Dissertations / Theses on the topic 'CEP170'

Pendant le développement du cervelet, les progéniteurs des neurones granulaires (PNG) nécessitent la présence du cil primaire pour proliférer en réponse à Sonic Hedgehog (SHH). En effet, la prolifération dérégulée des PNGs peut conduire à la formation d'une tumeur pédiatrique maligne appelée SHH-médulloblastome (MB), de ce fait comprendre comment le cil primaire est régulé dans les PNGs est crucial.Nous montrons que le facteur de transcription Atoh1 contrôle la présence du cil primaire dans les PNGs in vitro et in vivo. En particulier, la suppression du cil primaire par l’inactivation génétique de gènes impliqués dans la ciliogenèse (par exemple, Kif3a ou Ift88) empêche Atoh1 de maintenir les PNGs en prolifération, ce qui indique qu’Atoh1 favorise l’expansion des PNGs en maintenant la présence du cil primaire. D’un point de vue moléculaire, Atoh1 contrôle la formation du cil primaire en régulant le bon positionnement peri-centrosomal des satellites centriolaires (SC), complexes protéiques essentiels pour la ciliogenèse. L'inactivation de Atoh1 dans les PNGs perturbe en effet la distribution subcellulaire des SCs, altérant ainsi inévitablement la ciliogenèse. Cette nouvelle fonction de Atoh1 est gouvernée par la régulation transcriptionnelle directe d'un composant clé des SCs, Cep131. L’expression ectopique de Cep131 dans les PNGs restore les effets liés à l'inactivation d'Atoh1, rétablissant la localisation correcte du SC et comme conséquence la présence d’un cil primaire.De plus, nous avons montré que cette voie Atoh1-SC-cil primaire-SHH contrôlant la prolifération des PNGs est également conservée dans le contexte du SHH-MB, où Atoh1 est surexprimée et essentielle pour sa formation et sa maintenance.Ces données révèlent un mécanisme par lequel la ciliogenèse est régulée dans des progéniteurs de neurones, offrant de nouvelles informations sur la neurogenèse dans le cervelet et sur la pathogenèse du SHH-MB<br>Cerebellar granule neuron progenitors (GNPs) require the primary cilium to proliferate in response to Sonic Hedgehog (SHH) during cerebellar development. As aberrant proliferation of GNPs may lead to SHH-type medulloblastoma (SHH-MB), a pediatric brain tumor, understanding which mechanisms control ciliogenesis in GNPs represents a major interest in the field. Here, we show that the proneural bHLH transcription factor Atoh1 controls the presence of primary cilia in GNPs both in vitro and in vivo, thus maintaining GNPs responsive to the mitogenic effects of SHH. Indeed, loss of primary cilia induced via knockdown of specific ciliary components (e.g. Kif3a and Ift88) abolishes the ability of Atoh1 to keep GNPs in proliferation in vivo. Mechanistically, Atoh1 controls ciliogenesis by regulating the proper peri-centrosomal clustering of centriolar satellites (CS), large multiprotein complexes working as essential machineries for ciliogenesis. Knockdown of Atoh1 in GNPs perturbs CS subcellular distribution, leading to impairment of ciliogenesis. Luciferase reporter assays and chromatin immunoprecipitation experiments indicate that Atoh1 can directly regulate the expression of Cep131, a key CS core component. Importantly, ectopic expression of Cep131 in GNPs depleted of Atoh1, is sufficient to restore proper CS localization and consequent primary cilia formation, indicating that the Atoh1-Cep131-CS axis is responsible for ciliogenesis in GNPs.In addition, we further demonstrated that these functions of Atoh1 are conserved in the context of SHH-MB, where Atoh1 is typically overexpressed and acts as a lineage-dependent transcription factor.These data reveal a mechanism whereby ciliogenesis is regulated in neuron progenitors providing novel insights into cerebellar neurogenesis and pathogenesis of SHH-MB

