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Most intracranial aneurysms morphologic studies focused on characterization of size, location, aspect ratio, relationship to the surrounding vasculature and hemodynamics. However, the spatial orientation with respect to the gravity direction has not been taken into account although it could trigger various hemodynamic conditions. The present work addresses this possibility. It was divided in two parts: 1) the orientations of 18, 3D time-of-flight MRI (3D TOF MRI), scans of saccular aneurysms were analyzed. This investigation suggested that there was no privileged orientation for cerebral aneurysms. The aneurysms were oriented in the brain as follows: 9 – down, 9 – up; 11 – right, 7 – left; 6 – front, 12 – back. 2) Based on these results, subsidiary in vitro experiments were performed, analyzing the behavior of red blood cells (RBCs) within a silicone model of aneurysm before and after flow diverter stent (FDS) deployment in the parent vessel. These experiments used a test bench that reproduces physiological pulsatile flow conditions for two orientations: an aneurysm sack pointing either up (opposite to gravitational force) and down (along the gravitational force). The results showed that the orientation of an aneurysm significantly affects the intra-aneurysmal RBCs behavior after stenting, and therefore that gravity can affect the intra-aneurysm behavior of RBCs. This suggests that the patient׳s aneurysm orientation could impact the outcome of the FDS treatment. The implementation of this effect in patient-specific numerical and preoperative decision support techniques could contribute to better understand the intrasaccular biological and hemodynamic events induced by FDS., SCOPUS: ar.j, info:eu-repo/semantics/published

Type 1 Diabetes (T1D) is an autoimmune disease affecting around 0.1-0.8% of the population worldwide and is characterized by a progressive destruction of insulin-producing beta cells. Pro-inflammatory cytokines released by immune cells around the islets contribute for the “first wave” of beta cell apoptosis. Cytokine-mediated activation of the transcription factor nuclear factor kappa (NF-κB) contributes to beta cell demise in T1D. This is unusual, since NF-κB has anti-apoptotic effects in other cells. NF-κB is activated in most cells via the canonical pathway, while its activation via the non-canonical NF-κB pathway is restricted to few cell types, such as maturing/differentiating immune cell and osteoclasts. We have now observed that IL-1β+IFN-γ induces an atypical activation of the non-canonical NF-κB pathway in beta cells. This activation depends on different crosstalk mechanisms between the canonical and non-canonical NF-κB pathways, including the down-regulation of the E3 ligase Fbw7, which targets the p100 for proteasomal degradation, and up-regulation of another E3 ligase, βTrCP, which in turn induces cleavage of p100 to p52, a hallmark step in the non-canonical NF-κB activation. Importantly, cytokine-mediated activation of the non-canonical pathway regulates the expression of late NF-κB dependent genes, such as Ccl5, Ccl19, Ccl12, Fas that regulate both pro-inflammatory and pro-apoptotic responses and are implicated in beta cell loss in T1D. This atypical activation of the non-canonical NF-κB pathway by pro-inflammatory cytokines in beta cells constitutes a novel “feed-forward” mechanism that may explain the particular pro-apoptotic effect of this transcription factor in beta cells. Besides regulation of pro-death responses, NF-κB activation in beta cells triggers the expression of the ubiquitin-editing protein A20, encoded by TNFAIP3. A20 restricts NF-κB signalling and possess anti-apoptotic activities in beta cells. Importantly, genome-wide association st, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

