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Hop/Stip1/Sti1 is thought to be essential as a co-chaperone to facilitate substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Despite this proposed key function for protein folding and maturation, it is not essential in a number of eukaryotes and bacteria lack an ortholog. We set out to identify and to characterize its eukaryote-specific function. Human cell lines and the budding yeast with deletions of the Hop/Sti1 gene display reduced proteasome activity due to inefficient capping of the core particle with regulatory particles. Unexpectedly, knock-out cells are more proficient at preventing protein aggregation and at promoting protein refolding. Without the restraint by Hop, a more efficient folding activity of the prokaryote-like Hsp70-Hsp90 complex, which can also be demonstrated in vitro, compensates for the proteasomal defect and ensures the proteostatic equilibrium. Thus, cells may act on the level and/or activity of Hop to shift the proteostatic balance between folding and degradation., Hop, also known as Stip1 or Sti1, facilitates substrate transfer between the Hsp70 and Hsp90 molecular chaperones. Characterization of proteostasis-related pathways in STIP1 knock-out cell lines reveals that in eukaryotes Stip1 modulates the balance between protein folding and degradation.

The recombinant antibody H90-10 detects the endogenous human heat-shock protein 90 beta (Hsp90β) by immunoprecipitation (IP) and Western blotting.

miR-22 is one of the most abundant miRNAs in the liver and alterations of its hepatic expression have been associated with the development of hepatic steatosis and insulin resistance, as well as cancer. However, the pathophysiological roles of miR-22-3p in the deregulated hepatic metabolism with obesity and cancer remains poorly characterized. Herein, we observed that alterations of hepatic miR-22-3p expression with non-alcoholic fatty liver disease (NAFLD) in the context of obesity are not consistent in various human cohorts and animal models in contrast to the well-characterized miR-22-3p downregulation observed in hepatic cancers. To unravel the role of miR-22 in obesity-associated NAFLD, we generated constitutive Mir22 knockout (miR-22KO) mice, which were subsequently rendered obese by feeding with fat-enriched diet. Functional NAFLD- and obesity-associated metabolic parameters were then analyzed. Insights about the role of miR-22 in NAFLD associated with obesity were further obtained through an unbiased proteomic analysis of miR-22KO livers from obese mice. Metabolic processes governed by miR-22 were finally investigated in hepatic transformed cancer cells. Deletion of Mir22 was asymptomatic when mice were bred under standard conditions, except for an onset of glucose intolerance. However, when challenged with a high fat-containing diet, Mir22 deficiency dramatically exacerbated fat mass gain, hepatomegaly, and liver steatosis in mice. Analyses of explanted white adipose tissue revealed increased lipid synthesis, whereas mass spectrometry analysis of the liver proteome indicated that Mir22 deletion promotes hepatic upregulation of key enzymes in glycolysis and lipid uptake. Surprisingly, expression of miR-22-3p in Huh7 hepatic cancer cells triggers, in contrast to our in vivo observations, a clear induction of a Warburg effect with an increased glycolysis and an inhibited mitochondrial respiration. Together, our study indicates that miR-22-3p is a master regulator of the lipid and glucose metabolism with differential effects in specific organs and in transformed hepatic cancer cells, as compared to non-tumoral tissue.

Background The molecular chaperone TRAP1, the mitochondrial isoform of cytosolic HSP90, remains poorly understood with respect to its pivotal role in the regulation of mitochondrial metabolism. Most studies have found it to be an inhibitor of mitochondrial oxidative phosphorylation (OXPHOS) and an inducer of the Warburg phenotype of cancer cells. However, others have reported the opposite, and there is no consensus on the relevant TRAP1 interactors. This calls for a more comprehensive analysis of the TRAP1 interactome and of how TRAP1 and mitochondrial metabolism mutually affect each other. Results We show that the disruption of the gene for TRAP1 in a panel of cell lines dysregulates OXPHOS by a metabolic rewiring that induces the anaplerotic utilization of glutamine metabolism to replenish TCA cycle intermediates. Restoration of wild-type levels of OXPHOS requires full-length TRAP1. Whereas the TRAP1 ATPase activity is dispensable for this function, it modulates the interactions of TRAP1 with various mitochondrial proteins. Quantitatively by far, the major interactors of TRAP1 are the mitochondrial chaperones mtHSP70 and HSP60. However, we find that the most stable stoichiometric TRAP1 complex is a TRAP1 tetramer, whose levels change in response to both a decline and an increase in OXPHOS. Conclusions Our work provides a roadmap for further investigations of how TRAP1 and its interactors such as the ATP synthase regulate cellular energy metabolism. Our results highlight that TRAP1 function in metabolism and cancer cannot be understood without a focus on TRAP1 tetramers as potentially the most relevant functional entity.

The recombinant antibodies AF291and AE391 against the standard HA tag detect an HA-tagged protein exogenously expressed in human cells by Western blotting. However, similarly to one of the original monoclonal antibodies, they also recognize a number of unrelated endogenous proteins.

Complex patterns of protein-protein interactions (PPInts) are involved in almost all cellular processes. This has stimulated the development of a wide range of methods to characterize PPInts in detail. Methods with fluorescence resonance energy transfer can be technically challenging and suffer from several limitations, which could be overcome by switching to luminescence resonance energy transfer (LRET) with lanthanide ions such as Tb3+. With LRET, energy transfer between PPInt partners works over a larger distance and with less topological constraints; moreover, the long-lived luminescence of lanthanides allows one to bypass the short-lived background fluorescence. We have developed a novel LRET method to investigate PPInts between partners expressed as fusion proteins with genetically encoded donor and acceptor moieties. Upon UV excitation of a tryptophan within a lanthanide binding peptide, the Tb3+ luminescence is harnessed to excite either a green or a red fluorescent protein. We demonstrate the usefulness of the LRET assay by applying it to analyze the interactions of the molecular chaperones HSP70 and HSP90 with their common co-chaperone HOP/Sti1. We recapitulate the previously described interaction specificities between the HSP70/HSP90 C-termini and tetratricopeptide repeat domains of HOP/Sti1 and demonstrate the impact of single point mutants on domain-domain interactions.

Dendrogenin A (DDA) is a newly discovered cholesterol metabolite with tumor suppressor properties. Here, we explored its efficacy and mechanism of cell death in melanoma and acute myeloid leukemia (AML). We found that DDA induced lethal autophagy in vitro and in vivo, including primary AML patient samples, independently of melanoma Braf status or AML molecular and cytogenetic classifications. DDA is a partial agonist on liver-X-receptor (LXR) increasing Nur77, Nor1, and LC3 expression leading to autolysosome formation. Moreover, DDA inhibited the cholesterol biosynthesizing enzyme 3β-hydroxysterol-Δ8,7-isomerase (D8D7I) leading to sterol accumulation and cooperating in autophagy induction. This mechanism of death was not observed with other LXR ligands or D8D7I inhibitors establishing DDA selectivity. The potent anti-tumor activity of DDA, its original mechanism of action and its low toxicity support its clinical evaluation. More generally, this study reveals that DDA can direct control a nuclear receptor to trigger lethal autophagy in cancers., Dendrogenin A, cholesterol metabolite, has tumor suppressive properties but the mechanisms are unknown. Here the authors show that Dendrogenin A can induce autophagy-mediated cell death in both melanoma and acute myeloid leukaemia.

