Les voies de signalisation des MAP kinases (MAPK) conventionnelles jouent des rôles essentiels pendant le développement des lymphocytes T (LT) ainsi que lors de leur activation suite à la reconnaissance antigénique. En raison de ses différences structurelles ainsi que de son mode de régulation, ERK3 fait partie des MAPK dites non-conventionnelles. Encore aujourd’hui, les événements menant à l’activation de ERK3, ses substrats ou partenaires ainsi que sa fonction physiologique demeurent peu caractérisés. Nous avons entrepris dans cette thèse d’étudier le rôle de ERK3 lors du développement et de l’activation des LT en utilisant un modèle de souris déficient pour l’expression de ERK3. Nous avons premièrement établi que ERK3 est exprimée chez les thymocytes. Ensuite, nous avons évalué le développement thymique chez la souris ERK3-déficiente et nous avons observé une diminution significative de la cellularité aux étapes DN1, DP et SP CD4+ du développement des LT. La création de chimères hématopoïétiques ERK3-déficientes nous a permis de démontrer que la diminution du nombre de cellules observée aux étapes DN1 et DP est autonome aux thymocytes alors que le phénotype observé à l’étape SP CD4+ est dépendant de l’abolition simultanée de ERK3 dans l’épithélium thymique et dans les thymocytes. Une étude plus approfondie de l’étape DP nous a permis de démontrer qu’en absence de ERK3, les cellules DP meurent plus abondamment et accumulent des cassures doubles brins (DSB) dans leur ADN. De plus, nous avons démontré que ces cassures dans l’ADN sont réalisées par les enzymes RAG et qu’en absence de ces dernières, la cellularité thymique est presque rétablie chez la souris ERK3-déficiente. Ces résultats suggèrent que ERK3 est impliquée dans un mécanisme essentiel à la régulation des DSB pendant le réarrangement V(D)J de la chaîne  du récepteur des cellules T (RCT). Dans le deuxième article présenté dans cette thèse, nous avons montré que ERK3 est exprimé chez les LT périphériques, mais seulement suite à leur activation via le RCT. Une fois activés in vitro les LT ERK3-déficients présentent une diminution marquée de leur prolifération et dans la production de cytokines. De plus, les LT ERK3-déficients survivent de façon équivalente aux LT normaux, mais étonnamment, ils expriment des niveaux plus faibles de la molécule anti-apoptotique Bcl-2. Ces résultats suggèrent que la prolifération réduite des LT ERK3-déficients est la conséquence d’une altération majeure de leur activation. Ainsi, nos résultats établissent que ERK3 est une MAPK qui joue des rôles essentiels et uniques dans le développement thymique et dans l’activation des lymphocytes T périphériques. Grâce à ces travaux, nous attribuons pour la toute première fois une fonction in vivo pour ERK3 au cours de deux différentes étapes de la vie d’un LT. / Classical MAP kinases (MAPK) play essential roles during T cell development and activation. ERK3 is a member of the MAPK family for which no physiological function has been described yet. Also, ERK3 is an atypical MAPK since its structure and mode of regulation are different from the conventional MAPK. Even today, the events leading to ERK3 activation and its substrates or partners are still largely unknown. We have studied in this thesis the role of ERK3 during T cell development and activation by using a mouse model in which ERK3 is not expressed. First, we have established that ERK3 is expressed in thymocytes. Next, we have evaluated thymic development in ERK3-deficient mice and we have observed a significant decrease in cell number at DN1, DP and CD4SP stages of T cell development. ERK3-deficient hematopoietic chimeras revealed that the DN1 and DP phenotype are T-cell autonomous, while abrogation of CD4SP development requires ERK3-deficiency in both thymocytes and thymic epithelium. By investigating further the DP stage, we have shown that ERK3-deficient DP thymocytes are more prone to apoptosis and also accumulate DNA double-strand breaks (DSBs). Moreover, we have shown that the increase DSBs are the direct consequence of RAG activity and that abolition of RAG almost restored thymic cellularity in ERK3-deficient mice. These results suggest that ERK3 is involved in an essential mechanism of DBSs regulation during TCR recombination. In the second article presented in this thesis, we have shown that ERK3 is expressed in peripheral T cell, but only when their TCR is activated. Also, ERK3-deficient T cells presented a strong reduction in proliferation and cytokine secretion following in vitro stimulation. Moreover, activated T cells lacking ERK3 are not more prone to death and surprisingly, they are unable to up-regulate the expression of the anti-apoptotic molecule Bcl-2 following TCR stimulation. These results suggest that the reduced proliferation of ERK3-deficient T cells is a consequence of their defective activation. Collectively, our results unveil essential and unsuspected roles for ERK3 in T cell development and activation. With this study, we establish for the first time an in vivo function for the atypical MAPK ERK3 in two different stages during T cell life.