Des études récentes menées chez l’homme et la souris ont suggéré que la diversité du répertoire TCR pourrait jouer un rôle dans la protection contre des pathogènes à haut pouvoir mutagène. Afin d’étudier le répertoire des lymphocytes T CD4, nous avons utilisé un modèle de souris TCRβ transgéniques exprimant une chaine β spécifique du peptide env122-141 dans le contexte du MHCII. Suite à l’immunisation des souris TCRβ transgéniques avec des cellules dendritiques pulsées avec le peptide env, une rapide prolifération et une restriction du répertoire des lymphocytes T Vα2 CD4 spécifiques est observée. L’analyse de la diversité du répertoire de ces cellules par séquençage à haut débit, a montré l’émergence d’un répertoire plus divers dans des souris déplétées en lymphocytes T régulateurs. Ces résultats suggèrent qu’en plus du rôle des Tregs dans le contrôle de la magnitude de la réponse immunitaire, ces cellules pourraient également contrôler la diversité du répertoire des lymphocytes T suite à une stimulation antigénique., Recent studies conducted in mice and humans have suggested a role for the TCR repertoire diversity in immune protection against pathogens displaying high antigenic variability. To study the CD4 T cell repertoire, we used a mouse model in which T cells transgenically express the TCRβ chain of a TCR specific to a MHCII-restricted peptide, env122-141. Upon immunization with peptide-pulsed dendritic cells, antigen-specific Vα2+ CD4+ T cells rapidly expand and display a restricted TCRα repertoire. In particular, analysis of receptor diversity by high-throughput TCR sequencing in immunized mice suggests the emergence of a broader CDR3 Vα2 repertoire in Treg-depleted mice. These results suggest that Tregs may play a role in the restriction of the CD4 T cell repertoire during an immune response, raising therefore the possibility that in addition to controlling the magnitude of an immune response, regulatory cells may also control the diversity of TCRs in response to antigen stimulation., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Together with the current antibiotic resistance crisis, bacterial persistence appears to play an increasingly important role in the frequent failure of antibiotic treatments. Persister cells are rare bacteria that transiently become drug tolerant, allowing them to survive lethal concentrations of bactericidal antibiotics. Upon antibiotic removal, persister cells are able to resume growth and give rise to a new bacterial population as sensitive to the antibiotic as the original population. Interest in persister cells seriously increased in the past few years as these phenotypic variants were shown to be involved in the recalcitrance of chronic infections, such as tuberculosis and pneumonia and in the well-known biofilm tolerance to antibiotics. Persistence has therefore been extensively studied throughout the last decade, which led to the discovery of large variety of different molecular mechanisms involved in persisters formation. However, the specific physiology of bacterial persisters remains elusive up to now, mainly because of the transient nature and the low frequencies of persister cells in growing bacterial cultures. This work aims to gain a better understanding of the physiology of Escherichia coli persisters by combining population analyses with single-cell observations.In the first part of this thesis, we developed an experimental method allowing for measuring persistence with increased reproducibility. The method was further refined, which allowed us to observe four distinct phases in the ofloxacin time-kill curve, suggesting the existence of a tolerance continuum at the population level at treatment time. Characterization of these four phases notably revealed that the growth rate and the intrinsic antibiotic susceptibility of the strain define the number of surviving cells at the onset of the persistence phase, while persister cells survival mainly relies on active stress responses (SOS and stringent responses in particular).We next investigated the mole, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Sepsis is an exaggerated inflammatory syndrome involving cell, organ and system dysregulation. This dysregulation is detrimental to the patient, mainly as it causes imbalances in the immune system. Monocytes and neutrophils are key cell players in the pathogenesis of sepsis; Two populations that follow divergent apoptotic fates; most importantly delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. These two subpopulations have recently been referred to as specific targets of leukocyte filtration, a noveltherapeutic approach in septic patients. As the molecular mechanisms driving the survival/death switches of these cells in sepsis is still controversial, we decided to investigate the role of a new family of proteins predicted to be involved in inflammation and cell death mechanisms; the ApoLs. We studied ApoL expression in whole blood leukocytes and purified populations of neutrophils and monocytes from healthy volunteers and patients with or without sepsis. We then correlated ApoL expression to the degree of inflammation (CRP levels) and cellular apoptosis. We showed a down regulation in ApoL expression in septic and non-septic patients as compared to healthy volunteers, whereas purified monocytes showed an up regulation of ApoL1 and ApoL2 expression in septic patients as compared to non-septic patients. Given that these cells behave differently in the septic frame, we aim to investigate and invalidate the role of ApoLs in conditions related to cell activation and death. Given the high degree of variability in human samples, we resorted to an in-vitro cell line, the PLB 985 myelomonoblastic cell line with granulocytic differentiation potential. Culturing these cells in RPMI 1640 in the presence of specific differentiation agents resulted in mature terminally differentiated cells with phenotypic and functional similarities to human neutrophils. This was determined by monitoring specific leukemic and granulocytic markers. T, Le sepsis est defini comme une reponse inflammatoire exageree, associee a une dysfonction d'organes faisant suite a un desequilibre du systeme immunitaire. En effet, les monocytes et les neutrophiles representent les acteurs cle dans la physiopathogenese du sepsis. Etant donne que les mecanismes moleculaires regulant la balance de survie / mort cellulaire de ces deux populations de cellules restent jusqu'a present controverses, nous nous sommes interesses a etudier l'implication d'une nouvelle famille de proteines: les apols, susceptibles d'intervenir dans le processus inflammatoire et de la mort cellulaire. Nous avons explore l'expression des apols dans les leucocytes totaux et dans les populations de neutrophiles et de monocytes purifiees a partir de donneurs sains et de patients septiques et non septiques. Par la suite, nous avons etablit le lien de correlation entre l'expression des apols, le degre d'inflammation systemique (dosage de la crp) et l'apoptose. Nos resultats ont demontre une baisse de l'expression des apols dans les leucocytes totaux el les neutrophiles purifies, chez les patients septiques et non septiques, en comparaison avec les donneurs sains. En revanche, nous remarquons une surexpression de l'apol1 et l'apol2 chez les patients septiques par rapport aux patients non septiques. Etant donne la grande variabilite dans les echantillons preleves, nous avons procede a la differenciation d'une lignee myelomonoblastique ayant un pouvoir de differenciation granulocytaire. La mise en culture de cette lignee dans les conditions de differenciation adequates, nous a permis d'obtenir des cellules ayant des similarites fonctionnelles et phenotypiques aux neutrophiles humains. Ce modele cellulaire va nous aider d'une part, a mieux comprendre l'implication des apols dans le controle de la viabilite cellulaire dans les conditions inflammatoires; et d'autre part, a caracteriser la / (les) cytokine(s) susceptibles de moduler l'expression des apols; et finalement a, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Delayed neutrophil apoptosis has been demonstrated in sepsis and may contribute to organ damage. It has recently been proposed that apolipoprotein L (ApoL) may be involved in programmed cell death, but the expression and functions of ApoLs in leukocytes (especially neutrophils) during sepsis and other inflammatory conditions are currently unknown. In this prospective observational study in a 36-bed university hospital medico-surgical intensive care unit (ICU), we included 78 adult ICU patients with (n = 41) or without (n = 37) sepsis and 47 healthy volunteers. We analyzed ApoL mRNA expression using quantitative polymerase chain reaction in whole blood leukocytes and protein expression in CD15 isolated neutrophils using western blotting. Neutrophil apoptosis was assessed using the APO-BRDU method. ApoL mRNA was downregulated in whole blood leukocytes and neutrophils in ICU patients compared to healthy volunteers and this effect translated at the protein level as indicated by western blot analysis of neutrophils. There was a negative correlation between ApoL expression in neutrophils and C-reactive protein (CRP) levels and a positive correlation between the number of apoptotic neutrophils and mRNA levels of ApoL1 and 2. The degree of neutrophil apoptosis in critically ill patients is therefore correlated with modified expression profiles of ApoLs., SCOPUS: ar.j, info:eu-repo/semantics/published

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and composed of proteins called nucleoporins. NPCs as such control the bidirectional traffic of proteins and RNAs between the nucleus and the cytoplasm in eukaryotic cells whereas individual nucleoporins were found to be implicated in other cellular processes such as, cell division, kinetochore assembly, gene expression and cell migration. A prime example for nucleoporin functional versatility can be seen in Nup153. Nup153 is since its discovery known to be a central player in nucleocytoplasmic transport, but additionally participates directly or indirectly, for example, in gene expression and cell cycle control. In this context, it was previously shown that altered levels of Nup153 led to mitotic abnormalities, particularly in cytokinesis and in the spindle assembly checkpoint (SAC). The SAC promotes accurate chromosome separation to ensure the faithful segregation of genetic material to daughter cells. Nup153 was found to interact with the SAC protein Mad1. In the present study, we have further dissected the interaction between Nup153 and Mad1 and investigated the function of the Nup153-Mad1 complex in human cells. By using the high resolution imaging technique “in situ proximity ligation assay”, we found that Nup153 and Mad1 interact with each other exclusively in the presence of a NE, from late mitosis to prophase. By in vitro binding assays, we have confirmed the direct interaction between Nup153 and Mad1 and furthermore identified two independent Nup153-binding sites in Mad1. We have also provided some evidence that Nup153 interacts also with SUMO-modified Mad1.It was previously shown that depletion of Nup153 had no obvious effect on Mad1 and SAC activity. In the present study, we have shown by time-lapse imaging microscopy that the depletion of Mad1 led to a delayed recruitment of Nup153 at the reforming NE during anaphase in living cells, which was often accompanied by a prolongation of, Les pores nucléaires sont des structures enchâssées dans l’enveloppe nucléaire et composées de protéines appelées les nucléoporines. Ces pores nucléaires contrôlent le trafic bidirectionnel des protéines et des ARNs entre le noyau et le cytoplasme dans les cellules eucaryotes tandis que les nucléoporines individuelles sont également impliquées dans d’autres processus cellulaires tels que la division cellulaire, l’assemblage des kinétochores, l’expression génétique et la migration cellulaire. Un exemple primordial de la versatilité fonctionnelle des nucléoporines peut être observé à travers Nup153. Depuis sa découverte, Nup153 est connue pour être un élément clé dans le transport nucléo-cytoplasmique, mais il a également été démontré qu’elle participait directement ou indirectement à l’expression génétique et au contrôle du cycle cellulaire. Dans ce contexte, nous avons montrés précédemment que des niveaux altérés de Nup153 menaient à des anomalies mitotiques, particulièrement en cytokinèse et dans le point de contrôle de l’assemblage du fuseau mitotique (SAC). Le SAC assure la ségrégation correcte du matériel génétique entre les cellules filles. Il a été montré que Nup153 interagit avec la protéine du SAC Mad1. Dans cette étude, nous avons utilisé une technique d’imagerie de haute résolution, « in situ proximity ligation assay » pour disséquer davantage l’interaction entre Nup153 et Mad1 dans les cellules humaines. Nous avons montré que ces deux protéines interagissent exclusivement au niveau de l’enveloppe nucléaire, depuis les dernières phases de la mitose jusqu’à la prophase. Par des expériences d’interaction in vitro, nous avons également identifiés sur Mad1 deux sites de liaison indépendants pour Nup153. Nous avons également fourni des indications que Nup153 interagit aussi avec une forme SUMOylée de Mad1. La déplétion de Mad1 menait à un recrutement tardif de Nup153 au niveau de l’enveloppe nucléaire en cours de reformation en anaphase dans les cellules vivant, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The apolipoprotein L (apoL) family has not yet been ascribed any definite patho-physiological function although the conserved BH3 protein domain suggests a role in programmed cell death. As repression of the regular apoptotic program is considered a hallmark of tumor progression, we investigated apoL expression in cancer. We show that the levels of one member of the family, apolipoprotein L1 (apoL1) is higher in papillary thyroid carcinoma compared to normal tissue. A combination of qRTPCR, immunohistochemistry and in situ hybridization allowed us to ascribe this increase to endogenous overexpression in carcinoma cells. Whether apoL1 plays an instrumental role in refraining cell death is the subject of ongoing molecular biology experiments., SCOPUS: ar.j, info:eu-repo/semantics/published