Phytoestrogens are a chemically diverse group of compounds made by plants that can have estrogenic effects in animals. Both tumorigenic and antitumorigenic effects have been reported. Although estrogens stimulate the growth of many breast tumors, there is a negative correlation between the incidence of breast cancer and the phytoestrogen-rich diet of certain Asian populations. To begin to resolve this paradox, we have analyzed the estrogenic properties of genistein and quercetin, two flavonoid phytoestrogens particularly abundant in soybeans. Trans-activation experiments with a transfected reporter gene for nuclear estrogen receptors (ER) show strong activation of the endogenous ER alpha by both phytoestrogens in two MCF7 human breast cancer cell lines. This is supported by the observation that the two phytoestrogens induce the down-regulation of ER alpha mRNA and protein levels. Using chimeric proteins consisting of the hormone binding domains of ER alpha and ER beta fused to the Gal4 DNA binding domain, we have established that genistein and quercetin are full estrogenic agonists of both ER isoforms. Ligand binding experiments with purified ER alpha and ER beta confirm that the two phytoestrogens are ER ligands. At concentrations that are sufficient to obtain substantial transcriptional activity, they stimulate the proliferation of two ER alpha-dependent breast cancer cell lines. At high concentrations, such as those reached with a soy-rich diet, genistein and quercetin are strong cytotoxic agents that even kill ER-independent HeLa cells. Thus, the mode of action of phytoestrogens and the balance between being risk or chemopreventive factors for breast cancer may depend on the dietary load.

The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation. Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae. Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation. Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

Cancer is the number one cause of mortality worldwide. Breast cancer is the most common type of cancer diagnosed in both sexes and the major cause of cancer-related mortality in women. The vast majority of breast tumors (~70%) express estrogen receptor α (ERα) and depend on estrogen for their growth. ERα is a hormone receptor and when it binds to its ligand (estrogen), it can promote tumor progression as a transcription factor. ERα-mediated growth is also possible in the absence of estrogens, through direct or indirect activation of ERα by kinases such as the mitogen-activated protein kinases (MAPKs) ERK1/2. Owing to the strong dependency on the estrogen-ERα axis, targeting ERα with antagonists, for example tamoxifen or fulvestrant, or inhibiting the production of estrogens with aromatase inhibitors have been therapeutic approaches for ERα+ breast tumors for decades. Tamoxifen has now been successfully used in the clinical setting for over 40 years to treat ERα+ tumors. However, around one third of the patients treated with tamoxifen ultimately develop resistance and face recurrence of the disease. In order to overcome this challenge, it is very important for new biomarkers and potent therapeutic targets to emerge for the early detection of tamoxifen-resistance and ultimately for most efficient treatment. For this reason, a whole-genome CRISPR/Cas9 knockout screen was conducted in our laboratory to identify possible new biomarkers of tamoxifen resistance as well as novel therapeutic targets for breast cancer. Seven genes, namely FIBP, STK11, PTPN12, SPRED2, UBE2R2, CUX1 and LZTR1, whose depletion yielded a similar profile of tamoxifen- and fulvestrant-resistant phenotypes, were chosen for further validation in this project. Later on, we decided to focus on SPRED2 for a more in-depth investigation of the underlying molecular mechanisms that might confer tamoxifen resistance after its depletion. We found that SPRED2 deficiency leads to hyperactivation of ERK1/2, which in turn leads to an increase in ERα activity and eventually resistance to tamoxifen. In this study, we propose a therapeutic regimen of ERK1/2 inhibitor, ulixertinib, in combination with tamoxifen for the treatment of those ERα+ breast cancers characterized by low expression levels of SPRED2. Identification of SPRED2 as novel biomarker of tamoxifen resistance brings us closer to personalized medicine, where treatments are chosen based on the genetic signature of each individual patient.

Heat shock protein (Hsp) 70 and Hsp90 are an evolutionarily conserved class of molecular chaperones and are major contributors to the maintenance of protein homeostasis. They fold nascent and misfolded proteins called “clients” and they collaborate with other proteins, called co-chaperones. One of these co-chaperones is the Hsp70-Hsp90 organizing protein (Hop) which is only found in eukaryotic cells. Hop interacts with Hsp70 and Hsp90 to form the Hsp70-Hop-Hsp90 ternary complex. An upregulation or overexpression of Hop is positively correlated with several cancers. In this work, we investigated whether Hop is an oncoprotein. We could not verify nor disprove an implication of Hop in cancer in isogenic pairs of normal and oncogenically transformed RPE1 cells by Hop knock-down (KD). Also, our preliminary results showed that Hop KD in normal RPE1 cells renders them resistant to Hsp90 inhibition whereas this is not the case for transformed RPE1 cells. Hop is also prone to post-translational modifications such as phosphorylation. Here we showed that Hop mutants with the phosphomimic (Y354E) and the non-phosphorylatable (Y354F) form of tyrosine 354 (Y354) exhibit weaker interactions with Hsp70 and Hsp90 isoforms compared to Hop WT. Even though we could not identify the kinase responsible for the phosphorylation of Y354, we showed that the association of the Hsp90 client and tyrosine kinase c-Abl with the ternary complex is increased in presence of Y354E Hop and Y354F Hop and that its activation is halved in presence of Y354E Hop. Taken together, our data suggest that substitutions of Y354 may lead to a slowdown in the transfer and the activation of c-Abl.

Metastasis is one of the major causes of endocrine resistance and mortality in breast cancer patients. The epithelial to mesenchymal transition (EMT) enables the metastatic spread of cancer cells. EMT-promoting transcription factors determine a continuum of different EMT states. In contrast, estrogen receptor α (ERα) contributes to the maintenance of the epithelial phenotype of breast cancer cells and is crucial for effective hormonal therapies. Determining whether and how the EMT modulates ERα signaling at early stage of EMT could drive the discovery of novel therapeutic approaches to prevent or to fight metastasis. We have discovered that during EMT initiation the expression of ZEB1 modulates ERα-mediated transcription induced by estrogen or cAMP signaling in breast cancer cells. We discovered that a cooperative interaction between ZEB1 and ERα is important for modulating tissue tropism of breast cancer cells towards bone metastasis.

Hematologic malignancies are the most frequent cancers in the pediatric population. Despite the high cure rate, treatments are highly toxic on the long-term basis, damaging different organ systems. In addition, medications do not display the same efficacy in every patient. Some of them do not respond at all, leading to possible therapeutic failure. Improving quality of life of the patients by reducing treatment-related toxicities (TRTs) and increasing the effectiveness of already existing drugs is an important field of research that requires special attention. Busulfan is one of the alkylating agents used in the myeloablative conditioning regimen prior to hematopoietic stem-cell transplantation (HSCT), a medical procedure to treat blood cancers. The enzymatic family of glutathione S-transferases (GSTs) is responsible for the metabolism of this medication and other electrophilic chemotherapeutic drugs. As reported in the literature, there is an association between genotypes of GSTs subtypes and TRTs after infusion with electrophilic agents. In addition, an overexpression of GSTs in tumor tissues is associated with chemotherapeutic drug resistance. Besides their role as detoxifier, GSTs regulate mitogen-activated phosphate kinase (MAPK) pathways. Considering these two roles, the main biological hypothesis is that the use of a drug combination, constituted of Busulfan and a GST inhibitor will increase the efficacy of the alkylating agent.