Cilia and flagella are hair-like projections found on the surface of virtually every vertebrate cell. These microtubule-based organelles are historically known for their striking motility, a valuable tool for the manipulation of fluid environments. In addition, immotile (or 'primary') cilia play critical roles in cell signaling. More than ten human diseases have been linked to cilia function, with pleiotropic phenotypes including obesity, kidney and liver disease, skeletal abnormalities, situs defects, mental retardation, and sterility. In this dissertation, I first examine the function of Cep290, a putative master regulator of cilia biology, which is mutated in five human ciliopathies. I found that the zebrafish Cep290 protein was localized in a cell-type specific fashion to two distinct ciliary compartments: transition zones and centriolar satellites. Through morpholino knockdown, I demonstrated that Cep290 regulates the length of photoreceptor, Kupffer’s vesicle, and spinal canal cilia, while it was dispensable for normal cilia length in other tissues. Rescue of Cep290 associated cilia length defects by overexpression of cilia membrane proteins implicated Cep290 in cilia vesicle trafficking. Unexpectedly, I found that Cep290 deficiency in Kupffer’s vesicle and spinal canal resulted in cilia paralysis, accounting for left right asymmetry and hydrocephalus phenotypes, and identifying a novel function for Cep290 in dynein arm assembly. In the second chapter I identify and characterize three novel ciliopathy genes. We performed a small-scale morpholino screen to test the function of predicted cilia proteins. Three genes essential to cilia motility were identified: c21orf59, ccdc65, and c15orf26. Parallel studies in other systems revealed that C21orf59 was a component of the flagellar matrix required for the assembly of outer dynein arms, while Ccdc65 was part of the dynein regulatory complex, which regulates ciliary beat patterns. Importantly, we discovered that both C21ORF59 and CCDC65 were mutated in patients diagnosed with the human motile cilia disorder primary ciliary dyskinesia, identifying two novel human disease genes. Taken together, this work analyses multiple requirements for the assembly of motile and primary cilia and highlights the utility of the zebrafish system in investigations of cilia biology, particularly in the discovery and characterization of human disease genes.

碩士<br>國立交通大學<br>生物科技學系<br>104<br>Microtubule is the major cytoskeleton in neuron, and regulates various crucial neuron developmental processes. To understand how microtubule cytoskeleton influences neuronal development, we have previously utilized quantitative proteomics to examine microtubule-associated proteomes in mouse embryonal carcinoma cells (P19 cells) before and after neuronal differentiation. One of the proteins on microtubules exhibiting substantial and significant increase upon neuronal differentiation is centrosomal protein 170 (Cep170). This suggests that it plays a role in regulating neuronal microtubules. In mitotic cells, Cep170 locates at subdistal appendages of mother centriole and has been shown to play a role in microtubule organization. However, whether Cep170 participates in microtubule organization in post-mitotic neurons remain elusive. To answer this question, we depleted endogenous Cep170 in both P19 cell-derived neurons and primary hippocampal neurons. Surprisingly, Cep170 knockdown did not significantly affect neuronal differentiation or neuronal morphogenesis. In contrast, overexpressing Cep170 caused a significant increase in neurite length in both P19 cell-derived neurons and mouse hippocampal neurons and. Furthermore, we observed that exogenously expressed Cep170 localized as puncta along neurites. We also attempted to determine the domain of Cep170 that promotes neurite elongation by overexpressing truncated Cep170 in hippocampal neurons. Our results suggest that Cep170 plays an important role in post-mitotic cells and its increase on microtubules promotes neurite formation and elongation.