Glutathione (GSH) is the keystone of the cellular response toward oxidative stress. Elevated GSH content correlates with increased resistance to chemotherapy and radiotherapy of head and neck (HN) tumors. The purpose of the present cross-sectional study was to evaluate whether the expression of glutamate-cysteine ligase (GCL) accounts for the increased GSH availability observed in HN squamous cell carcinoma (SCC). For that purpose, the messenger (m)RNA levels of the modifier (M) and catalytic (C) subunits of GCL and its putative regulators (namely, nuclear factor erythroid 2-related factor 2, heme oxygenase-1 and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha) were monitored in 35 surgical resections of untreated HNSCC. The localization of GCLM was evaluated using in situ hybridization and immunohistochemistry. GCLM expression was significantly increased in tumor samples, compared with normal mucosa, both at the mRNA and protein level (P=0.029), but the pathway of GCLM activation remains to be elucidated. Protein expression of GCLM was detected in the cytoplasm and nucleus. GCLM and the proliferation marker Ki-67 displayed a similar distribution, being both mainly expressed at the periphery of tumor lobules. The present study reported increased expression of GCL and the rate-limiting enzyme of GSH synthesis, within HNSCC. The nuclear localization of GCLM and the concomitant expression of Ki-67 suggested that the localization of GSH synthesis contributes to the protection against oxidative stress within hotspots of cell proliferation., SCOPUS: ar.j, info:eu-repo/semantics/published

There is increasing evidence that the effect of chemotherapy on tumor rejection is not cell autonomous but relies on the immune system. Indeed, several reports have shown that human and murine tumors respond to chemotherapeutic agents more efficiently when the host immune system is intact. In particular, we have shown that cyclophosphamide treatment of DBA/2 mice bearing P815 mastocytoma induces rejection and long term protection in a CD4- and CD8-dependent manner. We used this tumor model, as it is poorly immunogenic, expresses tumor-associated P1A and tumor-specific P1E antigens, encoded by germline and mutated genes, respectively, and allows the identification of some tumor-specific CD8+ T cells.We have previously reported that tumor regression correlates with selective infiltration of CD8+ T cells specific for P1E/H-2Kd antigen in tumor bed upon cyclophosphamide treatment. Unexpectedly, the proportion of CD8+ T cells specific for the tumor-associated antigen P1A in the context of H-2Ld decreases concomitantly, indicating that cyclophosphamide alters the repertoire of CD8+ T cells recognizing tumor antigens. Using P1A KO mice, we found that preferential activation of CD8+ T cells to P1E is not solely due to thymic negative selection. The major role of “mutated” antigens in tumor resistance has been recently highlighted in humans and raises an interesting question about the immune mechanisms of tumor rejection. Additionally to its effect on the specific immune response, cyclophosphamide promotes tumor infiltration by effector memory (P1E/H-2Kd)+ CD8+ T cells which are characterized by higher expression of KLRG1 and Eomes. Our data point to a role of IL-15 and type 1 IFNs for their development, as increased levels of IL-15 and IRF7 were measured in tumor after cyclophosphamide. IFNAR1 blockade interferes with the tumor rejection in 50% of mice and decreases the (P1E/H-2Kd)+ CD8+ T cell infiltration induced by cyclophosphamide, suggesting a role of this cytokine in, Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Le ribosome est un macro complexe moléculaire en charge de la synthèse de toutes lesprotéines de la cellule. Depuis les premiers essais de reconstruction in vitro d’un ribosome procaryote,des générations de chercheurs se sont succédées durant plusieurs décennies pour tenter d’éluciderles voies par lesquelles les ribosomes sont assemblés par la cellule. Un regain d’intérêt pour l’étude dela biogenèse du ribosome est advenu récemment suite à la mise en évidence de maladies liées à desmutations dans des acteurs de la biogenèse du ribosome mais également, et surtout, suite à la miseen évidence du rôle fondamental du ribosome dans les processus de transformation oncogénique.Le ribosome est composé de deux sous-unités, une petite et une grande, formées, ellesmêmes,d’un assemblage intriqué d’ARN ribosomique et de protéines ribosomiques. La formation d’unribosome, appelée biogenèse du ribosome, est un processus complexe, intégré et extrêmementhiérarchisé impliquant, chez la levure, plus 200 facteurs accessoires. Bien que les principes sousjacentsà la biogenèse du ribosome eucaryote établis à partir d’études réalisées chez la levure semblentconservés chez l’Homme, de nombreux éléments suggèrent qu’elle y soit plus complexe. Ainsi, latransposition directe chez l’Homme des connaissances acquises chez la levure quant à la biogenèse duribosome serait, tout du moins partiellement, inexacte.Au cours de ma thèse, j’ai poursuivi différents objectifs s’intégrant tous dans le cadre de labiogenèse du ribosome eucaryote. D’une part, chez la levure, j’ai poursuivi la caractérisation du rôlede la protéine Las1 dans la biogenèse de la grande sous-unité ribosomique. D’autre part, chezl’Homme, mon objectif premier a été de participer à l’identification de nouveaux facteurs accessoiresimpliqués dans la biogenèse du ribosome et à la caractérisation fonctionnelle de certains d’entre eux.Dans un second temps, je me suis concentré sur l’implication de deux particules ribonucléoprotéiquesnucléolai, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Le trypanosome sud américain, Trypanosoma cruzi, transmis par un insecte hématophage de type triatome est le protozoaire connus pour causer la maladie de Chagas chez l’Homme. Le cycle de vie de ce parasite alterne à la fois sur le type d’hôte, insecte ou hôte vertébré, et sur la forme :trypomastigote pour la forme quiescente et amastigote et épimastigote pour les formes prolifératives. Concernant la forme, seuls les parasites épimastigotes évoluent et prolifèrent dans le tube digestif des triatomes et possèdent un système endocytaire actif nécessaire à leur besoin énergétique. Toutefois, cette endocytose est restreinte à deux sites membranaires, la poche flagellaire et le cytostome, à partir desquels se créent des cargos endocytaires. Ces cargos endocytaires fusionnent ensuite avec un réseau vésiculaire endosomal qui délivre son contenu dans des réservosomes, compartiments similaires aux lysosomes.Chez le trypanosome africain (Trypanosoma brucei brucei), l’endocytose ne se réalise qu’au niveau de la poche flagellaire. Certaines protéines appartenant à cette voie endocytaire sont modifiées par de longues chaines de résidus poly-N-acétyllactosamine (pNAL) de manière post-traductionnelle. Initialement, il a été proposé que ces résidus puissent agir en tant que signal de tri dans le processus d’endocytose chez ces parasites.En nous basant sur les travaux qui ont été réalisés chez le trypanosome africain, nous nous sommes proposés d’approfondir les connaissances sur la voie endocytaire du trypanosome sud américain (Trypanosoma cruzi) qui est beaucoup moins étudié. Pour ce faire, à l’aide de deux lectines, la tomatolectine et la lectine de Griffonia simplicifolia qui présentent respectivement une affinité pour les résidus pNAL et les résidus N-acétylglucosamine (GlcNAc) en fin de chaine, nous avons pu enrichir et caractériser par LC-MS², 173 glycoprotéines putatives dont plus de 13% sont localisées dans la voie endocytaire. Parmi les protéines identifiées, en plus des nom, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Apolipoprotéines L est une famille nouvellement caractérisée en humain sans une fonction patho- physiologique définitive. Ces protéines sont classiquement considérées être impliquées dans le transport et métabolisme des lipides, principalement due à l'association de son premier membre de la famille sécrétée l’apolipoprotéine L1 aux particules des lipoprotéines de haute densité. Néanmoins, le reste des membres sont des protéines intracellulaires (absence de domaine de peptide signal). Apolipoprotéine L1 a été initialement identifiée comme l'élément clé du facteur trypanolytique dans le sérum humain. L'exploration de la séquence des différents apolipoprotéines L a révélé un domaine distinct «B cell lymphoma-2 homology domain 3» ayant des similitudes structurelles et fonctionnelles avec le domaine B cell lymphoma-2 homology domain 3 des protéines de la famille B cell lymphoma-2. Ainsi la découverte de ce domaine peut contribuer à la compréhension de la fonction et rôle des apoLs dans différents mécanismes et processus tels que la mort cellulaire programmée, la prolifération cellulaire, le métabolisme cellulaire .Notre étude visait à caractériser les fonctions de patho- physiologique du premier membre de la famille «apolipoprotéine L1 ». L’expression de l’apolipoprotéine L1 ARNm, à partir de 48 carcinomes papillaires de la thyroïde, a été évaluée par des études à haut débit et normalisée à un pool de tissus normal de la thyroïde. Une confirmation de PCR en temps réel valide ainsi la surexpression d’apoL1 dans 91,67 % des cas testés. Le niveau élevé de l’apolipoprotéine L1 ARNm est en corrélation avec une expression protéique élevée dans les échantillons histologiques (70%), et détermine que les cellules folliculaires de la thyroïde dans la zone de la tumeur sont les cellules principales responsables de l’expression spécifique de l’apolipoprotéine L1. Nous avons étudié l'expression apolipoprotéine L1 dans le modèle de cancer pour approfondir notre compréhension des relat, Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