Les vésicules extracellulaires (VE), étant issues de tout type cellulaire et retrouvées dans les liquides biologiques circulants, peuvent représenter une source prometteuse de biomarqueur non-invasif. Nous avons quantifié des VE provenant du plasma de patients atteints de leucémie myéloïde aiguë (LMA), afin de les comparer aux VE provenant du plasma de donneurs sains, en utilisant la cytométrie en flux basée sur la détection de fluorescence. Nous avons expérimenté l'utilisation potentielle du CD81 en tant que marqueur pan-vésiculaire, néanmoins, les concentrations en VE CD81+ mesurées chez les patients LMA et chez les donneurs sains sont variables et s'avèrent être influencées par l'anticoagulant. Nous avons ensuite exploré l'utilisation potentielle du CD34 en tant que marqueur des VE provenant des blastes LMA. Une corrélation a été constaté entre la concentration en globules blancs et la concentration en VE CD34+ chez les patients LMA. Nos résultats nous permettent d'envisager l'utilisation des VE CD34+ comme biomarqueur non-invasif pour le diagnostic et le suivi des patients LMA.

The molecular chaperone TRAP1 has been studied for almost two decades, yet, the mechanistic basis underlying its function in the mitochondria still remains poorly understood. Recent studies have presented it as an inhibitor of OXPHOS and an inducer of the Warburg phenotype, but multiple reports have conflicted with this paradigm. This study seeks to reconcile previous reports in a cohesive model by proposing that TRAP1 does not necessarily down or upregulate OXPHOS, but, is actually required to maintain OXPHOS homeostasis. Using multiple CRISPR KO cell lines, real time OCR analyses with different carbon sources, metabolomics and various proteomic approaches to understand interactions, this study provides a platform to understand TRAP1 function in mitochondria. We show that TRAP1 induces a metabolic rewiring independent of its ATPase activity, requires an ATP-bound state to interact with various mitochondrial proteins and forms an oligomerized protein complex that may be the functional unit of TRAP1.

Following the identification of two new KIAA2022 variants in two subjects with non-autoimmune diabetes. In order to determine if Kiaa2022 could play a role in diabetes we performed in vitro studies in insulin-secreting INS-1E cells. Additionally, we generated Kiaa2022 knock out mice, which showed a reduced amount of proliferating β-cells. Interestingly, besides a pancreatic phenotype a variety of other phenotypes was observed in mutant and non-mutant progeny. RNAseq showed that down-regulated genes in Kiaa2022 mutant islets are associated to stress response and the disposition of epigenetic marks. We also observed an upregulation of LINE1 elements and not only in the pancreas, but also in the brain and testis. Activated LINE1 elements can cause genomic instability and genetic variation through retrotransposition into the genome. This suggests a mechanism by which genomic instability could be induced in Kiaa2022 mutant mice, which could cause phenotypic variability in all progeny.

Molecular chaperones are abundant and essential molecular machines that support cellular proteostasis through the folding and structural maintanance of proteins. The heat-shock protein 90 family of proteins is an evolutionarily conserved class of molecular chaperones, important in the structural maintenance of hundreds of substrate proteins. In this work, we have investigated the nuclear localization of nucleocytoplasmic Hsp90 isoforms and the function of the mitochondrial isoform TRAP1 as a molecular chaperone.

Nos résultats confirment que l'activité transcriptionnelle de ERα dépend de la phosphorylation de CREB1 par la PKA active, et de plus, nous montrons que l'activation de ERα peut varier en fonction du stimulus de déclenchement. En outre, nous montrons qu'en fonction du stimulus, deux protéines, les protéines de choc thermique 90 β (HSP90β) et l'arginine méthyltransférase associée au coactivateur (CARM1) peuvent participer de différentes manières à l'activité du complexe nucléocapsidique ERα-pCREB1 et que leur l'activité dépend du recrutement des gènes cibles pCREB1 à ERa. Dans le contexte clinique, la synergie entre CREB1 activé et ERα est cruciale pour la survie des tumeurs mammaires. Les deux facteurs de transcription activés sont inhibiteurs de l'apoptose, principalement en contrôlant la perméabilité de la membrane mitochondriale et en orchestrant la localisation et l'activation subséquente des protéines anti-pro-apoptotiques.

Cancer is regarded by many as the “disease of the century” and, as such, sparked more and more interest among researchers. Solid evidence indicates that one of the core features of cancer as a whole, is the ability of cancer cells to modify their metabolism and reshape their environment. As our understanding of cancer metabolism and how it affects surrounding cells grows, more and more metabolic pathways are regarded as potential targets in immunotherapies as they affect components of the immune system. L-arginine metabolism is one of these pathways; arginine is a semi-essential amino acid, it is metabolised by two competing enzymes, arginase and nitric oxide synthase and is involved in numerous cellular function including immunity. For decades, evidence has been gathered regarding the involvement of arginase 1 (Arg1), one of the two isoforms of arginase, in the impairment of immune functions. Moreover, both isoenzymes have been identified as upregulated in numerous cancer types. While most of the focus has been put on Arg1 due to it being the most expressed of the two in numerous immune cells, arginase 2 (Arg2) has been found to be a key player of T-cell function and the T-cell anti-tumour response. Here, we show, using flow cytometry, qPCR and ELISA, that deleting Arg2 in mice has an effect on multiple aspects of T-cell biology. We observed differences in activation kinetics in both CD8+ and CD4+ T-cells indicating that Arg2-/- T-cells are activated faster than WT T-cells. Additionally, we observed that Arg2-/- CD8+ T-cells shed CD62L faster and produce more IFNγ but do not produce more perforin compared to WT CD8+ T-cells. Our results also show that, while there is no difference in proliferation in the CD8+ lineage, Arg2-/- CD4+ T-cells proliferate more and produce more IL-2 than WT CD4+ T-cells. Finally, we also unveiled a link between L-arginine concentration and the mitochondrial mass of CD8+ T-cells. Globally, the results of this work show that the deletion of Arg2 allows for a better T-cell response in line with previous observations that Arg2 acts as an immune brake in the context of T-cell mediated immune responses.

The availability of a wide range of reporter proteins, which can easily be quantitated, has had a major impact on many fields of biomedical research. In some experiments with tissue culture cells, it is necessary to control for differences in transfection efficiency and in other expression parameters. This requirement has been very conveniently met with the popular dual luciferase assay. Its disadvantages are the requirement for cell lysis, the inability to analyze the same cells repeatedly, and the cost, at least in its most commonly used commercial format. Here we describe a novel dual reporter assay with the naturally secreted luciferase from Gaussia princeps as the main reporter protein and a secreted version of the red fluorescent protein mCherry as internal standard. After first measuring mCherry fluorescence in the medium, an enzyme buffer with coelenterazine as substrate is added to the same sample to trigger a glow-type luminescence of the luciferase. The simple and cheap assay can easily be adapted to a variety of experimental situations. As a case in point, we have developed a panel of Gaussia luciferase reporter genes for transcriptional activation assays with estrogen and glucocorticoid response elements, and with response elements for fusion proteins with the Gal4 DNA binding domain for use in mammalian cells. Our secreted dual reporter assay should be an attractive alternative to the currently available commercial kits.

