京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21645号 / 医博第4451号 / 新制||医||1034(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 羽賀 博典, 教授 小西 靖彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Les anomalies du développement sexuel recouvrent un spectre phénotypique large. Les hommes XX présentent dans la majorité des cas un développement testiculaire normal, lié à la présence SRY sur un des chromosomes X. Dans 10% des cas, aucune cause n’est retrouvée. Chez les femmes, l’origine de l’insuffisance ovarienne prématurée (IOP) n’est identifiée que dans 20% des cas. L’objectif de cette thèse a été d’identifier de nouveaux mécanismes moléculaires impliqués dans le développement gonadique, testiculaire et ovarien, ainsi que dans son fonctionnement. L’étude d’une cohorte de patients DSD 46,XX SRY négatifs a mis en évidence une duplication de la région RevSex dans un désert génique en amont de SOX9 chez quatre patients. Ceci a permis de redéfinir la région minimale impliquée dans l'activation de l'expression de SOX9 à une taille maximale de 41.9 kb et de proposer un mécanisme permettant cette expression chez des hommes XX. Le séquençage d’exome chez dix patients de la cohorte n’a pas mis en évidence de mutations dans des gènes d’intérêts. L’ensemble de ces résultats pose la question du rôle des régions régulatrices dans la survenue des DSD.L’analyse d’une cohorte de patientes ayant une IOP a permis d'identifier des délétions incluant le gène CPEB1. Des études précédentes chez la souris ont montré son implication dans le développement folliculaire. Le séquençage du gène CPEB1 dans la cohorte n'a pas mis en évidence de mutation pathogène. Ce travail a permis de montrer que la délétion impliquant le gène CPEB1est une cause rare mais récurrente d'IOP et concerne environ 1% des patientes. Une microdélétion contenant le gène CASP3 un gène de la voie des caspases impliquée dans la régulation du pool folliculaire a également été identifiée chez une patiente. L'ensemble de ces résultats montre l'intérêt de l'étude génétique des patients présentant une anomalie du développement de la gonade ou de son fonctionnement par des techniques d'étude globale du génome. / Disorders of Sex Development (DSD) can be identified in new-born and during infancy but also in adults because of infertility. Most 46,XX testicular DSD have a normal testicular development due to the presence of the SRY gene at the tip of one of their X chromosome. However, the genetic causes of 46,XX-SRY negative testicular DSD remain poorly defined. In women, disorders of gonadal development can be responsible for primary ovarian insufficiency (POI) and genetic causes are identify in only 20% of cases. The aim of this thesis was to identify molecular mechanisms involved in gonadal development and in its functioning. The cohort study of 46,XX testicular DSD identified four patients with a duplication in the previously reported RevSex region located about 550 kb upstream of SOX9. One duplication allowed us to refine the minimal region associated with 46,XX-SRY negative DSD to a 40.7–41.9 kb element. Exome sequencing of 10 patients from the cohort did not show any mutation in genes implicated in DSD or in new candidate genes. These results raise questions about the role of the regulatory sequences in the onset of DSD.The cohort study of POI patients identified three patients carrying a microdeletion including CPEB1 a good candidate gene for POI as study in mice showed the implication of CPEB1 in follicular development. Sequencing CPEB1 gene did not identified any mutation. Therefore, heterozygous deletion of CPEB1 gene leading to haploinsufficiency could be responsible for POI in humans. This microdeletion is rare but recurrent and was identified in about 1% of patients with POI. Another microdeletion containing CASP3 gene that belongs to the caspase family, which is implicated in the regulation of the follicular pool, was identified in a patient. Further studies are needed to confirm the role of CASP3 in POI. These results demonstrate the importance of genetic study of patients presenting with DSD or POI using whole genome techniques.

