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For many years epigenetics was defined by three pillars: DNA methylation, histone modifications, and ncRNA regulation. But recently, the development of next-generation sequencing and more sensitive mass spectrometry unveiled the presence of novel mRNA modifications. This new field of RNA epigenetics was then referred to as epitranscriptomics.During the golden age of epitranscriptomics, many new mRNA modifications were identified. Thanks to its abundance, m6A persists until now as the reference, with the discovery of complete enzymatic machinery (writers, readers, and erasers) as well as functions in physiology and pathology. However, the very existence of the other mRNA modifications remains under debate due to the high error rates in the sequencing technologies, the low specificity of new methods developed, and the poor reproducibility between the different epitranscriptomics studies. Thus, the aim of this thesis is to dive into the identification of new mRNA modifications through the prism of the writers and readers.In the first part of the thesis, by using the RNA biotin pull-down method, we uncovered promising readers from two mRNA modifications: FUBP3 for inosine and CSDE1 for m5U. Then, we further described the direct binding of FUBP3 to hyper-edited oligonucleotides.In the second part of the thesis, we developed and optimized new sequencing techniques, based on the TRMT2A writer to study the distribution of m5U in mRNA. Here, we provide the first transcriptome-wide list of around one thousand transcripts that are bound and m5U modified by TRMT2A. Moreover, we challenged the recently described Nanopore technology to map m5U at a single-nucleotide level. Using two different bioinformatic tools, we obtained a list of confident m5U peaks that need to be confirmed by primer extension.Lastly in the third part, we confirmed that CSDE1 acts as a reader of the m5U modification. We also showed that the depletion of TRMT2A induces changes in the transcriptome and proteo, Durant de nombreuses années, l’épigénétique était définie par trois piliers :la méthylation de l’ADN, les modifications d’histone et la régulation par ARN non codant. Récemment, le développement de nouvelles technologies de séquençage et de spectrométries de masse plus sensibles a dévoilé la présence de nouvelles modifications d’ARNm. Ce nouveau domaine de recherche lié à l’épigénétique des ARN est appelé épitranscriptomique. Durant l’âge d’or de l’épitranscriptomique, une dizaine de nouvelles modifications d’ARN a été identifiée. Grâce à son abondance, m6A reste jusqu’à ce jour la référence avec la découverte d’une machinerie enzymatique complète (writers, readers et erasers) ainsi que des fonctions en physiologie et pathologie. Cependant, l’existence même des autres modifications d’ARNm reste débattue à cause notamment du haut taux d’erreur des techniques de séquençage et de la faible spécificité des nouvelles méthodes développés. Donc, le but de cette thèse est de plonger dans le monde des nouvelles modifications d’ARNm par le biais des writers et readers.Dans cette première partie de thèse, en utilisant la méthode du biotin pull-down, nous avons identifié des readers prometteurs pour deux modifications d’ARNm :FUBP3 pour l’inosine et CSDE1 pour m5U. Ensuite, nous avons caractérisé davantage la liaison directe de FUBP3 à un oligonucléotide hyper-édité.Dans la deuxième partie, nous avons développé et optimisé de nouvelles techniques de séquençage basées sur le writer TRMT2A afin d’étudier la distribution de m5U dans les ARNm. Ici, nous présentons la première liste transcriptomique d’environ mille transcrits qui sont liés et modifié m5U par TRMT2A. De plus, nous avons testé la récente technologie du Nanopore afin de cartographier la position de m5U au nucléotide près. En utilisant deux outils bioinformatiques, nous avons obtenu une liste de piques m5U qui doivent être prochainement confirmés par la méthode du primer extension. Enfin, dans la troisième partie, nous, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro. Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo, beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes.

In vitro differentiation of human induced pluripotent stem cells (iPSCs) into beta cells represents an important cell source for diabetes research. Here, we fully characterized iPSC-derived beta cell function in vitro and in vivo in humanized mice. Using a 7-stage protocol, human iPSCs were differentiated into islet-like aggregates with a yield of insulin-positive beta cells comparable to that of human islets. The last three stages of differentiation were conducted with two different 3D culture systems, rotating suspension or static microwells. In the latter, homogeneously small-sized islet-like aggregates were obtained, while in rotating suspension size was heterogeneous and aggregates often clumped. In vitro function was assessed by glucose-stimulated insulin secretion, NAD(P)H and calcium fluctuations. Stage 7 aggregates slightly increased insulin release in response to glucose in vitro .Aggregates were transplanted under the kidney capsule of NOD-SCID mice to allow for further in vivo beta cell maturation. In transplanted mice, grafts showed glucose-responsiveness and maintained normoglycemia after streptozotocin injection. In situ kidney perfusion assays showed modulation of human insulin secretion in response to different secretagogues. In conclusion, iPSCs differentiated with equal efficiency into beta cells in microwells compared to rotating suspension, but the former had a higher experimental success rate. In vitro differentiation generated aggregates lacking fully mature beta cell function. In vivo ,beta cells acquired the functional characteristics typical of human islets. With this technology an unlimited supply of islet-like organoids can be generated from human iPSCs that will be instrumental to study beta cell biology and dysfunction in diabetes., SCOPUS: ar.j, info:eu-repo/semantics/published