Au cours de ma thèse, j'ai étudié la famille des protéines TM9, d'abord chez Dictyostelium discoideum puis chez des cellules de mammifère. Nos résultats montrent que Phg1a, l'un des trois membres de la famille TM9 chez Dictyostelium transporte un ensemble de domaines transmembranaires (TMDs) hors du reticulum endoplasmique, et permet leur expression à la surface des cellules. Nous avons démontré que Phg1a s'associe de façon spécifique avec des TMDs riches en glycine, via des interactions intra- membranaires. Il existe quatre protéines TM9 chez les mammifères. TM9SF4, le plus proche orthologue humain de Phg1a, contrôle également le transport vers la surface de TMDs riches en glycine. Mes résultats plus récents ont montré que TM9SF4 interagit également avec des TMDs contenant un résidu chargé, et permet leur relocalisation du reticulum endoplasmique dans l'appareil de Golgi.

CD47 is a ubiquitously expressed immune checkpoint receptor playing a major role in recognition of self by phagocytes. CD47 interacts with SIRPα to generate an inhibitory signal blocking phagocytosis. Tumor cells have hijacked this immunosuppressive mechanism by overexpressing CD47, which efficiently protects them against attack by the body's immune system. Targeting CD47, with a blocking monoclonal antibody (mAb), promotes tumor elimination via antibody dependent cellular phagocytosis (ADCP) and antibody dependent cellular cytotoxicity (ADCC). In several in vivo models, blocking CD47 leads to tumor elimination, inhibits the development of metastasis and promotes the development of an antitumor immunological memory. Several anti-CD47 mAb are under clinical investigation for cancer treatment. However, the development of these antibodies is confronted with several hurdles. Due to the expression of CD47 also on healthy cells, mAb based therapies lead to toxicity, including severe anemia and thrombocytopenia, as well as rapid drug elimination. As a solution, we hypothesized that using not a mAb but a bispecific Ab format (biAb) pairing a high affinity tumor associated antigen (TAA) arm with a CD47 targeting arm of lower affinity could avoid these unwanted characteristics. Such a biAb with unbalanced affinities would allow to selectively target CD47 to the tumor cells. Both anti-human and anti-mouse CD47/TAA biAb were generated using the κλ body format, a fully human biAb composed of a common IgG heavy chain and two different light chains (i.e., kappa and lambda). We exploited light chain diversity to identify the TAA and CD47 targeting arms composing the biAbs. The resulting biAbs efficiently neutralized CD47 on TAA positive tumor cells, but interacted only weakly with healthy cells. Accordingly, the biAbs were not subject to CD47- mediated drug disposition and were well tolerated in vivo. The selection of a low affinity anti- CD47 arm was also primordial for biAb anti-tumor activity in a context of CD47 ubiquitous expression. The combination of targeted CD47 neutralization and Fc effector functions afforded by the biAb format resulted in potent killing by ADCP and ADCC in vitro, and translated into a potent anti-tumor activity in both xenograft and syngeneic in vivo models. In immunocompetent mouse models, biAb-mediated blockade of CD47 lead to tumor cell phagocytosis and the development of a long term immunological memory in vivo as mice were protected from tumor rechallenge. In vitro, we demonstrated that indeed cross-priming of anti-tumor CD8+ T cells had occurred. Taken together, my work confirmed that targeting CD47 is a powerful way to engage the innate and adaptive immune system to kill cancer. Thus, dual-targeting biAbs with unbalanced affinities are an elegant means to employ this strategy effectively yet safely in patients. With a better risk to benefit ratios, the biAb approach to CD47 targeting can be envisioned as part of combination therapies, for example synergizing with cyclophosphamide or anti-PD-L1 therapies. These studies also suggest that a similar bispecific design could be exploited for the targeting of other widely expressed receptors.

T cell lymphocytes play an essential role in the adaptive immunity. They arise from the hematopoietic compartment and reach the thymus, where they achieve their development through specialized maturation and selection steps. Immune system comprises specific and complex mechanisms in the thymus and the periphery aiming at preventing the generation and the survival of self-reactive T cells that could lead to autoimmune disorders. These mechanisms are known as central and peripheral tolerance and rely on different cellular actors such as medullary thymic epithelial cells (mTECs), dendritic cells (DCs), lymph node stromal cells (LNSCs) and regulatory T and B cells. Self-reactive T cells escaping thymic central tolerance are kept in check in the periphery through specific mechanisms that include T cell anergy, T cell deletion and Treg induction. Although conventional DCs (cDCs) have been first in line in mediating peripheral tolerance, increasing evidence demonstrates the contribution of other actors such as plasmacytoid dendritic cells (pDCs) in this process. pDCs are important linkers of the innate and adaptive immunity. They are characterized by their ability to secrete pro-inflammatory cytokines and large amount of type I interferon (IFN-I) upon pathogenic infections, but also express major Histocompatibility complex (MHC) and costimulatory molecules enabling them to interact with T cells. pDCs have been involved in the control of infections, but are also important actors of peripheral tolerance. The diversity of their functions, depending on the context in which they will evolve, has associated pDCs to pro-immunogenic and tolerogenic scopes. In the lab, we are principally interested in the contribution of pDCs in Multiple sclerosis, a progressive inflammatory demyelinating disease of the central nervous system (CNS). To address the role of pDCs in MS, we performed our studies in the murine model of Experimental autoimmune encephalomyelitis (EAE). Using genetically modified mice harbouring specific abrogation of Ag-presenting function in pDCs, my co-workers previously demonstrated that myelin Ag presentation by pDCs promotes the expansion of regulatory T cells (Tregs) that inhibit encephalitogenic TH1 and TH17 cell priming in secondary lymphoid organs (SLOs). In this manuscript I report our recent findings supporting that pDCs display tolerogenic functions during the priming and the effector phase of EAE development and are able to control and dampen the disease. The first study investigates the interplays between pDCs and Tregs and shows that Ag-specific MHCII interactions with Tregs licenced tolerogenic features in steady-state pDCs by inducing their expression of the indoleamine-2,3 dioxygenase (IDO1). In EAE context, Treg-educated IDO+ pDCs are required to confer suppressive functions to Tregs which promote the inhibition of encephalitogenic T cell priming in draining LNs, resulting in attenuated EAE. In the second study, we explore the therapeutic effect of pDCs transfer in EAE mice after disease onset. We show that the transfer of immature MOG35-55 pre-loaded pDCs during EAE acute phase leads to substantial reduction of CNS inflammation and significant amelioration of disease clinical scores. We demonstrate that pDC-protection relies on the massive recruitment of endogenous immature pDCs in the inflamed spinal cord via the Chemerine/CMKLR1 axis. Endogenous pDC recruitment is required to down-modulate CNS inflammation, encephalitogenic TH1 and TH17 cell responses and EAE severity. Overall this work supports previous findings showing the importance of pDCs in the regulation of CNS autoimmunity and unravel these cells for potential use, by targeting different particular functions, in the development of future therapies to treat MS patients.