Dans le contexte de la physiologie comparée de la reproduction, les amphibiens sont peu étudiés. Le travail réalisé durant cette thèse visait à analyser des marqueurs de différenciation testiculaire chez l'urodèle Pleurodeles waltl, dont le déterminisme génétique du sexe (ZZ/ZW) peut être influencé par la température. Nos études ont d'abord porté sur le gène sox9 marqueur de la différenciation testiculaire chez les vertébrés supérieurs. Le gène cloné chez le pleurodèle montre une bonne conservation par rapport aux autres vertébrés. Son expression plus élevée dans la gonade mâle n'apparaît que tardivement suggérant qu'il n'est probablement pas impliqué dans les stades précoces de la différenciation testiculaire. En outre, son expression dans le mésonéphros rend difficile son utilisation comme marqueur de différenciation testiculaire. Nous avons ensuite étudié l'Amh, hormone testiculaire impliquée dans la régression des canaux de Müller chez de nombreux vertébrés. Son expression spécifique de la gonade, précocement plus élevée chez les larves ZZ que les ZW en font un excellent marqueur de la différenciation testiculaire. Le fait que les pleurodèles mâles voient les canaux de Müller persister malgré la présence d'Amh suggère que la fonction primaire de cette hormone était en relation avec la différenciation gonadique et que la fonction de régression des canaux de Müller n'est apparue que secondairement au cours de l'évolution. Ces marqueurs ont été mis à profit pour caractériser le phénotype gonadique lors d'inversions sexuelles ou lors de développements en absence de cellules germinales. Ils ont permis de montrer que les cellules germinales ne semblent pas jouer de rôle dans la différenciation gonadique du pleurodèle / In the context of comparative physiology of reproduction, amphibians are poorly studied. This work was dedicated to the analysis of testis differentiation markers in the newt Pleurodeles waltl, which shows a ZZ/ZW genetic mode of sex determination that can be affected by temperature. First, we studied sox9, a testis differentiation marker well characterized in many higher vertebrates. The gene cloned in Pleurodeles shows a good level of identity with other vertebrates. The testis-enriched expression appears late during the testis differentiation process indicating that it is probably not involved in the early steps of testis differentiation. Its use as a marker of testicular differentiation proved difficult since it is expressed not only in the gonads but also in the mesonephros. Then, we studied amh, a testis hormone responsible for müllerian duct regression in many vertebrates. Its early expression in the gonad, significantly higher in male than in female larvae makes it an excellent marker for testis differentiation. Since in Pleurodeles waltl, Müllerian ducts persist in males, it suggests that during the course of evolution, the function of Amh on the regression of Müllerian ducts appeared secondarily after its role in gonadal differentiation. These markers have been used to characterize the gonadal phenotype during sex reversal, or in gonads developed in the absence of germ cells. They showed that these cells do not seem to play a role in gonadal differentiation of Pleurodeles waltl

Medulloblastomas are the most frequent malignant childhood brain tumors, arising in the posterior fossa of children. The overall 5-year survival is 70%, although children often suffer severe long-term side effects from standard medical care. To improve progression-free survival and quality of life for these children, finding new therapeutic targets in medulloblastoma is imperative. Medulloblastoma is divided in to four molecular subgroups (WNT, SHH, Group 3 and Group 4) based on key developmental pathways essential for the initiation and maintenance of tumor development. The MYC family of proto-oncogenes regulates cell proliferation and differentiation in normal brain. Aberrant expression of MYC proteins occurs commonly in medulloblastoma. Our studies on Group 3 medulloblastoma identify the transcription factor SOX9 as a novel target for the E3 ubiquitin ligase FBW7, and show that increased stability of SOX9 confers an increased metastatic potential in medulloblastoma. Moreover, SOX9-positive cells drive distant recurrences in medulloblastoma when combining two regulatable TetON/OFF systems. MYCN depletion leads to increased SOX9 expression in Group 3 medulloblastoma cells, and the recurring tumor cells are more migratory in vitro and in vivo. Segueing to treatment of medulloblastoma, we show that BET bromodomain inhibition specifically targets MYC-amplified medulloblastoma cells by downregulating MYC and MYC-transcriptional targets, and that combining BET bromodomain- and cyclin-dependent kinase- inhibition improves survival in mice compared to single therapy. Combination treatment results in decreased MYC levels and increased apoptosis, and RNA-seq confirms upregulation of apoptotic markers along with downregulated MYC target genes in medulloblastoma cells. This thesis addresses novel findings in transcription factor biology, recurrence and treatment in Group 3 medulloblastoma, the most malignant subgroup of the disease.