Advancements in oncology have improved the overall survival rate of cancer patients. However, chemotherapeutic regimens can be gonadotoxic and induce infertility. To ensure good quality of life after treatment, preservation of fertility is highly recommended. Among the available techniques, ovarian tissue cryopreservation (OTC) is an attractive approach that can be performed without postponing cancer treatment or after first-line chemotherapy. It is also the only available option for children. This technique consists of freezing the cortical tissue followed by grafting after remission in order to restore fertility. The major limitation is the risk of recurrence of the disease if cancer cells are present in the stored tissue. As an alternative, in vitro follicle growth until maturation with subsequent fertilization of the oocyte appears to be a promising option for all patients. However, this procedure is challenging and has poor efficacy in humans. To optimize this method, a better understanding of the mechanisms regulating primordial follicle activation (PFA) in vitro as well as the factors that modulate it is essential. This project aimed to investigate two signaling pathways, the PI3K/AKT/mTOR and Hippo signaling pathways, during spontaneous and chemotherapy induced-PFA in vitro using mouse model. The impact of a first-line chemotherapy exposure was further studied in ovarian tissue from adults and children exposed (or not) to chemotherapy before OTC. Finally, we assessed the impact of two inhibitors, everolimus (EVE) and verteporfin (VERT), to control these pathways in a short term culture system using mouse model. Mouse experiments demonstrated that the removal of post-natal day 3 mouse ovaries from their physiological environment impaired the Hippo signaling pathway, inducing spontaneous PFA. After tissue fragmentation, alteration of both signaling pathways occurred in mouse model. To evaluate the effect of chemotherapy on subsequent follicular activation, we, Grâce aux progrès diagnostiques et thérapeutiques, la survie des patientes atteintes d’un cancer a grandement augmenté ces dernières décennies. Néanmoins, certains agents chimiothérapeutiques peuvent être gonadotoxiques et mener à une infertilité. Afin d’assurer une qualité de vie adéquate aux patients après rémission, il est grandement recommandé de préserver leur fertilité. Parmi les techniques disponibles en clinique, la cryopréservation de tissu ovarien est une procédure intéressante puisqu’elle peut être réalisée sans devoir postposer le traitement ou même après une première cure de chimiothérapie. De plus, elle est la seule option disponible pour les enfants. Elle consiste en la congélation du cortex ovarien en vue d’une greffe après rémission, pour autant que le tissu ne contienne pas de cellules néoplasiques. Afin de palier à cette limitation, la culture du tissu ovarien in vitro afin d’obtenir des ovocytes matures fertilisables est une alternative prometteuse actuellement en développement en recherche. Cependant, ce système de folliculogenèse in vitro montre de grandes limitations dû à une activation massive des follicules en culture. Afin d’améliorer cette technique, il est essentiel d’avoir une meilleure compréhension des mécanismes régulant l’activation des follicules in vitro. De plus, les impacts de la procédure de congélation du tissu et d’un éventuel traitement antérieur doivent être considérés. Les objectifs de ce projet sont d’étudier deux voies de signalisations, la voie PI3K/AKT/mTOR et Hippo, durant l’activation folliculaire spontanée et induite en modèle murin. L’impact du traitement chimiothérapeutique a quant à lui été étudié plus en détail en comparant le tissu de patientes adultes et prépubères qui ont eu de la chimiothérapie ou non avant la cryopréservation. Enfin, nous avons tenté de contrôler ces deux voies de signalisations in vitro en utilisant deux inhibiteurs, l’everolimus (EVE) et la verteporfine (VERT), en modèle animal. Nos analys, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

DNAJC3, also known as P58IPK, is an Hsp40 family member that interacts with and inhibits PKR-like ER-localized eIF2α kinase (PERK). Dnajc3 deficiency in mice causes pancreatic β-cell loss and diabetes. Loss-of-function mutations in DNAJC3 cause early-onset diabetes and multisystemic neurodegeneration. The aim of our study was to investigate the genetic cause of early-onset syndromic diabetes in two unrelated patients, and elucidate the mechanisms of β-cell failure in this syndrome., info:eu-repo/semantics/published

The global rise in type 2 diabetes results from a combination of genetic predisposition with environmental assaults that negatively affect insulin action in peripheral tissues and impair pancreatic β-cell function and survival. Nongenetic heritability of metabolic traits may be an important contributor to the diabetes epidemic. Transfer RNAs (tRNAs) are noncoding RNA molecules that play a crucial role in protein synthesis. tRNAs also have noncanonical functions through which they control a variety of biological processes. Genetic and environmental effects on tRNAs have emerged as novel contributors to the pathogenesis of diabetes. Indeed, altered tRNA aminoacylation, modification, and fragmentation are associated with β-cell failure, obesity, and insulin resistance. Moreover, diet-induced tRNA fragments have been linked with intergenerational inheritance of metabolic traits. Here, we provide a comprehensive review of how perturbations in tRNA biology play a role in the pathogenesis of monogenic and type 2 diabetes., info:eu-repo/semantics/published

Rare diseases are life-threatening or chronically debilitating low-prevalent disorders caused by pathogenic mutations or particular environmental insults. Due to their high complexity and low frequency, important gaps still exist in their prevention, diagnosis, and treatment. Since new drug discovery is a very costly and time-consuming process, leading pharmaceutical companies show relatively low interest in orphan drug research and development due to the high cost of investments compared to the low market return of the product. Drug repurposing–based approaches appear then as cost- and time-saving strategies for the development of therapeutic opportunities for rare diseases. In this article, we discuss the scientific, regulatory, and economic aspects of the development of repurposed drugs for the treatment of rare neurodegenerative disorders with a particular focus on Huntington’s disease, Friedreich’s ataxia, Wolfram syndrome, and amyotrophic lateral sclerosis. The role of academia, pharmaceutical companies, patient associations, and foundations in the identification of candidate compounds and their preclinical and clinical evaluation will also be discussed., SCOPUS: re.j, info:eu-repo/semantics/published

The thyroid gland is a critical organ whose development is a complex morpho-genetic process during which naïve endodermal cells acquire thyroid cell identity, bud out of the endoderm, and move toward their final location organising into hormone-producing 3D structures named follicles. Defects during any of these developmental stages lead to congenital hypothyroidism. Congenital hypothyroidism represents the most common congenital endocrine disorder in humans, affecting around 1 in every 2000-5000 live births. To date, the cause of most CH cases remains unexplained due to poor understanding of the molecular regulation of thyroid development. Although recent studies started to describe the external signals driving thyroid specification, little is known about the intrinsic factors driving the thyroid specification, the different steps of its morphogenesis, its functional maturation or maintaining the thyroid function once it is fully developed.To deal with this situation, we decided to take two different approaches. In the first, we analysed, at a single-cell resolution, the transcriptome of the adult thyroid gland and its surrounding tissue. Analysis of this dataset revealed the existence of two thyroid cell populations co-existing in the thyroid gland. Interestingly, these two populations showed differential expression of thyroid functional markers (such as slc5a5, duox, duoxa or tpo) and of pax2a. To confirm this observation and better characterise these two populations, we developed a new generation of a pax2a reporter line using Crispr/Cas9-based knock-in strategy. Using this line, we were able to observe the distribution of these two populations and show that they are stochastically distributed across the thyroid follicles forming the adult gland. Overall, these analyses lead us to believe that the thyroid gland consists of two functionally distinct populations of thyroid follicular cells, of which the less active population might consist of resting thyroid cells, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

La phosphoinositide phosphatase SHIP2 est une protéine capable de moduler le PI(3,4,5)P3, le PI(4,5)P2 et le PI(3,4)P2 qui sont des phosphoinositides importants lors des remaniements membranaires de la cellule. La régulation de la composition en phosphoinositides des membranes permettant l’assemblage de complexes protéiques est primordiale pour la génération de protrusions essentielles pour la migration et l’invasion cellulaire. D’autre part, SHIP2 possède des propriétés d’échafaudage permettant sa participation à plusieurs complexes multi-protéiques et à son ancrage à des localisations subcellulaires spécifiques. Plusieurs études ont montré que SHIP2 pouvait jouer un rôle dans le développement de certains cancers dont le cancer du sein. Au cours de ce travail, nous avons mis en évidence deux nouveaux partenaires de SHIP2 :IRSp53 et CIN85. Ces deux protéines participent respectivement à la formation d’invadopode et à l’endocytose. IRSp53 et CIN85 lient la même région C-terminale riche en prolines (PRR-II) de SHIP2, mais toutefois pas par les mêmes séquences. Cette région contient plusieurs motifs consensus permettant l’interaction de SHIP2 avec plusieurs autres partenaires connus comme l’intersectine, une protéine impliquée dans l’endocytose et Mena, un partenaire commun de SHIP2 et d’IRSp53 impliqué dans l’invasion cellulaire. Nous avons également montré que la mutation de la F1053 située dans la PRR-II de SHIP2 est, essentielle pour la liaison à l’intersectine, impliquée dans la stabilité de la liaison avec Mena et IRSp53 mais pas impliquée dans la liaison de CIN85. De plus, nous montrons qu’à la différence d’IRSp53 qui n’en possède qu’un, CIN85 possède trois domaines SH3 mais ne doit lier que deux motifs dans la PRR-II de SHIP2 afin d’assurer sa complète interaction.Dans les cellules dérivées du cancer du sein MDA-MB-231, nous avons montré que Mena n’est pas nécessaire pour l’interaction entre SHIP2 et IRSp53. Cependant, nous avons observé que l’absence de Mena d, Doctorat en Sciences biomédicales et pharmaceutiques (Médecine), info:eu-repo/semantics/nonPublished

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