L’aide fournie aux lymphocytes B par les lymphocytes T CD4+ est cruciale pour une réponse humorale de qualité. Les lymphocytes T helper folliculaires (Tfh) jouent un rôle majeur dans l’aide apportée aux lymphocytes B au sein des centres germinatifs et peu de choses sont connues sur la capacité d’aide des autres populations de cellules T CD4+. Le but de notre étude est d’évaluer la capacité d’aide aux lymphocytes B des lymphocytes T helper de type 2 (Th2), une population considérée, à l’origine, comme responsable de l’aide apportée aux lymphocytes B in vivo ainsi que d’analyser le rôle de l’axe IL6 / STAT3 dans la différenciation des lymphocytes Tfh et leur plasticité phénotypique. Nous montrons que les lymphocytes Th2 co-expriment des formes actives des facteurs de transcription STAT6 et STAT3 et que l’expression de STAT3 est requise pour la fonction d’aide aux lymphocytes B des lymphocytes Th2. Nous avons également pu montrer que la voie de signalisation IL-6 / STAT3 durant le développement des lymphocytes Tfh s’oppose à l’expression des gènes associés au programme de différenciation Th2., Option Biologie moléculaire du Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Particular intra-aneurysmal blood flow conditions, created naturally by the growth of an aneurysm or induced artificially by implantation of a flow diverter stent (FDS), can potentiate intra-aneurysmal thrombosis. The aim of this study was to identify hemodynamic indicators, relevant to this process, which could be used as a prediction of the success of a preventive endovascular treatment., SCOPUS: ar.j, info:eu-repo/semantics/published

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Background: It is a known fact that blood flow pattern and more specifically the pulsatile time variation of shear stress on the vascular wall play a key role in atherogenesis. The paper presents the conception, the building and the control of a new in vitro test bench that mimics the pulsatile flows behavior based on in vivo measurements. Methods: An in vitro cardiovascular simulator is alimented with in vivo constraints upstream and provided with further post-processing analysis downstream in order to mimic the pulsatile in vivo blood flow quantities. This real-time controlled system is designed to perform real pulsatile in vivo blood flow signals to study endothelial cells' behavior under near physiological environment. The system is based on an internal model controller and a proportional-integral controller that controls a linear motor with customized piston pump, two proportional-integral controllers that control the mean flow rate and temperature of the medium. This configuration enables to mimic any resulting blood flow rate patterns between 40 and 700 ml/min. In order to feed the system with reliable periodic flow quantities in vivo measurements were performed. Data from five patients (1 female, 4 males; ages 44-63) were filtered and post-processed using the Newtonian Womersley's solution. These resulting flow signals were compared with 2D axisymmetric, numerical simulation using a Carreau non-Newtonian model to validate the approximation of a Newtonian behavior. Results: This in vitro test bench reproduces the measured flow rate time evolution and the complexity of in vivo hemodynamic signals within the accuracy of the relative error below 5%. Conclusions: This post-processing method is compatible with any real complex in vivo signal and demonstrates the heterogeneity of pulsatile patterns in coronary arteries among of different patients. The comparison between analytical and numerical solution demonstrate the fair quality of the Newtonian Womersley's appr, SCOPUS: ar.j, info:eu-repo/semantics/published

