L’hématopoïèse est un processus actif, ordonné, et hautement régulé faisant intervenir des étapes de prolifération, de différenciation et d’apoptose et permettant la production de toutes les cellules sanguines matures à partir d’un nombre restreint de cellules souches hématopoïétiques. La dérégulation des mécanismes intervenant dans l’hématopoïèse induit le développement d’hémopathies, notamment de leucémies. De nombreux facteurs de transcription et microARN (miARN) ont été identifiés en tant que des régulateurs essentiels à l’établissement des différents lignages hématopoïétiques. Mon travail de thèse a porté sur l’étude du rôle des miARN dans la régulation de la différenciation macrophagique humaine. Nous avons reproduit le processus de différenciation macrophagique in vitro à partir de monocytes issus du sang périphérique traités avec du CSF-1 (colony stimulating factor-1). Suite à l’analyse du profil d’expression des miARN au cours du processus de différenciation, notre projet s’est orienté sur l’étude de miR-142-3p dont le taux d’expression diminue le plus fortement au cours de cette différenciation. Nous avons montré que miR-142-3p forme une boucle d’auto-régulation négative avec EGR2 (early growth response 2), un facteur de transcription connu pour réguler positivement la différenciation macrophagique. Cette boucle est essentielle au bon déroulement de la différenciation. Par ailleurs, nous avons observé une altération de cette boucle de régulation dans les monocytes de patients atteints d’une LMMC (leucémie myélomonocytaire chronique) suggérant que ce mécanisme puisse être impliqué dans la leucémogenèse. Au cours de ce projet, nous avons également initié une étude in vivo via l’utilisation du modèle que représente le Poisson-Zèbre. L’hématopoïèse du Poisson-Zèbre est très similaire à celle des mammifères que ce soit au niveau des populations hématopoïétiques ou des mécanismes de régulation impliqués. L’inhibition de l’expression du miR-142a-3p, homologue du miR-142-3p humain, se traduit par une absence de monocytes et de macrophages au niveau de l’ICM (intermediate cell mass), organe primaire de l’hématopoïèse, ainsi que par une diminution de l’expression de la myéloperoxydase, marqueur des granulocytes chez le Poisson-Zèbre. Ainsi, miR-142-3p semble être un inducteur de la formation des granulocytes et monocytes. / Hematopoiesis is an active process, orderly and highly regulated, involving proliferation, differentiation and apoptosis steps, and allowing the production of mature blood cells from a restricted number of hematopoietic stem cells. Deregulation of mechanisms involved in hematopoiesis leads to the development of leukemias. Many transcription factors and microRNAs (miRNAs) have been identified as essential regulators in the establishment of different hematopoietic lineages. My thesis investigated the role of miRNAs in the regulation of human macrophage differentiation. We examined macrophage differentiation in vitro, from peripheral blood monocytes treated with CSF-1 (colony stimulating factor-1). After the analysis of miRNAs expression profile, our project has focused on the study of miR-142-3p whose expression levels decreased most strongly during macrophage differentiation. We showed that miR-142-3p involved in a negative feedback loop with EGR2 (early growth response 2), a transcription factor known to favor macrophage differentiation. This molecular circuitry is necessary for the normal processus of differentiation. Furthermore, we observed an alteration of this regulation circuitry in monocytes of CMML (chronic myelomonocytic leukemia) patients, suggesting that this mechanism may be involved in leukemogenesis. During this project, we also initiated a study in vivo through the use of the zebrafish model. Zebrafish hematopoiesis is very similar to that in mammals both at the level of hematopoietic populations or regulatory mechanisms involved. The inhibition of miR-142a-3p expression, homolog of the human miR-142-3p, gave rise to an absence of monocytes and macrophages in ICM (intermediate cell mass), the primary organ of hematopoiesis and a decreased expression of myeloperoxidase, a marker of granulocytes in the zebrafish. Thus, miR-142-3p appears to be an inducer of granulocytes and monocytes formation.