The developmental processes of lymphopoiesis generate mature B lymphocytes from hematopoietic stem cells through increasingly restricted intermediates. Networks of transcription factors regulate these cell fate choices and are composed of both ubiquitously expressed and B lineage-specific factors. E-protein transcription factors are encoded by the three genes E2A, E2-2 (SEF2-1), and HEB. The E2A gene is required for B cell development and encodes the alternatively spliced proteins E12 and E47. During B lymphocyte development, the cells have to pass several checkpoints verifying the functionality of their antigen receptors. Early in the development, the expression of a pre-B cell receptor (pre-BCR) with membrane-bound immunoglobulin (Ig) heavy chain protein associated with surrogate light chain (SLC) proteins is a critical checkpoint that monitors for functional Ig heavy chain rearrangement. Signaling from the pre-BCR induces survival and a limited clonal expansion. Here it is shown that pre-BCR signaling rapidly down-regulates the SLCs l5 and VpreB and also the co-receptor CD19. Ca2+ signaling and E2A were shown to be essential for this regulation. E2A mutated in its binding site for the Ca2+ sensor protein calmodulin (CaM), and thus with CaM-resistant DNA binding, makes l5, VpreB and CD19 expression resistant to the inhibition following pre-BCR stimulation. Thus, Ca2+ down-regulates SLC and CD19 gene expression upon pre-BCR stimulation through inhibition of E2A by Ca2+/CaM. A general negative feedback regulation of the pre-BCR proteins as well as many co-receptors and proteins in signal pathways from the receptor was also shown. After the ordered recombination of Ig heavy chain gene segments, also Ig light chain gene segments are recombined together to create antibody diversity. The recombinations are orchestrated by the recombination activating gene (RAG) enzymes, other enzymes that cleave/mutate/assemble DNA of the Ig loci, and the transcription factor Pax5. A key feature of the immune system is the concept that one lymphocyte has only one antigen specificity that can be selected for or against. This requires that only one of the alleles of genes for Ig chains is made functional. The mechanism of this allelic exclusion has however been an enigma. Here pre-BCR signaling was shown to down-regulate several components of the recombination machinery including RAG1 and RAG2 through CaM inhibition of E2A. Furthermore, E2A, Pax5 and the RAGs were shown to be in a complex bound to key sequences on the IgH gene before pre-BCR stimulation and instead bound to CaM after this stimulation. Thus, the recombination complex is directly released through CaM inhibition of E2A. Upon encountering antigens, B cells must adapt to produce a highly specific and potent antibody response. Somatic hypermutation (SH), which introduces point mutations in the variable regions of Ig genes, can increase the affinity for antigen, and antibody effector functions can be altered by class switch recombination (CSR), which changes the expressed constant region exons. Activation-induced cytidine deaminase (AID) is the mutagenic antibody diversification enzyme that is essential for both SH and CSR. The AID enzyme has to be tightly controlled as it is a powerful mutagen. BCR signaling, which signals that good antibody affinity has been reached, was shown to inhibit AID gene expression through CaM inhibition of E2A.  SH increases the antigen binding strength by many orders of magnitude. Each round of SH leads to one or a few mutations, followed by selection for increased affinity. Thus, BCR signaling has to enable selection for successive improvements in antibodies (Ab) over an extremely broad range of affinities. Here the BCR is shown to be subject to general negative feedback regulation of the receptor proteins as well as many co-receptors and proteins in signal pathways from the receptor. Thus, the BCR can down-regulate itself to enable sensitive detection of successive improvements in antigen affinity. Furthermore, the feedback inhibition of the BCR signalosome and most of its protein, and most other gene regulations by BCR stimulation, is through inhibition of E2A by Ca2+/CaM. Differentiation to Ab-secreting plasmablasts and plasma cells is antigen-driven. The interaction of antigen with the membrane-bound Ab of the BCR is critical in determining which clones enter the plasma cell response. Genome-wide analysis showed that differentiation of B cells to Ab-secreting cell is induced by BCR stimulation through very fast regulatory events, and induction of IRF-4 and down-regulation of Pax5, Bcl-6, MITF, Ets-1, Fli-1 and Spi-B gene expressions were identified as immediate early events. Ca2+ signaling through CaM inhibition of E2A was essential for these rapid down-regulations of immediate early genes after BCR stimulation in initiation of plasma cell differentiation.

Les voies de signalisation des MAP kinases (MAPK) conventionnelles jouent des rôles essentiels pendant le développement des lymphocytes T (LT) ainsi que lors de leur activation suite à la reconnaissance antigénique. En raison de ses différences structurelles ainsi que de son mode de régulation, ERK3 fait partie des MAPK dites non-conventionnelles. Encore aujourd’hui, les événements menant à l’activation de ERK3, ses substrats ou partenaires ainsi que sa fonction physiologique demeurent peu caractérisés. Nous avons entrepris dans cette thèse d’étudier le rôle de ERK3 lors du développement et de l’activation des LT en utilisant un modèle de souris déficient pour l’expression de ERK3. Nous avons premièrement établi que ERK3 est exprimée chez les thymocytes. Ensuite, nous avons évalué le développement thymique chez la souris ERK3-déficiente et nous avons observé une diminution significative de la cellularité aux étapes DN1, DP et SP CD4+ du développement des LT. La création de chimères hématopoïétiques ERK3-déficientes nous a permis de démontrer que la diminution du nombre de cellules observée aux étapes DN1 et DP est autonome aux thymocytes alors que le phénotype observé à l’étape SP CD4+ est dépendant de l’abolition simultanée de ERK3 dans l’épithélium thymique et dans les thymocytes. Une étude plus approfondie de l’étape DP nous a permis de démontrer qu’en absence de ERK3, les cellules DP meurent plus abondamment et accumulent des cassures doubles brins (DSB) dans leur ADN. De plus, nous avons démontré que ces cassures dans l’ADN sont réalisées par les enzymes RAG et qu’en absence de ces dernières, la cellularité thymique est presque rétablie chez la souris ERK3-déficiente. Ces résultats suggèrent que ERK3 est impliquée dans un mécanisme essentiel à la régulation des DSB pendant le réarrangement V(D)J de la chaîne  du récepteur des cellules T (RCT). Dans le deuxième article présenté dans cette thèse, nous avons montré que ERK3 est exprimé chez les LT périphériques, mais seulement suite à leur activation via le RCT. Une fois activés in vitro les LT ERK3-déficients présentent une diminution marquée de leur prolifération et dans la production de cytokines. De plus, les LT ERK3-déficients survivent de façon équivalente aux LT normaux, mais étonnamment, ils expriment des niveaux plus faibles de la molécule anti-apoptotique Bcl-2. Ces résultats suggèrent que la prolifération réduite des LT ERK3-déficients est la conséquence d’une altération majeure de leur activation. Ainsi, nos résultats établissent que ERK3 est une MAPK qui joue des rôles essentiels et uniques dans le développement thymique et dans l’activation des lymphocytes T périphériques. Grâce à ces travaux, nous attribuons pour la toute première fois une fonction in vivo pour ERK3 au cours de deux différentes étapes de la vie d’un LT. / Classical MAP kinases (MAPK) play essential roles during T cell development and activation. ERK3 is a member of the MAPK family for which no physiological function has been described yet. Also, ERK3 is an atypical MAPK since its structure and mode of regulation are different from the conventional MAPK. Even today, the events leading to ERK3 activation and its substrates or partners are still largely unknown. We have studied in this thesis the role of ERK3 during T cell development and activation by using a mouse model in which ERK3 is not expressed. First, we have established that ERK3 is expressed in thymocytes. Next, we have evaluated thymic development in ERK3-deficient mice and we have observed a significant decrease in cell number at DN1, DP and CD4SP stages of T cell development. ERK3-deficient hematopoietic chimeras revealed that the DN1 and DP phenotype are T-cell autonomous, while abrogation of CD4SP development requires ERK3-deficiency in both thymocytes and thymic epithelium. By investigating further the DP stage, we have shown that ERK3-deficient DP thymocytes are more prone to apoptosis and also accumulate DNA double-strand breaks (DSBs). Moreover, we have shown that the increase DSBs are the direct consequence of RAG activity and that abolition of RAG almost restored thymic cellularity in ERK3-deficient mice. These results suggest that ERK3 is involved in an essential mechanism of DBSs regulation during TCR recombination. In the second article presented in this thesis, we have shown that ERK3 is expressed in peripheral T cell, but only when their TCR is activated. Also, ERK3-deficient T cells presented a strong reduction in proliferation and cytokine secretion following in vitro stimulation. Moreover, activated T cells lacking ERK3 are not more prone to death and surprisingly, they are unable to up-regulate the expression of the anti-apoptotic molecule Bcl-2 following TCR stimulation. These results suggest that the reduced proliferation of ERK3-deficient T cells is a consequence of their defective activation. Collectively, our results unveil essential and unsuspected roles for ERK3 in T cell development and activation. With this study, we establish for the first time an in vivo function for the atypical MAPK ERK3 in two different stages during T cell life.

T cells, which constitute an essential arm in the adaptive immunity, complete their development in the thymus through a process called thymopoiesis. However, thymic involution can be induced by a couple of factors, which impairs T cell functions and is slow to recover. Therefore, understanding how thymopoiesis is regulated may lead effort to accelerate thymic recovery and improve immune functions in thymocyte-depleted patients. In this project, we identified scavenger receptor BI (SR-BI), a high density lipoprotein (HDL) receptor, as a novel modulator in thymopoiesis. In mice, absence of SR-BI causes a significant reduction in thymus size after puberty and a remarkable decrease in thymic output. Consequently, SR-BI-null mice show a narrowed naïve T cell pool in the periphery and blunted T cell responses, indicating that the impaired thymopoiesis due to SR-BI deficiency leads to compromised T cell homeostasis and functions. The impaired thymopoiesis of SR-BI-null mice is featured by a significant reduction in the percentage of earliest T progenitors (ETPs) but unchanged percentages of other thymocyte subtypes, suggesting that SR-BI deficiency causes a reduction in progenitor thymic entry. Further investigations reveal that SR-BI deficiency impairs thymopoiesis through affecting bone marrow progenitor thymic homing without influencing the lymphoid progenitor development in bone marrow. Importantly, SR-BI-null mice exhibit delayed thymic recovery after sublethal irradiation, indicating that SR-BI is also required for thymic regeneration. Using bone marrow transplantation models, we elucidate that it is non-hematopoietic rather than hematopoietic SR-BI deficiency that results in the defects in thymopoiesis. However, SR-BI deficiency-induced hypercholesterolemia is not responsible for the impaired thymopoiesis. Using adrenal transplantation models, we found that absence of adrenal SR-BI is responsible for the impaired thymopoiesis, as shown by that adrenalectomized mice transplanted with SR-BI-null adrenal gland display reduced thymus size, decreased percentage of ETPs and delayed thymic regeneration compared with those transplanted with wild-type adrenal. Altogether, results from this study elucidate a previously unrecognized role of SR-BI in thymopoiesis. We reveal that SR-BI expressed in adrenal gland is critical in maintaining normal T cell development and enhancing thymic regeneration, providing novel links between adrenal functions and T cell development.