The estrogen receptor α (ERα) is a transcription factor that can be directly activated by estrogen or indirectly by other signaling pathways and plays a central role in breast carcinogenesis. We previously reported that activation of the unliganded ERα by cAMP is mediated by phosphorylation of its coactivator CARM1 by protein kinase A (PKA), allowing CARM1 to bind ERα directly. This being insufficient by itself to activate ERα, we looked for additional factors that could be involved in the response of ERα to cAMP. In order to do that we have developed a set of gene knockdown and overexpression experiments in ERα- positive breast cancer cell lines and tested their functional importance for ERα signaling in transactivation assays and for the expression of endogenous ERα target genes in response to cAMP and estrogens. We have investigated the recruitment of these factors to ERα and to chromatin. We have tested different inhibitors of the main catalytic activities of the ERα transcriptional complex to gain insights on their efficacy to disrupt the recruitment of coactivators to ERα and reduce proliferation of breast cancer cells. Finally, we have investigated the expression of certain of these proteins in ERα-positive breast tumors looking for the correlation with the outcome in breast cancer. We identified the histone H3 demethylase LSD1 as a substrate of PKA and an important mediator of this signaling crosstalk as well as of the response to estrogen. Surprisingly, ERα engages not only LSD1, but its partners of the CoREST corepressor complex, the repressor element-1 silencing transcription factor (REST), and the molecular chaperone Hsp90. The recruitment of Hsp90 to promote ERα transcriptional activity suggests that it might be involved as an assembly factor or scaffold. In a breast cancer cell line, which is resistant to the anti-estrogens tamoxifen (OHT) because of constitutively activated PKA, drug combinations partially rescue OHT sensitivity. In ERα-positive breast cancer patients, high expression of the genes encoding some of these factors correlates with poor prognosis. We believe that understanding the molecular mechanisms that trigger activation of ERα by cAMP are important to establish differences or similarities from the estrogen-activated pathway, which might be relevant in the context of breast cancer. Due to the existing correlation of increased PKA activity and OHT resistance in breast cancer the discovery of druggable proteins implicated in the signaling crosstalk of cAMP and ERα might be important in the improvement of the efficacy of endocrine therapy for these tumors.

HSP90 is an essential ATP-dependent molecular chaperone that facilitates the folding, the maturation and the activation of a wide range of proteins, called ‘clients'. Among the list of HSP90 clients there is transcription factors especially hormone receptors and kinases. Therefore HSP90 regulates numerous cellular processes such as: cell proliferation, cell survival and immunity. Our study aimed to understand the regulation of Hsp90 during skeletal muscle differentiation. We showed that the HSP90α mRNA levels remain constant during C2C12 myoblast differentiation, whereas the HSP90α protein levels were downregulated. Furthermore, we observed that HSP90β mRNA and protein levels remained constant during differentiation. Inhibition of the proteasomal and lysosomal degradation machinery allowed us to maintain some HSP90α protein during late stages of C2C12 cell differentiation. Taken together, these results suggest that the HSP90α protein is degraded by the proteasome and the lysosome during muscle differentiation. In the future, HSP90α ubiquitination needs to be confirmed in order to clarify the detailed mechanisms of degradation. Moreover, the regulation of key proteins for myogenesis by HSP90β should be investigated.

Centrosomes are the microtubules organizing centers of most eukaryotice cells. Centrosomes are formed of a protein matrix called the pericentriolar material and two cylindrical shape structures formed of microtubules and associated proteins called centrioles. Centrosomes are important to ensure normal bipolar spindle formation during cell division. The daughter centriole elongates from its proximal end perpendicularly to the mother centriole. At the distal end of the new forming centriole, a complex formed of CCP110 and Cep97 stops probably the elongation by acting as distal cap. In previous work our lab has found a putative Cep97 homologous protein in C. elegans as an interactor of the multifunctional polo-like kinase 1 (PLK1). In this work we screen for interactors of the Cep97 C. elegans homologous protein R02F11.4. We have found four interactions; one homotypic interaction, an interaction with SAS-5 and interactions with two proteins which are candidates to be functional homologous of CCP110.

Xenotransplantation represents an attractive alternative to overcome the human organ shortage in clinical transplantation. Nevertheless, delayed cell-mediated xenograft rejection by natural killer (NK) cells and cytotoxic T lymphocytes (CTL) remains an obstacle for successful xenotransplantation. By contrast, tolerogenic DC represents a promising tool for the promotion of transplantation tolerance. The main goal of the present thesis was to evaluate whether tolerogenic DC could be used as cell-based therapy to protect xenografts. We demonstrated that human monocyte-derived DC, generated in the presence of rapamycin (Rapa-DC) or IL-10 (IL-10-DC), could modulate human anti-pig NK cell and CTL responses in vitro. Finally, we investigated the potential of autologous bone marrow-derived DC, differentiated in the presence of IL-10, to prevent xenograft rejection in a rat-to-mouse islets xenotransplantation model. When co-transplanted with rat islets under the kidney capsule, IL-10-DC significantly prolonged islets xenograft survival. Recipients of IL-10-DC demonstrated higher infiltration of CD4+ FoxP3+ T-cells in islets xenograft.

Afin de mieux comprendre les mécanismes cellulaires impliqués dans le développement du cancer de l'ovaire, nous nous sommes intéressés à la protéine GRP78. Dans les cellules cancéreuses, la GRP78 du RE est relocalisée à la surface cellulaire et participe au développement tumoral. Afin d'apporter des informations sur les mécanismes de transport de la GRP78 du RE à la membrane plasmique, nous avons investigué le rôle d'un potentiel partenaire : la protéine Par-4. Nous confirmons l'implication de Par-4 dans, 1) le transport de la GRP78 à la membrane plasmique et, 2) le développement tumoral et la réponse au taxol dans le cancer de l'ovaire. Nous avons également investigué l'implication des différents domaines de la GRP78 dans son transport à la membrane plasmique et montrons que le SBD joue un rôle important. Enfin, nous suggérons que la forme transmembranaire au niveau du RE ne serait pas l'« origine » de la forme transmembranaire à la surface cellulaire.

Data indicate a role for TGF-β1 expression in regulatory B cell functions, although this mechanism has not been tested in autoimmune neuroinflammation. Transgenic mice deficient for TGF-β1 expression in B cells (B–TGF-β1–/–) were tested in EAE induced by recombinant mouse MOG (rmMOG) or adoptive transfer EAE (at-EAE). In rmMOG EAE, B–TGF-β1–/– mice showed an earlier disease onset compared to littermate controls. Exacerbated EAE susceptibility was associated with augmented CNS T helper cell responses. Additionally, selective B cell TGF-β1–deficiency increased the frequencies and activation of myeloid DCs. Lack of TGF-β1 production by B cells did not influence the EAE course in at-EAE. Collectively our data suggest that in rmMOG EAE B cells can regulate the function of APCs, and in turn encephalitogenic Th1/17 responses, via TGF-β1. An in vitro study of human B cells further indicated that TGF-β1 is downregulated when B cells are activated.