Articular cartilage lines the surfaces of load bearing joints and has limited capabilities for self-repair due to its alymphatic and avascular structure. Attempts at making repairs to this tissue has resulted in substandard materials and/or causing further injury to the patient making this tissue a prime candidate for tissue engineering studies incorporating adult stem cells. These studies have given rise to some answers and many more questions including a search for alternative stem cell sources and what biochemical changes the cells undergo during the differentiation of these stem cells into chondrocytes, the cells which make up articular cartilage. Recently, periodontal dental ligament stem cells (PDLs) have come to the forefront as a practical alternative to other adult stem cells as well as the involvement of the mitogen-activated protein kinases (MAPKs) in stem cell differentiation via mechanical stimulation. During dynamic unconfined compression, levels of p42/44 MAPK increased by 50% (p<0.05). Additionally, the expression of the chondrogenic differentiation factor SRY (sex determining region Y)-box 9 (SOX-9) increased by 3-fold (p<0.05) as well as the chondrocyte marker aggrecan by over 2-fold after 4h of dynamic unconfined compression. Addition of the p42/44 phosphorylation inhibitor PD98059, along with compression, yielded no change in SOX-9 or aggrecan expression levels from basal levels in uncompressed controls. Inhibition of p38 MAPK or JNK phosphorylation during unconfined compression had no effect on the elevated expression of SOX-9 and aggrecan as compared to compressed cells without the addition of an inhibitor. It is therefore the overall findings of this study that PDLs possess the ability to differentiate into chondrocytes by mechanical compression and this differentiation is mediated by the p42/44 MAPK cascade.

Components of the molecular pathway underlying gonadogenesis in organisms with temperature-dependent sex determination (TSD) have been retained from genetic sex determination. Furthermore, although much of this network has been conserved, new functions for these genes have evolved in this different mode of sex determination. We find that the transcription factors Sox9 and Dmrt1 and the hormone Mis are involved in the formation of a testis and/or the repression of an ovary at a male-producing temperature. While Mis expression may be maintained by Sox9, the initial upregulation of Mis in the developing testis is most likely modulated by some other upstream factor. Dmrt1 appears to play an upstream role in testis sex determination. We provide evidence that the transcription factor Dax1 and the signaling molecule Wnt4, cloned for the first time in an organism with TSD, play roles in gonadogenesis in both sexes. Finally, we show that the transcription factor FoxL2 and the signaling molecule Rspo1 are involved in the formation of an ovary and/or the repression of a testis at a female-producing temperature. In the first investigation of Rspo1 in any organism exhibiting TSD, we demonstrate it is involved upstream in ovarian sex determination. Complementary to descriptive studies, we optimize a whole organ culture system in which gonad explants develop in vitro for up to three weeks. We show that expression of the sex-determining network in isolated gonads mimics in ovo patterns, revealing an endogenous temperature-sensing mechanism that does not require other embryonic tissues. Ectopic expression of Sox9 reveals a possible positive feedback regulation of Dmrt1. The use of this culture system opens the door to functional manipulation of the gonad at the molecular level and is suitable for a myriad of future studies. This work makes strides in elucidating the molecular network underlying gonadogenesis in an organism exhibiting TSD, and invites investigation of the evolution of gene function. The data lend insight into the changing roles of molecules in sex determination across diverse taxa, and into the evolution of developmental pathways in general. / text

No description available.

Type 1 diabetes (T1D) is a chronic disease characterized by an autoimmune-mediated destruction of insulin-producing pancreatic beta cells. Environmental factors such as viruses play an important role in the onset of T1D and interact with predisposing genes. Recent data suggest that viral infection of human islets leads to a decrease in insulin production rather than beta cell death, suggesting loss of beta cell identity. We undertook this study to examine whether viral infection could induce human b cell dedifferentiation. Using the functional human b cell line EndoC-bH1, we demonstrate that polyinosinic-polycytidylic acid (PolyI:C), a synthetic double-stranded RNA that mimics a byproduct of viral replication, induces a decrease in beta cell–specific gene expression. In parallel with this loss, the expression of progenitor-like genes such as SOX9 was activated following PolyI:C treatment or enteroviral infection. SOX9 was induced by the NF-kB pathway and also in a paracrine non–cell-autonomous fashion through the secretion of IFN-a. Lastly, we identified SOX9 targets in human b cells as potentially new markers of dedifferentiation in T1D. These findings reveal that inflammatory signaling has clear implications in human beta cell dedifferentiation.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21420号 / 医博第4410号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 羽賀 博典, 教授 武田 俊一, 教授 坂井 義治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM