergo a phenotypic switch into highly proliferative duct-like structures through a two-stage process entailing beta-cell death and the dedifferentiation of the resulting cells. The transformed islets were no longer immunoreactive for islet cell hormones, but now expressed markers of pancreatic duct epithelial cells. Pharmacologic inhibition of signal transduction demonstrated that the balance in signalling activity between ERK/Akt and JNK/caspase-3 appears to be an important regulator of islet cell death and differentiation. / In the second published study, quiescent adult human islets were induced to undergo a similar phenotypic switch into highly proliferative duct-like structures in a process that implicated glucagon- and somatostatin-expressing cells, and was characterized by a loss of expression of islet-specific hormones and transcription factors as well as a temporally-related rise in expression of markers of stermness and duct epithelium. Short-term treatment of these primitive duct-like structures with the islet neogenic factor INGAP 104-118 induced their scalable reversion back to islet-like structures in a P13-kinase-dependent manner. These neoislets resembled freshly isolated human islets with respect to the presence and topological arrangement of the four endocrine cell-types, islet gene expression and hormone production, insulin content and glucose-responsive insulin secretion. The demonstration that adult human islets are able to regenerate themselves establishes a new paradigm in the context of tissue regeneration and diabetes therapy. / These original findings may have important clinical implications for understanding and controlling pancreatic carcinogenesis and islet neogenesis in the adult human pancreas.

Translation is the vital process by which the information contained in messenger RNAs (mRNA) is used to synthesise proteins. This is a highly regulated process that involves a complex machinery and tight control at every step. In eukaryotes, translation initiation is the rate-limiting step and the most tightly controlled. The translation of an mRNA is preceded by multiple post-transcription steps including, splicing, export and chemical modification including the addition of a 5'-end cap structure. The initiation factor eIF4E binds to this cap structure while in the nucleus, and initiates the translation by recruiting the ribosome after export to the cytoplasm. Controlling the availability of eIF4E within the various compartments of the cell has a direct effect on the efficiency of translation initiation and indirectly on the rate of proliferation of the cell. On the other hand, disturbing the level of eIF4E synthesis could lead to various pathologies especially tumour development. eIF4E requires multiple binding partners to fulfill its mission. The factor 4E-T is involved in eIF4E transport, while eIF4G enhances the binding of eIF4E to the cap structure. eIF4E can be inactivated upon sequestration by the 4E Binding Protein (4E-BP) who competes with eIF4G for binding of eIF4E. / The localization of eIF4E inside the cell is clearly critical for its normal function. It is known that many external stresses can influence cellular translation and this prompted us to investigate the effect of such stresses on the cellular localization of eIF4E and to study the role of eIF4E-binding partners under these conditions. The present work demonstrates that, during heat shock and oxidative stress, 4E-BP plays an essential role in controlling the localization of eIF4E to the stress response cytoplasmic foci, known as the stress granules (SGs). In addition, eIF4E is partially retained in the nucleus during heat shock but not in oxidative stress. These observations suggest that upon stress the cellular translation mechanism is delayed or even stopped by reducing the availability of eIF4E to the translation complex. On the other hand, we show that eIF4E nuclear translocation upon poliovirus infection is correlated with eIF4G cleavage. This translocation could favour the shutdown of host cell protein synthesis by reducing the cap dependent translation and preventing mRNA circularization. / In our study, we focused on the role of eIF4E as a key player for cellular survival under stressful conditions. Therefore, identifying reagents that induce the relocalization of eIF4E to the nucleus or to SGs could help in the development of anti-proliferative drugs. / La traduction est un processus vital par lequel l'information contenue dans l'ARN messager est utilisée pour la synthèse protéique. Chaque étape de ce procédé est strictement régulé grâce à l'implication d'une machinerie complexe hautement contrôlée. Chez les eucaryotes, l'initiation est l'étape limitante et la mieux contrôlée du processus de traduction. Surviennent ensuite, les étapes dites post-traductionnelles qui incluent, l'épissage, l'exportation nucléaire et les modifications biochimiques. Le facteur de transcription eIF4E lie la coiffe du messager en 5' au niveau du noyau, permettant son exportation au cytoplasme et l'initiation de la traduction grâce au recrutement des sous-unites ribosomales. Le contrôle d'eIF4E dans les différentes parties cellulaires est crucial pour l'efficacité traductionnelle, mais génère également un effet indirect sur la prolifération et division cellulaire. Aussi, un défaut de synthèse ou de niveau d'expression d'eIF4E amènent de nombreuses anomalies, spécifiquement lors du développement tumoral. eIF4E requiert de nombreux partenaires pour agir efficacement. Le facteur 4E-T est impliqué dans le transport d'eIF4E tandis que eIF4G favorise sa liaison a la coiffe. eIF4E est connu pour être inactive par séquestration de 4E-BP, principal compétiteur pour la liaison a la coiffe. / La localisation d'eIF4E au sein de la cellule est critique pour son bon fonctionnement. De nombreux facteurs de stress sont connus pour influencer la traduction cellulaire. Je me suis donc concentre à étudier les effets des facteurs de stress dans la localisation cellulaire d'eIF4E et le rôle respectif de chacun de ses partenaires dans ces conditions. J'ai ainsi démontré que durant un choc de chaleur et un stress oxydatif, 4E-BP joue un rôle essentiel dans le contrôle de la localisation d'eIF4E au niveau des granules de stress. De plus, eIF4E est partiellement retenu au noyau lors d'un stress de chaleur mais pas au cours d'un stress oxydatif. Ces observations suggèrent que lors d'un stress, la machinerie traductionnelle est retardée ou arrêtée par une baisse de la disponibilité d'eIF4E a ce complexe protéique. D'un autre coté, j'ai montré que la translocation nucléaire d'eIF4E au cours d'une infection au Poliovirus est corrélée avec le clivage d'eIF4G. Cette translocation pourrait favoriser l'arrêt de la synthèse protéique de la cellule hote en réduisant la traduction dépendant de la coiffe et en prévenant la circularisation du messager. / En conclusion, eIF4E est un élément clé dans la survie cellulaire associée aux conditions de stress. De plus, l'identification de réactifs pouvant induire la relocalisation d'eIF4E au noyau ou aux granules de stress aiderait au développement de composes anti-prolifératifs.