Le récepteur des glucocorticoïdes (GR) est exprimé de façon ubiquitaire et son activation affecte la transcription de 10 à 20 % des gènes. Les glucocorticoïdes exercent aussi leurs effets de manière indirecte en interagissant avec un grand nombre d'autres voies de signalisation. Ce travail a pour but de valider l'implication de deux protéines du cytosquelette, MpRip et Ckap5, dans la régulation de GR et de commencer à clarifier la nature de leur interaction. Nous avons montré que MpRip exerce un effet inhibiteur sur GR tandis que pour Ckap5 les résultats ont été plus nuancés et n'ont pas permis de trancher de manière sûre, mais un effet activateur a plus souvent été observé. Il semble également que ces effets impliquent le domaine de liaison du ligand de GR mais les données doivent être confirmées avant de conclure de manière certaine et définitive.

Le récepteur des estrogènes α (ERα) peut être activé par nombreux signaux, même en absence de son ligand, l'hormone estradiol (E2). Nous avions montré précédemment que l'activation de ERα par cAMP dépend de la phosphorylation de la arginine methyltransférase CARM1 par cAMP/PKA. Nous avons constaté que Hsp90β est recruté avec CARM1 et ERα aux promoteurs des trois gènes-cibles de ERα formant un complexe qui participe au contrôle de la transcription par le récepteur actif. Nous avons confirmé que, selon le mode d'activation de ERα, l'expression génique et les sites de recrutement de ERα diffèrent et induisent des réponses cellulaires différentes et que les gènes régulés par l'activation de ERα par cAMP/PKA sont liés à des processus comme l'apoptose et la prolifération cellulaire. Dans ce contexte, nous avons repéré deux gènes, RALBP1 et NFkB2, qui pourraient être importants pour le développement de la résistance au tamoxifène, car NFKB2 est lié à la prolifération cellulaire et RALBP1 à l'apoptose.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease mainly characterized by an overactivation of autoreactive lymphocyte which leads to the production of autoantibodies recognizing self-nucleic acids. This results in the formation of excessive immune complexes that settle in several organs and lead to inflammation and functional damages such as glomerulonephritis in the kidney. In order to investigate the mechanisms involved in SLE pathology we focused our studies on B cell and monocyte implication in our model of lupus-prone mice. We explored the role of the endosomal Toll-like receptor 8 (TLR8) in development of disease in lupus-prone mice and the key molecules involved in B cell differentiation and plasma cell survival, such as APRIL and its receptors TACI and BCMA. Our researches demonstrated two interesting pathways, one which is dependent to endosomal TLR and another which implicates TACI-dependent APRIL signaling. These pathways should be further explored with the hope that a deeper understanding could help to the development of novel effective therapies to treat human SLE.

Adipose derived mesenchymal stem cells (AT-MSCs) are multipotent stem cells able to self-renew and differentiate to mesodermal lineage. In vitro expansion is necessary to achieve sufficient number of cells. We used non-activated Platelet Rich Plasma (nPRP), as an autologous and efficient medium supplement. We showed that nPRP could increase the proliferation of AT-MSCs. Additionally, the platelets were viable up to 10 days of culture and produced growth factors over time. We showed that nPRP induces main proliferation cascades in AT-MSC, namely AKT, ERK and Smad2 signalling pathways. Based on our in vitro results, we postulated that PRP might have an impact on fat graft survival using murine model. Although CT-Scan analysis showed that PRP did not ameliorate fat graft volume after 30 and 90 days, angiogenesis increased in PRP-mixed fat graft when compared to control group. Analysis revealed that the rate of surviving adipocytes increased comparing to the control group.

The protozoan parasite Plasmodium falciparum (Pf) has to cope with perpetual environmental stresses during its whole lifecycle. As an adaptation to this, the parasite resorts to a sophisticated system of molecular chaperones. Hsp90 is one of the most important molecular chaperones that help regulate numbers of biological processes such as signal transduction, cell cycle control, hormone signaling, transcription, heat shock response, etc. Additional evidence suggested that Hsp90 is involved in the development of the parasite and its pathogenesis during the infection. The Hsp90 inhibitor geldanamycin has been found to be effective against infection by Plasmodium yoelli in a mouse model. These results suggested that Hsp90 inhibitors may have great potential as new antimalarial agents. As most of the known antimalarials intervene during the blood-stages of the infection, the design of antimalarial drugs should maximally evade human off targets on which impact may potentially elicit toxicity. But many drug targets are actually essential proteins that are functionally and structurally conserved. This fact hinders the therapeutic exploitation of many promising drug targets. Hsp90 might be among them. Hsp90s are highly conserved between Pf and human. There are only three substitutions found in the drug-binding pocket, which account for a 92% of conservation in protein sequences. Such high degree of homology hinders the discovery of species-specific inhibitors. Is there still a chance for Hsp90 to become an antimalarial target? The work presented in this thesis is a “hunt” for selective inhibitors against PfHsp90 using structure-based drug design (SBDD). We have elaborated a strategy of SBDD based on comparative analyses of the drug-binding pocket between PfHsp90 and its human counterparts. Furthermore, we have established several in vivo and in vitro systems as tools for step-wise drug validation. The combination of the efforts allowed us to discover a lead molecule, IND31119, that inhibits exclusively PfHsp90 while bypassing its highly similar human orthologs. Our work illustrates a new trend in drug discovery that involves a highly dynamic cooperation across biology, chemistry and computer science. Our publication also represented the first report that confirmed the feasibility of designing selective inhibitors against PfHsp90. My thesis can pave the way for translational research into PfHsp90 inhibitors as new weapons in the fight against malaria.

Within skeletal muscles, myogenic stem cells - satellite cells - will proliferate and then, differentiate to build/re-build muscle fibers. In vitro, myoblasts derived from human satellite cells can be induced to differentiate after medium switch. Using this cellular model, we previously showed that one of the first steps of myoblast differentiation is a hyperpolarization of myoblasts' membrane. This change of membrane potential is followed by a calcium entry and the expression of myogenic transcription factors. More precisely, myoblast hyperpolarization relies on a potassium channel, Kir2.1 which is maintained inactive during proliferation. Here, EGF receptor, was identified as a stimulator of proliferation and an inhibitor of differentiation. EGFR expression is physiologically regulated in myoblasts and decreases during differentiation, in particular via the proteasome. Absence of EGFR results in a decrease of Kir2.1 phosphorylation, activation of this channel favoring calcium entry, expression of muscle-specific transcription factors and proteins.

The steroid hormones estrogens have an influence on many physiological and developmental processes in vertebrates. The estrogen receptor α (ERα), which is a ligand-regulated transcription factor, is one of the effectors of estrogens. Besides hormonal signals, the regulation of ERα function is mediated by a large number of factors acting at different steps in the ERα signaling pathway. We sought to define the degree of dependence of ERα activity toward the nuclear transport receptor Exportin-5 (Exp-5), which exports various RNA and protein species out of the nucleus. Our interest was oriented on Exp-5 since previous work from our laboratory suggested that in the budding yeast the Exp-5 ortholog Msn5p was essential for proper function of the human ERα. We used different experimental strategies that were principally based on the depletion of Exp-5 coupled with assays monitoring the activity of ERα in tissue culture cells. In addition, we evaluated the impact of Exp-5 knockdown on the subcellular localization of ERα. Finally, we employed an in vitro approach to determine whether these two proteins physically interact. In this context, we also used bioinformatics in order to identify proteins that interact both with ERα and with Exp-5. Overall, although we did not collect solid enough data to draw firm conclusions, we obtained several results hinting at the involvement of Exp-5 in the regulation of ERα activity. Chez les vertébrés, les oestrogènes (des hormones stéroïdes) ont une influence sur de nombreux processus physiologiques et développementaux. Le récepteur aux oestrogènes ERα (de lʼanglais « estrogen receptor α »), qui est un facteur de transcription régulé par des ligands, est lʼun des récepteurs produisant une réponse cellulaire après liaison aux oestrogènes. En plus des signaux hormonaux, la régulation de lʼactivité de ERα peut être faite par une large panoplie de facteurs agissant à différents niveaux de la voie de signalisation de ce récepteur. Nous avons cherché à définir le degré de dépendance de lʼactivité de ERα par rapport au récepteur de transport nucléaire Exportin-5 (Exp-5), lequel exporte diverses espèces dʼARN et de protéines en-dehors du noyau. Nous étions intéressés par Exp-5 car certains travaux précédemment effectués au sein de notre laboratoire indiquaient que dans la levure, lʼorthologue de Exp-5, cʼest-à-dire Msn5p, était nécessaire pour assurer le bon fonctionnement de ERα. Nous avons utilisé différentes stratégies expérimentales, principalement axées sur la déplétion de Exp-5 couplée à des tests de lʼactivité de ERα dans des cellules en culture. Nous avons également évalué lʼimpact du knockdown de Exp-5 sur la localisation subcellulaire de ERα. Finalement, nous avons employé une approche in vitro pour déterminer si ces deux protéines interagissent physiquement. Dans ce contexte, nous avons aussi utilisé une méthode bioinformatique dans le but dʼidentifier des protéines qui interagissent avec ERα et Exp-5. Dans lʼensemble et bien que nous nʼayons pas obtenu de données assez solides pour tirer des conclusions de manière assurée, certains de nos résultats suggèrent que Exp-5 est impliqué dans le régulation de lʼactivité de ERα.

Metabolic liver diseases include disorders ranging from hepatic steatosis to steatohepatitis and cirrhosis. Finally, hepatocellular carcinoma can occur as an end stage of these disorders. With obesity, the metabolic syndrome and alcohol consumption, hepatic viral infections are one of the major causes of metabolic liver disorders. Among them, hepatitis C virus (HCV) is of particular importance. The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has been reported to be downregulated in the liver of obese rats and patients having hepatic steatosis. Additionally, liver-specific PTEN knockout mice develop steatosis in the liver then evolving into steatohepatitis and hepatocellular carcinoma. In this work, we showed that PTEN protein expression is downregulated by HCV infection in patients having genotype-induced steatosis and that this downregulation can trigger lipid droplet accumulation and insulin resistance. We also established that PTEN downregulation by HCV is able to improve viral release from infected cells.

TRAP1 est un chaperon moléculaire de la famille des Hsp90. Nous avons généré des cellules et des souris n'exprimant pas TRAP1 afin d'étudier sa fonction et avons proposé un rôle dans la régulation du métabolisme énergétique. TRAP1 est localisé dans les mitochondries de cellules de mammifères, même après différents stress. Les méthodes biochimiques indiquent une localisation dans la matrice avec une fraction dans la membrane mitochondriale interne. Dans les fibroblaste en culture, la déplétion de TRAP1 induit une diminution de la prolifération, une augmentation de la taille cellulaire et du noyau, un enrichissement des protéines de la glycolyse et du cycle de Krebs, ainsi qu'une augmentation de la consommation d'oxygène et de l'activité métabolique. Chez les souris, la déplétion de TRAP1 induit une augmentation de la taille des cellules du foie et des reins, une diminution de la concentration sanguine de créatinine et de lactate et une diminution de performance au test du Rotarod.

One of the central features of the metabolic syndrome is insulin resistance which is described as the inability of insulin to stimulate glucose uptake. This disorder has been intensively investigated in adipocytes and skeletal muscles. Insulin resistance also affects the myocardium however the involved mechanisms remain elusive. Therefore, we aimed to investigate the cellular mechanisms contributing to impaired glucose metabolism in cultured adult rat cardiomyocytes (ARC). Firstly, we demonstrated that ERK1/2 activation prevents the formation of the microtubule organizing center and disrupts microtubule architecture leading to altered stimulated-glucose transport in ARC. Secondly, we found that exposure of cardiomyocytes to fatty acids and peroxisome proliferator activated receptors agonist was accompanied by impaired stimulated glucose transport. Thirdly, we showed that cardiotrophin-1 is involved in regulation of myocardial glucose metabolism in a dose dependant manner. In summary, this thesis provides new insights into the mechanisms regulating glucose metabolism in cardiomyocytes.

Ma thèse porte sur l'étude des interactions entre les bactéries pathogènes et l'hôte qu'elles infectent. L'utilisation de l'amibe Dictyostelium discoideum, un phagocyte professionnel, m'a permis d'analyser d'une part la pathogénicité bactérienne, et d'autre part les mécanismes par lesquels une cellule phagocytaire tue les bactéries. J'ai étudié l'évolution de la virulence bactérienne de souches cliniques de Pseudomonas aeruginosa, au cours de l'infection aiguë ou chronique. Mes résultats montrent que la virulence diminue chez environ 50% des patients atteints de mucoviscidose, mais pas chez les patients intubés, et ce indépendamment de l'apparition de mutations dans le gène lasR (un régulateur du quorum-sensing). Par mutagenèse aléatoire de D. discoideum, j'ai dévoilé l'implication de plusieurs nouveaux gènes dans les interactions amibe-bactérie. J'ai montré que la protéine Kil2 est indispensable pour tuer efficacement Klebsiella pneumoniae. Cette P-ATPase semble transporter du magnésium à l'intérieur du phagosome, ce qui active les enzymes protéolytiques responsables de l'élimination des bactéries.

The nuclear receptor superfamily includes steroid hormone receptors (SHR), which are activated by hormones and regulate many key physiological processes. However, their deregulation is implicated in a variety of pathological disorders and diseases, including many types of cancer. MicroRNAs are non-conding RNAs of about 20-25 nucleotides and in the recent years, they have come to represent a major layer of regulation of gene expression, contributing to regulation of many physiological processes in a vast number of eukaryotic organisms. In metazoans, microRNAs regulate the gene expression mainly through the partial sequence complementarity present in the 3ʼUTR of the target mRNA. MicroRNAs are typically known to repress expression of their target genes, however in some cases, they are shown to upregulate gene expression. Whereas there is a plethora of literature for miRNA mediated repression of gene expression, upregulation mediated by miRNAs is quite poorly studied with only a few examples described. Here we report that miR-219 is potentially involved in upregulation of the estrogen receptor alpha (ERα) expression. Furthermore, our results indicate that this phenomenon may involve FXR1 and AUF1p45, proteins known to be involved in RNA metabolism. In the second part of this study, we focus on another SHR, Glucocorticoid receptor (GR). The 3ʼUTR of GR mRNA harbours target sites for many miRNAs. We tested several of them and found that miR-22 directly targets the 3ʼUTR of GR mRNA at two distinct sites and represses GR expression.