In recent years, the genetic landscape of B-cell derived lymphoid malignancies, including chronic lymphocytic leukemia (CLL), has been rapidly unraveled, identifying recurrent genetic mutations with potential clinical impact. Interestingly, ~30% of all CLL patients can be assigned to more homogeneous subsets based on the expression of a similar or “stereotyped” B-cell receptor (BcR). Considering that biased distribution of genetic mutations was recently indicated in specific stereotyped subsets, in paper I, we screened 565 subset cases, preferentially assigned to clinically aggressive subsets, and confirm the SF3B1 mutational bias in subset #2 (45%), but also report on similarly marked enrichment in subset #3 (46%). In contrast, NOTCH1 mutations were predominantly detected in subsets #1, #8, #59 and #99 (22-34%). This data further highlights a subset-biased acquisition of genetic mutations in the pathogenesis of at least certain subsets. Aberrant NF-κB signaling due to a deletion within the NFKBIE gene previously reported in CLL warranted extended investigation in other lymphoid malignancies. Therefore, in paper II, we screened 1460 patients with various lymphoid malignancies for NFKBIE deletions and reported enrichment in classical Hodgkin lymphoma (27%) and primary mediastinal B-cell lymphoma (PMBL) (23%). NFKBIE-deleted PMBL cases had higher rates of chemorefractoriness and inferior overall survival (OS). NFKBIE-deletion status remained an independent prognostic marker in multivariate analysis. EGR2 mutations were recently reported in advanced stage CLL patients; thus, in paper III we screened 2403 CLL patients for mutations in EGR2. An overall mutational frequency of 3.8% was reported and EGR2 mutations were associated with younger age, advanced stage and del(11q). EGR2 mutational status remained an independent marker of poor outcome in multivariate analysis, both in the screening and validation cohorts. Whole-genome sequencing (WGS) of 70 CLL cases, assigned to poor-prognostic subsets #1 and #2 and indolent subset #4, were investigated in Paper IV and revealed a similar skewing of SF3B1 mutations in subset #2 and NOTCH1 mutations in subset #1 to that reported in Paper I. Additionally, an increased frequency of the recently proposed CLL driver gene RPS15 was observed in subset #1. Finally, novel non-coding mutational biases were detected in both subset #1 and #2 that warrant further investigation.

This study investigated the role of tolerance induction in an inflammatory setting in regard to the early growth response genes Egr2 and Egr3. T cells robustly respond to pathogenic antigens during infection, but are tolerant to stimulation by self-antigens. The intrinsic mechanisms for self-tolerance in the periphery are still not clear. Egr2 and 3 are induced in tolerant T cells in response to antigen stimulation by NFAT-medicated tolerant signalling; however, their function in tolerant T cells is still unknown. The study demonstrated that Egr2 and 3, induced in tolerant T cells, are not directly involved in defective proliferation and IL-2 production, the hallmarks of T cell tolerance. However, they are essential for preventing inflammatory response of tolerant T cells. In the absence of Egr2 and 3, tolerant T cells show impaired proliferation and production of IL-2, but produce high levels of IFN-γ, a key inflammatory cytokine. This phenotype resembles CD4 T cells from autoimmune diseases such as lupus which show poor proliferative response, but hyper-inflammation. Our study demonstrated, for the first time, a distinctive mechanism to control inflammation from proliferative tolerance regulated by Egr2 and 3, which may be an important mechanism for the control of autoimmune diseases.

miR-17-92 cluster (Oncomir1) encodes seven microRNAs (miRNA, miR) regulating many biological processes including proliferation, differentiation or apoptosis. Overexpression of microRNAs encoded by miR-17-92 cluster is found in a number of tumors including acute and chronic myeloid leukemias (Dixon-McIver et al., 2008; Li et al., 2008; Venturini et al., 2007). Myeloid progenitors express miR-17-92 cluster at a high level, while macrophage differentiation associates with its downregulation. Our laboratory found, that miR-17-92 cluster is repressed by transcription factor Early growth response 2 (Egr2) upon differentiation of primary myeloid PUER progenitors, induced with transcription factor PU.1. Aim of this thesis is to further test the abovementioned data by preparing a reporter vectors set, carrying various fragments of miR-17-92 putative promoter, which enables us to study regulation of transcription of miR-17-92 cluster. This task complicated by presence of increased GC content of the miR-17-92 promoter was successfully accomplished resulting in amplification of eight fragments containing the various parts of miR-17-92 promoter including region -3.3 to 0 kb relative to the start of miR-17-5p sequence, that were inserted into pGL3 reporter vector. Transfection of pGL3 reporter vector carrying...

Les cellules de Schwann jouent un rôle crucial dans la réparation et la régénération des nerfs périphériques en soutenant la croissance axonale et en libérant des facteurs neurotrophiques essentiels. La capacité de convertir des fibroblastes en cellules de Schwann est particulièrement intéressante dans le contexte de lésion d’un nerf périphérique, où la restauration de la fonction nerveuse est un objectif critique. Cette étude examine la reprogrammation directe des fibroblastes en cellules de Schwann, en utilisant les facteurs de transcription SOX10 et EGR2 via la transduction lentivirale. Nous avons testé divers milieux de culture connus pour identifier les conditions de conversion optimales, et avons établi qu'une multiplicité d'infection de 300 assurait une reprogrammation robuste. Cependant, maintenir la viabilité cellulaire au-delà de dix jours a présenté un défi significatif. Pour résoudre ce problème, nous avons développé un nouveau milieu de culture, que nous avons appelé Schwann Cell Medium 4 (SCM4), incorporant de petites molécules connues pour être impliquées dans le développement des cellules de Schwann. SCM4 a considérablement amélioré l'expression des marqueurs clés des cellules de Schwann, y compris SOX10, EGR2, Growth Associated Protein 43, le récepteur neurotrophique P75, et la protéine zéro de la myéline, tout en améliorant la survie globale des cellules. De plus, SCM4 a favorisé une libération plus élevée de BDNF, un facteur neurotrophique crucial pour le soutien et le développement neuronal. Les résultats obtenus avec les cellules converties dans le SCM4 sont comparables à ceux obtenus avec des cellules de Schwann dérivées de cellules souches pluripotentes induites et des cellules de Schwann humaines primaires, démontrant que notre protocole produit des cellules s’apparentant aux cellules de Schwann. Ces résultats soulignent l'importance de conditions de culture optimisées pour la reprogrammation des cellules de Schwann et offrent des perspectives prometteuses pour de futures applications cliniques dans le traitement des maladies neurodégénératives et des lésions nerveuses périphériques. / Schwann cells play a crucial role in the repair and regeneration of peripheral nerves by providing support for axonal growth and releasing essential neurotrophic factors. The ability to convert fibroblasts into Schwann cells is particularly relevant in the context of peripheral nerve injury, where the restoration of nerve function is a critical goal. This study investigates the direct reprogramming of fibroblasts into Schwann cells, employing the transcription factors SOX10 and EGR2 through lentiviral transduction. We tested various culture media described in the literature to identify the optimal reprogramming conditions, and have determined that a multiplicity of infection of 300 ensured robust reprogramming. However, maintaining cell viability beyond ten days presented a significant challenge. To address this issue, we developed a new culture medium, which we termed Schwann Cell Medium 4 (SCM4), incorporating small molecules known to be involved in Schwann cell development. SCM4 markedly enhanced the expression of key Schwann cell markers, including SOX10, EGR2, Growth Associated Protein 43, P75 Neurotrophin Receptor, and Myelin Protein Zero, and also improved overall cell survival. Furthermore, SCM4 promoted a higher release of BDNF, a critical neurotrophic factor for neuronal survival and development. The results obtained with SCM4 were compared to those obtained from Schwann cells derived from induced pluripotent stem cells and primary human Schwann cells, demonstrating that our protocol produced a comparable cell product. These findings underscore the importance of optimized culture conditions for Schwann cell reprogramming and offer promising prospects for future clinical applications in the treatment of neurodegenerative diseases and peripheral nerve injuries.