During translation, the transfer RNAs (tRNAs) play the crucial role of adaptors between the messenger RNA and the amino acids. The tRNAs are first transcribed as pre-tRNAs which are then maturated. During this maturation, several nucleosides are modified by tRNA modification enzymes. These modifications are important for the functions of the tRNAs and for their correct folding. Many of the modifications are methylations of the bases or the ribose. Four families of tRNA methyltransferases are known, among which the SPOUT superfamily. Proteins of this superfamily are characterised by a C-terminal topological knot where the methyl donor is bound. With the exception of the monomeric Trm10, all known SPOUT proteins are dimeric and have an active site composed of residues of both protomers. Interestingly, depending on the organism, the same modification can be catalysed by completely unrelated enzymes. On the other hand, homologous enzymes can have different specificities or/and activities. These differences are well illustrated for the TrmJ and Trm10 enzymes.In the first part of this work we have identified the TrmJ enzyme of Sulfolobus acidocaldarius (the model organism of hyperthermophilic Crenarchaeota) which 2’-O-methylates the nucleoside at position 32 of tRNAs. This protein belongs to the SPOUT superfamily and is homologous to TrmJ of the bacterium Escherichia coli. A comparative study shows that the two enzymes have different specificities for the nature of the nucleoside at position 32 as well as for their tRNA substrates. To try to understand these shifts of specificity at a molecular level we solved the crystal structure of the SPOUT domains of the two TrmJ proteins.In the second part of this work, we have determined the crystal structure of the Trm10 protein of S. acidocaldarius. This is the first structure of a 1-methyladenosine (m1A) specific Trm10 and also the first structure of a full length Trm10 protein. The Trm10 protein of S. acidocaldarius is di, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Le cortex cérébral est composé d’un grand nombre de types de neurones organisés radialement en couches cellulaires et tangentiellement en aires corticales fonctionnellement distinctes. Son développement est régulé par des signaux fonctionnant comme morphogènes sécrétés par des centres organisateurs situés à la périphérie du télencéphale dorsal. Ces morphogènes contrôlent l’expression dans les progéniteurs corticaux de gènes codant pour des facteurs de transcription qui régulent la prolifération, la différenciation et la spécification des progéniteurs corticaux. Les cascades de gènes impliquées dans la mise en place du cortex cérébral dans lesquelles ces signaux et facteurs de transcription interviennent restent cependant actuellement mal connues.Les gènes Dmrt3 et Dmrt5 appartiennent à une famille de gènes fortement conservée évolutivement codant pour des facteurs de transcription à doigt de zinc connus pour leur rôle dans le développement sexuel. Des résultats obtenus dans le laboratoire ayant montré que Dmrt5 joue un rôle crucial dans la neurogenèse au niveau du système olfactif chez le xénope, j’ai voulu savoir, dans un premier temps, si la fonction de Dmrt5 dans la neurogenèse était une fonction ancestrale. Pour répondre à cette question, j’ai recherché des gènes Dmrts chez une espèce de Cnidaire, Nematostella vectensis, et ai étudié leur expression au cours du développement. L’un d’entre-eux, NvDmrtB, est fortement exprimé dans le système nerveux et s’est avéré être requis pour la différenciation des cellules nerveuses chez N. vectensis, suggérant que les gènes Dmrts avaient déjà un rôle dans la neurogenèse dans l’ancêtre commun des Bilatériens et des Cnidaires.Par ailleurs, d’autres travaux ont montré que chez la souris les gènes Dmrt5 et Dmrt3 sont exprimés dans le cerveau en développement au niveau du télencéphale dorsal. Afin d’approcher leur fonction dans le développement cortical, j’ai analysé leur expression au cours du développement embryonnaire, Doctorat en sciences, Spécialisation biologie moléculaire, info:eu-repo/semantics/nonPublished

African trypanosomes are a major plague in sub-Saharan Africa. They cause sleeping sickness in humans and nagana in cattle. These parasites are transmitted between their mammalian hosts by tsetse flies. They are adapting to their different environments through differentiation processes. These processes involve, amongst other things, the expression of different surface coats. These coats are made of procyclin protein at the insect midgut procyclic stage and of variant surface glycoprotein (VSG) at the mammalian bloodstream stage. At a given time, one VSG is expressed from a single VSG gene out of a repertoire of more than 1500 VSG genes present in the trypanosomes genome. The expressed VSG gene is always located at one of fifteen telomeric polycistronic transcription units called expression sites (ES). The VSG coat is changed regularly in a process called antigenic variation allowing trypanosomes to escape the immune response. The exact mechanism controlling the selection of the active ES is not yet known and controversies have been raised concerning the ES transcription control. Although several molecular factors involved in the ES monoallelic-expression have been identified, none of them seems to be a critical regulator.Thus during my thesis we decided to explore two aspects of ES expression: (A) deciphering the level at which this expression is controlled and (B) fishing for new protein factors controlling this expression.A) It is not even clear at which level the ES transcription control takes place. In particular, there has been debate on whether it is taking place at the transcription initiation or elongation level. Previous experiments generated contradictory conclusions and gave rise to two different models. The first model suggested that transcription initiation takes place in all ESs simultaneously. The second model suggested that transcription is initiated in only two ESs, one being fully active and a second being pre-active. These two models were, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

La tuberculose est une maladie contagieuse causée par les bactéries appartenant au complexe Mycobacterium tuberculosis. On estime près de neuf millions de nouveaux cas et un million de décès chaque année dans le monde. De plus, approximativement un tiers de la population mondiale est infecté de manière latente, donc à risque de développer la maladie. Le seul vaccin préventif jusqu’à présent disponible est le Bacille de Calmette-Guérin (BCG). Cependant, son efficacité contre la forme pulmonaire de la maladie, contagieuse et plus fréquente chez l’adulte, est extrêmement variable. Le développement de nouveaux vaccins prophylactiques contre la tuberculose est basé sur une stratégie de remplacement ou d’amélioration de l’actuel vaccin BCG. De nombreux candidats vaccins sous-unitaires sont évalués dans un protocole de vaccination de rappel après le BCG. Ce dernier est en effet administré à plus de 80% des nouveau-nés et des nourrissons des populations à haut risque.Le présent travail a eu pour but principal d’étudier une nouvelle approche de vaccination combinant le Bacille de Calmette-Guérin avec des vaccins sous-unitaires à ADN plasmidique dans différents modèles précliniques.Plusieurs hypothèses tentent d’expliquer la faible efficacité du vaccin BCG, comme la faible induction de réponses immunitaires de type cellulaire T CD8+, le déclin de l’immunité protectrice induite au cours du temps, ou son répertoire antigénique limité. Les vaccins à ADN plasmidique induisant de telles réponses, le travail proposé a consisté au développement d’un nouveau protocole de vaccination basé sur la coadministration par la voie intradermique du vaccin BCG formulé avec un vaccin à ADN plasmidique codant pour un antigène mycobactérien. Nous avons observé dans plusieurs modèles murins (adulte et néonatal) une augmentation significative des réponses cellulaires de type CD4+ Th1 et CD8+, ainsi que de la réponse humorale spécifique. L’immunogénicité de cette approche a également été analy, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Les ribosomes sont des macrocomplexes ribonucléoprotéiques sophistiqués, essentiels pour décoder l’information génétique et la traduire en protéines fonctionnelles. Chez les organismes eucaryotes, le ribosome est constitué de deux sous-unités, la petite (40S) et la grande (60S). Leur biogenèse est un processus fondamental, très complexe, qui mène à la synthèse et l’assemblage de 4 ARNr et 80 protéines ribosomiques (79 chez la levure). La biogenèse du ribosome a longtemps été étudiée chez Saccharomyces cerevisiae. Près de 20 ans de recherches ont été nécessaires à la communauté scientifique pour identifier les quelques 200 facteurs de synthèse du ribosome levurien. Alors que le schéma global de cette voie de biosynthèse semble conservé chez les organismes eucaryotes, de nombreux éléments suggèrent qu’elle serait plus élaborée chez l’homme et nécessiterait un plus grand nombre de facteurs que chez la levure. De plus, la caractérisation de nombreuses ribosomopathies, ou maladies du ribosome prédisposant aux cancers, a suscité un intérêt accru pour l’étude de la voie de biosynthèse du ribosome dans le paradigme expérimental le plus approprié, la cellule humaine.Au cours de ma thèse de doctorat, j’ai contribué à un projet systématique d’identification de facteurs d’assemblage (FA) du ribosome chez l’homme. Pratiquement, nous avons identifié 286 FA humains, dont beaucoup sont homologues aux facteurs levuriens connus, et 74 sont sans équivalent chez la levure. Par ailleurs, j’ai caractérisé en détail certains facteurs. En particulier, Trm112 pour lequel j’ai montré qu’il agit comme un stabilisateur de la méthyltransférase (MTase) Bud23, spécifique à l’ARNr 18S de la sous-unité levurienne 40S. J’ai également participé à la caractérisation de mutations à l’interface du complexe Bud23-Trm112. Enfin, j’ai contribué à l’étude de trois FA que nous avons identifiés chez l’homme, DIMT1L et WBSCR22-TRMT112. J’ai montré que ces protéines sont les orthologues des MTases levurie, Doctorat en sciences, Spécialisation biologie moléculaire, info:eu-repo/semantics/nonPublished