Cardiovascular disease is the leading cause of morbidity and mortality in the adult population in industrialized countries. It is now well-established that immuno-inflammatory processes are involved in several cardiovascular diseases, such as atherosclerosis and myocardial infarction. Chemokines are known to trigger and direct leukocyte trafficking from the blood to inflamed tissues through the binding to their cell-surface receptors. Chemokines and there receptors are involved in multiple inflammatory diseases such as atherosclerosis, rheumatoid arthritis, multiple sclerosis or chronic obstructive pulmonary disease, as well as HIV infectious disease, making them particularly attractive therapeutic targets. In this thesis, we investigated the role of the chemokine CCL5/RANTES and its receptors in atherogenesis and myocardial reperfusion injury. Using both pharmacological and genetic approaches, we showed that inhibition of CCL5/RANTES and its receptor CCR5 could represent an attractive therapeutic strategy to decrease atherosclerosis progression and limit damages due to the reperfusion of ischemic myocardium, in mice.

Immune cells are known to infiltrate human gliomas, but their implication in immunosurveillance is controversial. Since these infiltrates have been observed only at terminal stages of glioma development, it is not defined whether immunoediting occurs in these tumors. Alternatively, defective afferent immunity may delay a potent anti-tumor response, which is then overwhelmed by the tumor outgrowth. Finally, immune cells infiltrating gliomas may have poor anti-tumor competency, or may even promote tumor growth from early stages. In this study, a transgenic mouse model of spontaneous astrocytoma (GFAP-V12HA-ras or RasB8 mice) was analyzed to address these issues. Brain-infiltrating immune cells were characterized at different stages of tumor development. In addition, this model was used to determine whether the local immune balance in spontaneous gliomas could be tilted towards effector immune responses.

The transcription factor paired-box-6 (Pax6) plays a key role in the endocrine differentiation cascade of the pancreas. Mutations in Pax6 are associated with anomalies including diabetes. This study elucidates the impact of Pax6 in pancreatic a- and ß-cell function and differentiation. In a-cells, we found Pax6 to be critical for glucagon biosynthesis and processing, by directly and indirectly activating the glucagon gene through the transcription factors cMaf and NeuroD1, and the processing enzyme of proglucagon in a-cells PC2 and its molecular chaperon 7B2. In regard to ß-cells, the study foundsPax6 to control the cell function and differentiation through the control of genes coding for insulin-1, insulin-2, glucose-transporter-2, glucokinase, GLP-1-receptor, FFA receptor GPR40 as well as the transcription factors Pdx1, MafA and Nkx6-1. The study maps the complex network in which Pax6 regulates transcription of genes important for differentiation of the endocrine pancreas and plays a major role in the islet function.

The endogenous retroviral envelope glycoprotein, gp70, implicated in murine lupus nephritis is secreted by hepatocytes as an acute phase protein. To better understand the genetic basis of the expression of serum gp70, we analyzed the abundance of Xeno, PT or mPT gp70 RNAs in livers in various strains of mice. Our results demonstrated that the expression of different gp70 RNAs was remarkably heterogeneous among mouse strains and that serum gp70 production was regulated by multiple genes in physiological vs. inflammatory conditions. In addition, we observed a contribution of PT and mPT gp70s, in addition of Xeno gp70, to serum gp70. Furthermore, we observed an increased expression of intact mPT env RNA, regulated by the Sgp3 locus, in all lupus-prone mice, as compared with non-autoimmune strains of mice. Finally, we demonstrated that TLR7 played a critical role in the expression of gp70 and in the production of anti-gp70 autoantibodies. These data suggest that lupus-prone mice may possess a unique genetic mechanism responsible for the expression of mPT retroviruses, which could act as a triggering factor through activating TLR7 for the development of autoimmune responses in mice predisposed to SLE.

The steroid hormone 17β-estradiol (E2) regulates a number of developmental and physiological processes. The classical understanding of the estrogen signaling implies that the two nuclear receptors (ER), ERα and ERβ, mediate all the physiological responses of E2. ERs are transcription factors, which regulate the expression of their targets upon binding to E2. Estrogens also mediate a lot of rapid outcomes within a matter of seconds to minutes that cannot be explained by the classical understanding of estrogen signaling, which involves transcription of target genes mediated by the ERs. Whereas, many of the rapid effects of estrogen are attributed to the cytoplasmic pools of ERs, it has become clear in the recent years that there are other factors contributing to the rapid effects of estrogen and one of the emerging such factors is a G-protein coupled receptor, GPR30. MicroRNAs (miRNAs) are a new class of small non-coding RNAs, 20 to 25 nucleotides in length that regulate the expression of other genes. Upon binding the 3' untranslated region (UTR) of target mRNAs, miRNAs typically reduce their stability and/or translation. miRNAs are shown to be involved in regulating virtually every cellular process and their deregulation has been shown to contribute to many diseases. There are nearly 1,000 different miRNAs in humans, which regulate expression of thousands of other genes, reflecting that this is indeed a major layer of gene expression regulation. In my PhD, I had focused on these two different themes of estrogen signaling. In the first project, using a DNA microarray approach, we identified the target genes of the emerging new player in estrogen signaling, GPR30. In the second part, we have addressed some previously unexplained observations about the role of the 3ʼUTR of the ERα mRNA in regulating ERα levels and estrogen signaling.

L'infection chronique par le virus de l'hépatite B touche plus de 350 millions de personnes et constitue un facteur de risque majeur de l'hépatocarcinome. Dans les hépatocytes infectés, le virus synthétise la protéine régulatrice HBx dont la fonction exacte est inconnue. Son interaction avec la protéine cellulaire DDB1 est essentielle pour l'établissement de l'infection chez l'animal et son expression est associée au développement du cancer du foie durant l'infection chronique. HBx semble en effet favoriser la carcinogenèse en induisant une instabilité génétique dans les hépatocytes en division. Notre étude génétique et structurale a montré que HBx présente une structure commune à d'autres protéines virales et cellulaires se liant à DDB1 et jouant le rôle d'adaptateurs de substrats pour la DDB1-Cullin4A E3 ligase. Au travers de l'interaction avec ce complexe, HBx stimule l'expression du génome viral par un mécanisme différent de celui qui caractérise les activateurs de transcription cellulaire.

Dans ce travail, nous avons étudié comment est-ce que les contacts cellules-cellules influencent la sécrétion d'insuline des cellules β-pancréatiques. Cette étude nous a permis de mettre en évidence le double rôle des contacts intercellulaires dans la régulation de la sécrétion d'insuline puisque les cellules privées de contacts entre elles présentent une sécrétion basale trop élevée ainsi qu'une sécrétion stimulée trop faible en comparaison des cellules en contact les unes avec les autres. Nous montrons que les contacts physiques intercellulaires permettent de maintenir un niveau de calcium cytosolique stable, ce qui est indispensable pour une sécrétion basale adéquate et que, plus spécifiquement, l'engagement des molécules d'adhésion E-cadhérine entre cellules adjacentes permet d'optimiser la sécrétion d'insuline suite à une stimulation au glucose. Finalement, nous montrons que les cellules isolées restent capables de répondre au glucose une fois que leur sécrétion basale a été normalisée.