Human leukocyte antigen (HLA)-mismatched haematopoietic stem cell transplantation (HSCT) represents an important therapeutic option for patients lacking suitable donors. Delayed posttransplant immune recovery constitutes one of its major complications and is most pronounced in the T cellular compartment. A novel strategy to promote de novo thymopoiesis from donor derived HSCs and to accelerate T cellular reconstitution in patients after HSCT consists in the adoptive transfer of in vitro generated T cell progenitor cells. Identification of Notch1 as the key regulator of early T-lineage development has allowed the generation of Notch ligand-based culture systems, which provide a powerful tool to generate T-lymphoid progenitors in vitro. The efficacy of murine T-lymphoid progenitors to promote T cell reconstitution has been well demonstrated in conventional mouse models. In consistency, in vitro-generated human T cell progenitors were demonstrated to promote thymic recovery in humanized mice. Yet, positive effects of in vitro generated human T cell precursors on peripheral T cell reconstitution have not been demonstrated. Moreover currently used Notch-based co-culture systems consist of genetically modified murine cell lines. With view to establishing a clinically applicable system, feeder-cell-free Notch-ligand culture systems for the generation of T-lymphopoietic progenitors are warranted. During my PhD project I developed a new culture system based on the immobilized Notch ligand Delta-like-4 (DL-4). Exposure of human CD34+ cord blood cells to immobilized DL-4 enabled the in vitro generation of high number of T cell progenitors, which harboured the phenotype of immature early thymic progenitor cells (ETP) and prothymocytes (proT). ETP and proT cell generated during DL-4 culture upregulated essential genes involved in early T-lymphoid development (i.e. IL7Rα, PTα, RAG1 and BCL11b) and had undergone stage-specific recombination of the T cell receptor (TCR) locus in a similar way as in native human thymopoiesis. In limiting dilution analysis after secondary OP9/DL-1 co-culture, DL-4 progenitors displayed a highly increased T-lymphoid potential, which could be entirely attributed to the ETP and proT subset. When transferred into NOD/SCID/γc-/- mice, DL-4 primed T cell progenitors migrated to the thymus and accelerated intrathymic T cell differentiation and emergence of functional, mature and polyclonal αβ T cells in the periphery. In a co-transplantation approach, which more closely mimics a clinical setting, DL-4 progenitors and untreated CD34+ cells from HLA-disparate donors were simultaneously injected in the same recipient. This procedure allowed even more rapid and more robust T cell reconstitution. HLA-tracking of the distinct graft sources further showed, that DL-4 progenitors specifically reconstituted the T-lymphoid compartments. This work provides further evidence for the ability of in vitro-generated human T cell progenitors to promote de novo thymopoiesis and shows for the first time, that these cells accelerate peripheral T cell reconstitution in humanized mice. The availability of the efficient feeder-cell-free DL-4 culture technique represents an important step towards the future clinical exploitation translation of in vitro generated T-lymphoid progenitor cells to improve posttransplant immune reconstitution

The development of an organism and its adult homeostasis rely on regulatory mechanisms that control the underlying gene expression programs. In certain biological contexts, such as germ cell development, gene expression regulation is largely executed at the post-­‐transcriptional level. This relies on RNA-­‐binding proteins (RBPs), whose activity and expression are also heavily controlled. While the RNA-­‐binding potential of RBPs is currently of intense scrutiny, surprisingly little is known to date about the molecular mechanisms that control RNA-­‐binding proteins abundance in the context of germ cell development. This work identifies the molecular mechanisms that shape expression patterns of two evolutionarily conserved RNA-­‐binding proteins, CPB-­‐3 and GLD-­‐ 1, which belong to CPEB and STAR protein family, respectively. By focusing on their regulation in the C. elegans germ line, this work reveals an involvement of the proteasome in reducing levels of CPB-­‐3/CPEB and GLD-­‐1/STAR at the pachytene-­‐to-­‐diplotene transition during meiotic prophase I. Furthermore, it documents that CPB-­‐3 and GLD-­‐1 are targeted to proteasomal degradation by a conserved SCF ubiquitin ligase complex that utilises SEL-­‐10/Fbxw7 as a substrate recognition subunit. Importantly, destabilisation of both RBPs is likely triggered by their phosphorylation, which is regulated by the mitogen-­‐activated protein kinase, MPK-­‐1, and restricted to the meiotic timepoint of pachytene exit. Lastly, this work investigates the potential consequences of target mRNA regulation upon delayed RBP degradation. Altogether, the collected data characterise a molecular pathway of CPEB and STAR protein turnover, and suggest that MPK-­‐1 signaling may couple RBP-­‐mediated regulation of gene expression to progression through meiosis during oogenesis.

Signaling cascades, such as the extracellular signal-regulated kinase (ERK) pathway, play vital roles in early vertebrate development. Signals through these pathways are initiated by a growth factor or hormone, are transduced through a kinase cascade, and result in the expression of specific downstream genes that promote cellular proliferation, growth, or differentiation. Tight regulation of these signals is provided by positive or negative modulators at varying levels in the pathway, and is required for proper development and function. Two members of the dual-specificity phosphatase (Dusp) family, dusp6 and dusp2, are believed to be negative regulators of the ERK pathway and are expressed in both embryonic and adult zebrafish, but their specific roles in gametogenesis and embryogenesis remain to be fully understood. Using CRISPR/Cas9 genome editing technology, we generated zebrafish lines harboring germ line deletions in dusp6 and dusp2. We do not detect any overt defects in dusp2 mutants, but we find that approximately 50% of offspring from homozygous dusp6 mutants do not proceed through embryonic development. These embryos are fertilized, but are unable to proceed past the first zygotic mitosis and stall at the one-cell stage for several hours before dying by 10 hours post fertilization. We demonstrate that dusp6 is expressed in the gonads of both male and female zebrafish, suggesting that loss of dusp6 causes defects in germ cell production. Notably, the 50% of homozygous dusp6 mutants that complete the first cell division appear to progress through embryogenesis normally and give rise to fertile adults. The fact that offspring of homozygous dusp6 mutants stall at the one-cell stage, prior to activation of the zygotic genome, suggests that loss of dusp6 affects gametogenesis. Further, since only approximately 50% of homozygous dusp6 mutants are affected, we postulate that ERK signaling is tightly regulated and that dusp6 is required to keep ERK signaling within a range that is permissive for gametogenesis. Lastly, since dusp6 is expressed throughout zebrafish embryogenesis, but dusp6 mutants do not exhibit defects after the first cell division, it is possible that other feedback regulators of the ERK pathway compensate for loss of dusp6 at later stages.

Le virus HTLV-1 (Human T-cell Leukemia Virus Type 1) est l’agent étiologique de la Leucémie T de l’adulte (ATL) qui est caractérisée par la prolifération de cellules T CD4+ activées. L’absence de modèle animal fiable reproduisant la leucémogénèse associée à l’infection a ralenti la compréhension des étapes précoces du processus leucémogène et le développement de stratégies thérapeutiques efficaces. Récemment l’amélioration des modèles de souris humanisées a permis la reconstitution d’un système immunitaire humain dans des souris. L’injection de cellules souches hématopoïétiques purifiées à partir de sang de cordon humain dans des souris nouveau-nées de la lignée Rag2-/-γc-/- conduit à la formation de novo de cellules dendritiques, B et T humaines. Ces dernières étant la cible de l’infection par HTLV-1, nous avons infecté des souris humanisées avec des cellules productrices de HTLV-1. Cette inoculation conduit à l’infection stable des cellules humaines dans la souris humanisée et la formation de lymphome ou de leucémie à cellules T humaines activées. Cette infection altère le développement des cellules T dans le thymus conduisant à un phénotype plus mature des thymocytes. Ce modèle animal reproduisant l’infection et la pathogénèse associée nous a permis de suivre l’évolution de la clonalité du virus au sein des différents organes lymphoïdes. Basées sur ces observations, des tests préliminaires ont permis d’étudier une nouvelle approche thérapeutique potentiellement applicable en clinique humaine. Ce travail nous a également permis d’affiner le protocole conduisant à l’humanisation des souris afin d’obtenir une meilleure reconstitution humaine dans ce modèle. / Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

Durant l’ontogénie B, le locus des chaines lourdes d’immunoglobulines (IgH) subit trois processus de réarrangements géniques. Lors des phases précoces du développement B, indépendamment de la rencontre avec un antigène, les réarrangements VDJ permettent l’obtention d’un répertoire d’Ig fonctionnelles. Lors des phases tardives, l’hypermutationsomatique (SHM) permet l’augmentation de l’affinité de l’Ig pour son antigène tandis que larecombinaison isotypique (CSR) modifie ses fonctions effectrices. Ces évènements impliquent l’induction de lésions de l’ADN potentiellement oncogéniques, ce qui impose unerégulation très stricte. Cette régulation est assurée par divers éléments cis-régulateurs répartis tout au long du locus IgH, dont la région régulatrice en 3’ (3’RR). La 3’RR s’étend sur 30 kb et contient quatre activateurs transcriptionnels, les trois premiers formant une structure palindromique. Lors de ma thèse, j’ai utilisé plusieurs modèles murins porteurs de délétions de tout ou partie de la 3’RR pour étudier son rôle, ainsi que celui des différents éléments qui la compose lors des diverses étapes de l’ontogénie B. Nous avons pu déterminer comment la 3’RR régule précisément la CSR en ciblant spécifiquement la région switch acceptrice et caractériser le phénomène encore peu connu de CSR vers IgD. D’autre part, nous avons démontré l’importance de la 3’RR lors de la SHM et dans le développement des différentes sous populations lymphocytaires B. Enfin, la comparaison des résultats obtenus lors de l’analyse des différents modèles nous a permis de déterminer que la structure palindromique de la 3’RR est importante pour une SHM efficace, mais relativement dispensable lors de la CSR. / During B-cell development, the heavy chains locus (IgH) undergoes three genic rearrangement events. During the early stages, before encountering the antigen, VDJ rearrangements allow the generation of a functional Ig repertoire. During the late stages, somatic hypermutation (SHM) increases the affinity of the Ig for its antigen, while class switch recombination (CSR) modifies its effector functions. These events imply thegeneration of potentially oncogenic DNA lesions, and thus require a strict regulation. This regulation is assured by several cis-regulatory elements spread along the IgH locus, including the 3’ regulatory region (3’RR). The 3’RR extends on more than 30kb and contains four transcriptional enhancers, the first three displaying a palindromic conformation. During my PhD, I investigated several mouse models carrying deletion of part or totality of the 3’RR to investigate its role during B cell development. We demonstrated how she precisely regulates CSR by specifically targeting the acceptor switch region, and described the poorly known mechanism of CSR toward IgD. Otherwise, we have demonstrated its importance during SHM and in the correct development of the different B cell subpopulations. Finally, by comparing the results obtained during the analysis of the various mouse models, we have demonstrated that the palindromic structure of the 3’RR is required for optimal SHM, but not for CSR.

Excitotoxicity, a major hallmark of neurodegeneration associated with cerebral ischemia, is a result of accumulation of extracellular glutamate. This excess glutamate leads to hyperactivation of glutamate receptors such as the N-methyl-D-asparate (NMDA) receptors (NMDARs) following the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPARs). Excessive activation of NMDARs causes an influx of calcium, which can eventually activate apoptotic pathways and lead to death of neurons. Regulation of NMDAR subunit composition, localization, surface expression, and activity can balance cell survival via activation of either pro-death or pro-survival pathways after a course of an ischemic insult. Specifically, phosphorylation of different NMDAR subunits defines their activity and downstream signaling pathways. NMDARs are phosphorylated by multiple kinases and dephosphorylated by different phosphatases. Besides phosphatases and kinases, per se, phosphorylation of synaptic proteins that regulate kinase or phosphatase targeting and activity also mediate NMDAR phosphorylation. Spinophilin, a major synaptic scaffolding and protein phosphatase 1 (PP1) targeting protein, mediates substrate phosphorylation via its ability to bind PP1. Our studies focus on delineating the role of spinophilin in the regulation of phosphorylation and function of the GluN2B subunit of the NMDA receptor as well as the role of spinophilin in modulating glutamate-induced neurotoxicity. Interestingly, our data demonstrate that spinophilin sequesters PP1 away from GluN2B thereby enhancing phosphorylation of GluN2B at Ser-1284. These changes impact GluN2B protein interactions, subcellular localization, and surface expression, leading to alterations in the amount of calcium entering the neuron via GluN2B-containing NMDARs. Our data show that spinophilin biphasically regulates GluN2B function. Specifically, Ser-1284 phosphorylation enhances calcium influx through GluN2B containing NMDA receptors, but spinophilin leads to dramatic decreases in the surface expression of the receptor independent of Ser-1284 phosphorylation. Moreover, in spinophilin knockout mice, we observe less PP1 binding to GluN2B and less phosphorylation of Ser-1284, but more surface expression of GluN2B and greater levels of caspase activity. Together, these observations suggest a potential neuroprotective role for spinophilin by decreasing GluN2B-containing NMDA receptor-dependent surface expression and thereby decreasing intracellular calcium and neuronal cell death.