Mon travail de thèse a consisté à caractériser une sous-population de lymphocytes T régulateurs naturels de phénotype CD8+CD25+.Nous avons purifié les CD8+CD25+ nTregs et vérifié par cytométrie de flux leur expression en FOXP3 et CTLA-4. Puis nous avons pu montrer que ces cellules possèdent des propriétés suppressives dans un test d’inhibition de la prolifération de lymphocytes T activés allogéniquement. Les lymphocytes CD8+CD25+ nTregs expriment les gènes FOXP3, CTLA-4, GARP et CCL-4 et les cytokines IL-10 et TGF-β. Par contre, les gènes CD28, ICOS, FOXO1 et Helios sont sous-exprimés dans les nTregs CD8+CD25+ par rapport aux lymphocytes T CD8+CD25-. Nous avons établi une signature micro-ARN qui comprend 10 micro-ARNs différentiellement exprimés :7 micro-ARNs sous-exprimés "miR-9, -24, -31, -155, -210, -335 et -449 " et 3 micro-ARNs surexprimés " miR-214, -205 et -509". De plus, nous avons pu explorer la relevance biologique de cette signature micro-ARN en montrant dans un premier temps que les miRs "-31, -24, -210, -335" ciblent spécifiquement la région 3'UTR de FOXP3, de même les miR-9 et miR-155 ciblent la région 3'UTR de CTLA-4, et les miR-24, et -335 ciblent la région 3'UTR de GARP. Ceci a été fait par des expériences de co-transfections suivies d'une mesure de l'activité rapportrice luciférase. De plus, nous avons pu démontrer par des expériences de transduction lentivirale ex vivo, de cellules T primaires, que des micro-ARNs de la signature régulent l’expression de FOXP3, CTLA-4 et GARP dans les Tregs naturels CD8+CD25+ humains. Cette étude montre l'importance des micro-ARNs dans la régulation post-transcriptionnelle des gènes impliqués dans la fonction régulatrice des lymphocytes T régulateurs., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Extracellular nucleotides act as local intercellular messengers. In response to various stimuli, nucleotides can be released from the cytosol of damaged cells, exocytosis vesicles and efflux through membrane channel. In the extracellular fluids, nucleotides are rapidely degraded by ecto-nucleotidases, such as CD39,CD39L1 and CD73. Extracellular nucleotides activate the P2 receptor family .This family of receptors can be divided into 2 groups: P2X1-7R ( ionotropic receptors) and P2YR ( G protein-coupled receptors). Nowadays, there are eight accepted human P2Y receptors: P2Y1,2,4,6,11,12,13,14.To determine the physiological roles of P2Y receptor family, our laboratory generated different strains of P2Y knockout mice (P2Y4 ,6 and 13). In collaboration with A. Gartland ,I. And Arnett TR Orriss ( Sheffield and London University) ,it has been observed that the P2Y13R-/- mice exhibit an impaired bone tissue metabolism that leads to a reduction in the volume of the femoral trabecular bone and the number of trabeculae. This phenotype is correlated with the decrease in the number of osteoblasts at the endo-cortical bone surface .We therefore examined whether P2Y13 R activation was involved in the osteogenic differentiation of mesenchymal stem cells (MSCs). In the first part of our work we have shown that :Induction of the MSCs differentiation is associated with the release of ATP and its conversion to ADP (agonist of the P2Y13R). ATP release probably involves the pannexine1 while the conversion of ATP into ADP is probably due to the activity of the ecto-nucleotidase CD39L1.ADP stimulation of P2Y13 R+/+ (but not P2Y13 R-/-) adherent bone marrow stromal cells (BMSC) increased significantly the formation of alkaline phosphatase-colony forming units (CFU-ALP), as well as the expression of osteoblastic genes such as Osterix involved in the maturation of pre-osteoblasts into osteoblasts. The number of CFU-ALP obtained from P2Y13 R-/- BMSC and the level of osteoblastic g, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Regulation of gene expression occurs at several levels in a cell. While control of transcription is often viewed as the main source of regulation, it is now clear that post-transcriptional processes are essential to fine-tune protein availability. The presence of AU-rich elements (ARE) in the 3’ untranslated region (3’UTR) of many important mRNAs exemplifies one such process. AREs alter the mRNA translation or degradation status by recruiting ARE-binding proteins (ARE-BP). ARE-BPs of the TTP/TIS11 family bind to their cognate ARE-RNAs using their conserved tandem zinc-finger domain and induce rapid decay of their targets. This allows for proper regulation of cell proliferation, cell death and inflammation. In this regard, TTP/TIS11 are main regulators of gene expression, and as such are put under strict transcriptional, post-transcriptional as well as several layers of post-translational control.In this work, we aimed at elucidating the degradation mechanisms affecting TTP/TIS11. Using Drosophila as a model, we found that dTIS11 protein turnover is rapid due to continuous degradation by the proteasome. However, proteasomal recognition did not require ubiquitination of dTIS11 as non-ubiquitinable mutants were efficiently degraded by the proteasome. In addition, dTIS11 was digested by the 20S proteasome that lacks ubiquitin-recognition domains. Our results further indicate that intrinsically disordered regions (IDR) in dTIS11 may be responsible for proteasomal recognition. In fact, dTIS11 is predicted as disordered and possesses the main characteristics of intrinsically disordered proteins (IDP). We also identified dTIS11 N- and C-terminal domains as functional signals for degradation, potentially due to their destructuration. This ubiquitination-independent, disorder-dependent degradation process is conserved throughout evolution as dTIS11 mammalian counterpart, TTP, undergoes the same degradation by default pathway. In addition, we established that phosphorylatio, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Cardiovascular diseases are considered the first cause of death in westernized societies. They are directly linked to atherosclerosis, a clinical condition characterized by a thickening of the arterial wall. Atherosclerosis is in his turn linked to various genetic and environmental factors; among those factors are high oxidized LDL levels and endothelial dysfunction. In the present study, we have analyzed in vitro the effect of myeloperoxidase oxidized LDL on endothelial cells at the level of fibrinolysis and cell motility.In the first part of the work, we measured fibrinolysis in real time at the surface of endothelial cells. Our results suggest that myeloperoxidase oxidized LDL interferes with the regulation of fibrinolysis by endothelial cells by decreasing their pro-fibrinolytic activity. This effect was not related to a modification in expression of major regulators of fibrinolysis such as PAI-1 and t-PA. Our data link the current favorite hypothesis that oxidized LDL has a causal role in atheroma plaque formation with an old suggestion that fibrin may also play a causal role. A model that best explains our results would be as follows: oxidized LDL increases fibrin deposition on endothelial cells which will increase their permeability resulting in more oxidized LDL infiltration into the subendothelial space of the arterial wall initiating atherogenesis. In the second part of the work, we investigated the effect of myeloperoxidase oxidized LDL at the level of endothelial cell motility. We have shown that oxidized LDL is able to decrease cell migration, wound healing and tubulogenesis in endothelial cells. Those effects were not associated with any alteration at the level of neither cell viability nor proliferation. Subsequent gene expression analyses enabled us to link the oxidized LDL induced phenotypical changes in the cells to a change in expression of both microRNA-22 and Heme Oxygenase 1 genes. Our observations suggest a novel role of oxidized LDL not, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Nitrogen Catabolite Repression (NCR) is the regulatory pathway through which Saccharomyces cerevisiae reduces the expression of genes encoding components involved in the utilization of poor nitrogen sources when rich ones are available. Expression of NCR-sensitive genes is controlled by the negative regulator Ure2 and four DNA-binding GATA-like transcription factors: two activators (Gln3 and Gat1) and two repressors (Dal80 and Gzf3). In the presence of preferred nitrogen sources, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner, whereas upon growth under non-preferred nitrogen conditions, the GATA activators relocate to the nucleus and mediate the transcription of NCR-sensitive genes. Even though the Target of Rapamycin Complex 1 (TORC1) as well as several phosphatases are involved in regulating Gln3 and Gat1 subcellular localization, a detailed mechanistic understanding of the NCR process is still lacking. In the first part of this work, we have shown that class C and D VPS (vacuolar protein sorting) components, involved in Golgi-to-vacuole vesicular trafficking, are required for intact Gat1 and Gln3 nuclear localization in response to TORC1-inhibiting rapamycin treatment or upon shifting cells from rich to poor nitrogen conditions. The requirements of Vps proteins for Gln3 function are media-specific: a requirement after rapamycin treatment was observed in minimal but not in rich medium. Moreover, we have seen that a significant fraction of Gat1, like Gln3, is associated with light intracellular membranes. These observations support the view that GATA factor regulation in response to nitrogen signals seems to occur at intracellular compartments.In a second step, we confirmed an important role for the anabolic glutamate dehydrogenase (Gdh1) within NCR, through the control of Gat1 function. However, since we observed a strong correlation between the anabolic activity of Gdh1 and its NCR regulatory capacity, we do not exclude that an al, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The process of specific gene transcription by RNA polymerase II (Pol II) is initiated by thebinding of specific transcription factors to DNA. A global understanding of the mechanisms of genetranscriptional regulation of Saccharomyces cerevisiae goes through the description of the targets andthe behavior of those transcription factors.The GATA factors are specific transcription factors intervening in the regulation of NitrogenCatabolite Repression (NCR)-sensitive genes, a mechanism encompassing the transcriptionalregulations leading to the preferential use of good nitrogen sources of the growth medium of yeast inthe presence of less good nitrogen sources. Those 4 GATA factors involved in NCR comprise 2activators (Gat1 and Gln3) and 2 repressors (Gzf3 and Dal80).Generally speaking, the promoters of genes have always been described like the main place forthe integration of the transcription regulation signals relayed by the general and specific transcriptionfactors and the chromatin remodeling factors. Furthermore, the GATA factors have been described asintegrating the external signals of nitrogen availability thanks to their specific DNA binding toconsensus GATA sequences in the promoter of NCR-sensitive genes. The results presented hereintroduce many nuances to the model, notably implying new proteins but also new regions in theregulation process of the NCR-sensitive gene regulation. Indeed, the first goal of this work is todiscover and understand the mechanisms of NCR-sensitive gene regulation that will explain thevariations in their expression levels in the presence of various nitrogen sources and their dependencytowards the GATA factors.Strikingly, it appeared that GATA factor positioning was not limited to the promoter, butoccurred also in the transcribed region. It seems that the transcription factors may have been drivenby the general transcription machinery (Pol II). The binding of a chromatin remo, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

L’objectif de notre travail a été de caractériser le rôle de l’IL-6 dans la différenciation des lymphocytes Tfh et des lymphocytes Th2. Les lymphocytes Tfh ont pour fonction d’aider les lymphocytes B à produire des anticorps indispensables pour nous protéger contre divers pathogènes. Les lymphocytes Th2, quant à eux, sont spécialisés dans l’élimination de parasites extracellulaires tels que les helminthes.Dans un premier temps, nous avons voulu identifier les gènes dont l’expression est induite par l’IL-6, avec comme objectif une meilleure compréhension des mécanismes permettant aux lymphocytes T de se différencier en cellules Tfh.Au cours de notre travail, nous avons identifié le facteur de transcription, MyoR (Myogenic Repressor) comme étant exprimé au sein des lymphocytes T helper et dont l’expression est induite par l’IL-6. Nos observations expérimentales ont démontré que le facteur MyoR n’est pas indispensable pour la différenciation des lymphocytes Tfh, ni pour leur fonction. Cependant, l’expression de l’ARNm codant pour MyoR pourrait être utilisée comme un biomarqueur des cellules Tfh in vitro ou in vivo.Nous avons ensuite caractérisé la réponse immune induite in vivo par des cellules présentatrices d’antigènes issues de souris déficientes pour l’IL-6. Cette approche nous a permis de mettre en évidence le rôle immunosuppresseur de l’IL-6 sur le développement des réponses de type Th2. En effet, nous avons montré que l’injection de BMDCs (Bone Marrow derived dendritic cells) IL-6-/- dans des souris receveuses de type sauvage induisent une réponse Th2 augmentée in vivo.Nos résultats suggèrent que l’inhibition de la réponse Th2 par l’IL-6 in vivo et in vitro pourrait impliquer la présence d’un ou de plusieurs miRNAs.Cette inhibition pourrait être un mécanisme de rétrocontrôle afin d’éviter une exacerbation de la réponse immune Th2., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

Cardiovascular disease linked to atherosclerosis is the leading cause of death worldwide. Atherosclerosis is mainly linked to dysfunction in vascular endothelial cells and subendothelial accumulation of oxidized forms of LDL. In the present study, we investigated the role of myeloperoxidase oxidized LDL (Mox-LDL) in endothelial cell dysfunction. We studied the effect of proinflammatory Mox-LDL treatment on endothelial cell motility, a parameter essential for normal vascular processes such as angiogenesis and blood vessel repair. This is particularly important in the context of an atheroma plaque, where vascular wall integrity is affected and interference with its repair could contribute to progression of the disease. We investigated in vitro the effect of Mox-LDL on endothelial cells angiogenic properties and we also studied the signalling pathways that could be affected by analysing Mox-LDL effect on the expression of angiogenesis-related genes. We report that Mox-LDL inhibits endothelial cell motility and tubulogenesis through an increase in miR-22 and heme oxygenase 1 expression. Our in vitro data indicate that Mox-LDL interferes with parameters associated with angiogenesis. They suggest that high LDL levels in patients would impair their endothelial cell capacity to cope with a damaged endothelium contributing negatively to the progression of the atheroma plaque., SCOPUS: ar.j, info:eu-repo/semantics/published

Objective Plasma and synovial myeloperoxidase (MPO) and its products were strongly associated with osteoarthritis (OA) and rheumatoid arthritis (RA). In addition, it is well known that there is a link between oxidative stress and cytokines. The present study aims at investigating the link between synovial MPO (and its products), IL-18, which is involved in the degradation of articular cartilage in RA, and IL-8, which is involved in recruitment and activation of neutrophils during inflammation. Effects of the treatment of RA on the biological parameters were also investigated. Methods Patients (n=105) were studied including 39 patients with OA, 33 with RA and 33 with RA receiving a specific treatment. DAS-28 was calculated whereas MPO antigen/activity, neutrophils, chloro-tyrosine, homocitrulline, IL-8 and IL-18 were measured in synovial fluid (SF) and CRP was measured in serum. Results DAS-28 and CRP level were not significantly different between groups. MPO activity, and MPO, chloro-tyrosine and homocitrulline levels were significantly higher in SF of RA patients than OA patients. MPO specific activity (MPO activity/antigen ratio) was significantly lower in treated than in untreated RA patients as was IL-8. MPO activity and concentration were correlated with IL-8 and IL-18 in untreated but not in treated RA patients. Conclusions MPO level is related to IL-8 and IL-18 levels in untreated RA patients. A link has been shown between treatment and decrease of IL-8, MPO specific activity and homocitrulline in SF. The causal role of MPO in SF inflammation and how treatment can affect MPO specific activity need further investigations., JOURNAL ARTICLE, SCOPUS: ar.j, info:eu-repo/semantics/published

Background: In order to promote infection, the blood-borne parasite Trypanosoma brucei releases factors that upregulate arginase expression and activity in myeloid cells. Methodology/Principal findings: By screening a cDNA library of T. brucei with an antibody neutralizing the arginaseinducing activity of parasite released factors, we identified a Kinesin Heavy Chain isoform, termed TbKHC1, as responsible for this effect. Following interaction with mouse myeloid cells, natural or recombinant TbKHC1 triggered SIGN-R1 receptordependent induction of IL-10 production, resulting in arginase-1 activation concomitant with reduction of nitric oxide (NO) synthase activity. This TbKHC1 activity was IL-4Ra-independent and did not mirror M2 activation of myeloid cells. As compared to wild-type T. brucei, infection by TbKHC1 KO parasites was characterized by strongly reduced parasitaemia and prolonged host survival time. By treating infected mice with ornithine or with NO synthase inhibitor, we observed that during the first wave of parasitaemia the parasite growth-promoting effect of TbKHC1-mediated arginase activation resulted more from increased polyamine production than from reduction of NO synthesis. In late stage infection, TbKHC1- mediated reduction of NO synthesis appeared to contribute to liver damage linked to shortening of host survival time. Conclusion: A kinesin heavy chain released by T. brucei induces IL-10 and arginase-1 through SIGN-R1 signaling in myeloid cells, which promotes early trypanosome growth and favors parasite settlement in the host. Moreover, in the late stage of infection, the inhibition of NO synthesis by TbKHC1 contributes to liver pathogenicity.

MicroRNAs (miRNAs) are small (19-22-nt) single-stranded noncoding RNA molecules whose deregulation of expression can contribute to human disease including the multistep processes of carcinogenesis in human. Circulating miRNAs are emerging biomarkers in many diseases and cancers such as type 2 diabetes, pulmonary disease, colorectal cancer, and gastric cancer among others; however, defining a plasma miRNA signature in acute myeloblastic leukemia (AML) that could serve as a biomarker for diagnosis or in the follow-up has not been done yet., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

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The nucleo-cytoplasmic compartmentalization enables eukaryotic cells to develop sophisticated post-transcriptional regulations of gene expression. However, managing the exchanges of macromolecules between the two compartments also represents a formidable challenge for the cells. Nucleo-cytoplasmic exchanges rely on specialized soluble carriers and take place at nuclear pore complexes that span the nuclear envelope. Active nucleo-cytoplasmic transport of proteins, in particular, is performed mainly by a family of carriers called karyopherins, which includes about twenty members in mammals. Some of them, called importins, recognize nuclear localization signals (NLSs) in their substrates and convey them into the nucleus. Others, called exportins, recognize nuclear export signals (NESs) in their substrates and bring them back to the cytoplasm. Many RNA-binding proteins (RBPs) shuttle between the nucleus and the cytoplasm, where they can often fulfill different functions. RBPs also frequently localize into specialized microdomains that are not delimited by a membrane but in which specific factors are concentrated. Those include processing bodies and stress granules, which are cytoplasmic foci associated with mRNA decay, storage and translational repression. Post-transcriptional regulations mediated by RBPs can therefore be modulated rapidly and efficiently through changes in the localization of RBPs.The first part of this work focuses on the subcellular localization and nucleo-cytoplasmic transport of the Drosophila RBP dTIS11. Like its mammalian and yeast homologues, dTIS11 binds AU-rich elements in the 3’UTR of its target mRNAs, and stimulates their rapid deadenylation and decay. Here, we have observed that although dTIS11 appears to be located mostly in the cytoplasm, it is constantly shuttling in and out of the nucleus. We show that the export of dTIS11 from the nucleus depends on the CRM1 exportin and is mediated by a hydrophobic NES that encompasses residues, Doctorat en Sciences, info:eu-repo/semantics/nonPublished

FKBP12 proteins are able to inhibit TOR kinases or calcineurin phosphatases upon binding of rapamycin or FK506 drugs, respectively. The Trypanosoma brucei FKBP12 homologue (TbFKBP12) was found to be a cytoskeleton-associated protein with specific localization in the flagellar pocket area of the bloodstream form. In the insect procyclic form, RNA interference-mediated knockdown of TbFKBP12 affected motility. In bloodstream cells, depletion of TbFKBP12 affected cytokinesis and cytoskeleton architecture. These last effects were associated with the presence of internal translucent cavities limited by an inside-out configuration of the normal cell surface, with a luminal variant surface glycoprotein coat lined up by microtubules. These cavities, which recreated the streamlined shape of the normal trypanosome cytoskeleton, might represent unsuccessful attempts for cell abscission. We propose that TbFKBP12 differentially affects stage-specific processes through association with the cytoskeleton., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published