Contexte: Le centrosome est un petit organite bien connu pour son rôle dans l'établissement du fuseau bipolaire pendant la division cellulaire. Les déficiences de la fonction du centrosome donnent souvent lieu à des maladies humaines, y compris le cancer et la formation de kystes rénaux. Nous sommes intéressés à étudier la fonction d'une nouvelle protéine centrosomale nommée CEP78, identifiée dans un criblage protéomique pour de nouveaux composants centrosomaux. Méthodes et résultats : Le traitement des cellules avec le nocodazole, un agent qui dépolymérise spécifiquement les microtubules cytoplasmiques mais pas les microtubules stabilisés du centrosome, a montré que CEP78 est un composant centrosomal stable. La colocalisation de cette protéine avec d'autres marqueurs centrosomaux tels que CEP164, SAS6, Centrine, tubuline polyglutamylée et POC5, à différentes phases du cycle cellulaire a indiqué que CEP78 est précisément à l'extrémité distale des centrioles, mères et filles. Il existe deux pointts CEP78 au cours de l’interphase et les cellules passent par la mitose, procentrioles maturent, et le nombre de points de CEP78 augmente à 4 par cellule et, à la fin de la télophase chaque cellule fille possède 2 points CEP78. La caractérisation des domaines fonctionnels de CEP78 a montré que des répétitions riches en leucine sont nécessaires pour la localisation centrosomale de la protéine. En outre, nous avons constaté que la surexpression de CEP78 ne change pas le nombre de mères/procentrioles mais diminue le nombre et l'intensité des points de CEP170 (protéine d'appendice sous-distal) sans diminution du niveau d'expression de cette protéine. D'autres études ont montré qu'il n'y a pas d'interaction entre ces deux protéines. Enfin, la surexpression de CEP78 protège des microtubules contre la dépolymérisation en présence de nocodazole, ce qui suggère qu'il possède la capacité de lier les microtubules. Conclusion : Nos résultats suggèrent que CEP78 est destiné à l'extrémité distale des centrioles matures par ses répétitions riche en lecuine, où il pourrait être impliqué dans la maturation ou la régulation de l'assemblage ou de la rénovation de l'appendice sous-distal centriolaire, une structure connue dans la nucléation des microtubules et d'ancrage. Comprendre la fonction de Cep78 contribuera à éclaircir le rôle du centrosome dans le cycle cellulaire.<br>Background: The centrosome is a tiny organelle well-known for its role in establishing the bipolar spindle during cell division. Defects in centrosome function often give rise to human diseases including cancer and kidney cyst formation. We are interested in studying the function of one novel centrosomal protein named CEP78, identified in a proteomic screen for novel centrosomal components. Methods and results: Treatment of cells with nocodazole, a microtubule-depolymerizing agent that specifically depolymerizes cytoplasmic microtubules but not the stabilized centrosome microtubules, showed that CEP78 is a stable centrosomal component. Colocalization of this protein with other centrosomal markers such as CEP164, SAS6, Centrin, Polyglutamylated tubulin and POC5 at different phases of the cell cycle indicated that CEP78 specifically localizes to the distal end of the mother and daughter centrioles. There are 2 CEP78 dots during the interphase and as the cells go through mitosis, procentrioles mature, and the number of CEP78 dots increases to 4 dots per cell and by the end of telophase each daughter cell has 2 CEP78 dots. Characterization of CEP78 functional domains showed that Leucine-rich repeats are necessary for centrosomal localization of the protein. In addition, we found that overexpression of CEP78 did not change the number of centrioles and centrosomes but decreased the number and intensity of CEP170 dots (sub-distal appendage protein) without a decrease in the expression level of this protein. Further studies showed that there is no interaction between these 2 proteins. Finally, overexpression of CEP78 protects microtubules from depolymerization in the presence of nocodazole, suggesting its ability to bind microtubules. Conclusion: Our findings suggest that CEP78 is targeted to the distal end of mature centrioles via its lecuine-rich repeats, where it could be involved in centriolar maturation or regulation of sub-distal appendage assembly and/or remodeling, a structure known to nucleate and anchor microtubules. Understanding the function of CEP78 will shed light on the role of the centrosome in cell cycle.

碩士<br>國立陽明大學<br>腦科學研究所<br>107<br>Lissencephaly (smooth brain) is a human brain developmental disorder characterized by the lack of normal folding of the brain. Here we identified a nonsense mutation in the CEP170 gene, which results in the truncated of its product (p.R1259X), in a lissencephaly patient. CEP170 is localized on the subdistal appendage of the mother centriole and the centriole proximal end. In addition, CEP170 has also been reported to regulate the microtubule organization. However, the roles of CEP170 in brain development and how its p.R1259X mutation caused lissencephaly are still unclear. We hypothesize that p.R1259X mutation in CEP170 will decrease the centrosomal localization then affect the microtubule organization to cause neuronal migration defects. To investigate the function of CEP170 in migrating neurons, we delivered CEP170 shRNA into embryonic mice by in utero electroporation to knock down CEP170 expression in neural progenitor cells. CEP170 dysfunction led to neuronal migration delay and abnormal dendritic morphology at postnatal day 6. The p.R1259X mutation showed decreased localization to the centrosomes in culture cells. In addition the microtubule regrowth assay also observed that p.R1259X delayed microtubule nucleation. These results suggest that failure of CEP170 in centrosomal localization and microtubule regrowth may lead to migration defect. Our findings reveal the role of CEP170 in cortical development and provide novel mechanisms of the pathogenesis of lissencephaly.

碩士<br>國立交通大學<br>生物科技學系<br>108<br>Microtubule is the major cytoskeleton in neurons and involved in numerous developmental processes. In a previous study, quantitative proteomics has been used to examine microtubule-associated proteomes in mouse embryonal carcinoma cells before and after neuronal differentiation. The centrosome protein Cep170, which made up part of the mitotic centrosome, has been found to exhibit a significant increase on microtubules upon neuronal differentiation. Overexpressing Cep170 caused a significant increase in neurite length in both P19 cell-derived neurons and mouse hippocampal neurons, suggesting that Cep170 is involved in the regulation of neuronal morphogenesis. However, the exact mechanism of Cep170 in controlling the morphogenetic process of neurons remain elusive. In this study, we attempt to answer the aforementioned question by studying the effect of Cep170 overexpression on microtubule cytoskeleton as well as examining the localization of endogenous Cep170. We discovered that overexpressing Cep170 causes a significant increase in both axon and dendrite length in dissociated hippocampal neurons. Overexpressed Cep170 localizes as non-centrosomal puncta along both axons and dendrites, and displays variation in morphology in different regions of the neurite. In addition, overexpressed Cep170 is enriched at axon but not dendrite tips. Furthermore, overexpressing Cep170 in hippocampal neurons increases the microtubule polymerization rate. We also attempted to determine the localization of the endogenous Cep170 in neurons by immunofluorescence staining. Consistent with the overexpression study, endogenous Cep170 also localizes as non-centrosomal puncta along the neurite with a significant enrichment at the axon tip. Our results suggest that Cep170 localizes as non-centrosomal puncta in post-mitotic cells and its effect on microtubule polymerization rate may be the mechanism in promoting neurite elongation.

碩士<br>國立陽明大學<br>腦科學研究所<br>106<br>Abstract Background: During cerebellar development, granule neuron progenitors (GNPs) proliferate in the external granule layer (EGL) to expand the number of neurons. Abnormal GNP proliferation can give rise to medulloblastoma (MB), the most common malignant brain tumor in children. Interestingly, both GNP and MB cells proliferation requires primary cilia, short microtubule-based structures protruding from the cell membrane. Ablation of primary cilia in GNPs prevented MB formation. However, it is still not fully understood how primary cilia are regulated in GNPs. Previously, Cep70 has been shown to maintain primary cilia by inhibiting Hdac6, a deacetylase responsible for microtubule disassembly during the absorption of cilia. Therefore, we hypothesized that Cep70 is required for primary cilia maintenance in proliferating GNPs. Materials and Methods: We investigated the impacts of CEP70 overexpression and knockdown in GNPs by in vivo cerebellar electroporation. Lentiviruses were also used to manipulate the protein level in purified GNPs in vitro. Result: Here we showed that Cep70 expression is correlated with the presence of primary cilia in GNPs. We further investigated the effects of Cep70 expression in GNPs by in vivo cerebellar electroporation. We found that overexpression of Cep70 maintained more ciliated GNPs, while knockdown of Cep70 decreased ciliated GNPs in vivo. Importantly, treatment of SHH, a diffusible mitogen required for cerebellar development, enhanced Cep70 expression in purified GNPs in vitro, suggesting that Cep70 is tightly regulated by SHH. In addition, we discovered that knockdown of Atoh1, a transcription factor regulated by SHH, decreased Cep70 expression in GNPs in vitro. As Atoh1 maintained more ciliated GNPs, knocking down Cep70 abolished this effect, suggesting that Cep70 functions downstream of Atoh1 for ciliogenesis. Our results demonstrate that Cep70 is required for primary cilia existence in cycling GNPs during cerebellar development. This SHH-Atoh1-Cep70 axis keeps GNP proliferation in response to SHH signaling. Our study implied that Cep70 can be a promising target to treat MB.

Les centrosomes sont de petits organites qui régulent divers processus cellulaires comme la polarité ou la mitose dans les cellules de mammifères. Ils sont composés de deux centrioles entourés par une matrice péricentriolaire. Ces centrosomes sont les principaux centres organisateurs de microtubules. De plus, ils favorisent la formation de cils, des protubérances sur la surface des cellules quiescentes qui sont critiques pour la transduction du signal. Une grande variété de maladies humaines telles que les cancers ou les ciliopathies sont liées à un mauvais fonctionnement des centrosomes et des cils. C’est pourquoi le but de mes projets de recherche est de comprendre les mécanismes nécessaires à la biogénèse et au fonctionnement des centrosomes et des cils. Tout d'abord, j’ai caractérisé une nouvelle protéine centrosomale nommée nephrocystine - 5 (NPHP5). Cette protéine est localisée dans les cellules en interphase au niveau de la région distale des centrioles. Sa déplétion inhibe la migration des centrosomes à la surface cellulaire lors de l’étape précoce de la formation des cils. NPHP5 interagit avec la protéine CEP290 via sa région C-terminale qui est essentielle pour la ciliogenèse. Elle interagit également avec la calmoduline ce qui empêche son auto-agrégation. J’ai démontré que les domaines de liaison de NHPH5 à CEP290 et à la calmoduline, ainsi que son domaine de localisation centrosomale sont séparables. De plus, j’ai démontré que les protéines NPHP5 présentant des mutations pathogènes ne peuvent plus interagir avec CEP290 et ne sont plus localisées aux centrosomes, rendant ainsi ces protéines non fonctionnelles. Enfin, en utilisant une approche pharmacologique pour moduler les événements en aval dans la voie ciliogénique, j’ai montré que la formation des cils peut être restaurée même en absence de NPHP5. D’autre part, j’ai étudié le rôle de NPHP5 dans l'assemblage et le trafic du complexe BBSome dans le cil. Le BBSome est composé de huit sous-unités différentes qui s’assemblent en un complexe fonctionnel dont on sait peu de chose sur la régulation spatiotemporelle de son processus d'assemblage. J’ai précédemment montré que NPHP5 favorisait la formation des cils et que son dysfonctionnement contribuait au développement de néphronophtise (NPHP). Bien que la NPHP et le syndrome de Bardet-Biedl (BBS) soient des ciliopathies qui partagent des caractéristiques cliniques communes, la base moléculaire de ces ressemblances phénotypiques n’est pas comprise. J’ai constaté que NPHP5, localisé à la base du cil, contient deux sites de liaison distincts pour le BBSome. De plus, j’ai démontré que NPHP5 et son partenaire CEP290 interagissent de façon dynamique avec le BBSome pendant la transition de la prolifération à la quiescence. La déplétion de NPHP5 ou CEP290 conduit à la dissociation d’au moins deux sous-unités du BBSome formant alors un sous-complexe dont la capacité de migration dans le cil n’est pas compromise. J’ai montré que le transport des cargos vers le compartiment ciliaire par ce sous-complexe n’est que partiellement altéré. Enfin, j’ai également concentré mes recherches sur une autre protéine centrosomale peu caractérisée. La protéine centrosomale de 76 kDa (Cep76) a été précédemment impliquée dans le maintien d’une duplication unique des centrioles par cycle cellulaire, et dans une interaction avec la kinase cycline-dépendante 2 (CDK2). Cep76 est préférentiellement phosphorylée par le complexe cycline A/CDK2 sur le site unique S83. Cet événement est essentiel pour supprimer l'amplification des centrioles en phase S. J’ai démontré que Cep76 inhibe cette amplification en bloquant la phosphorylation de Plk1 au niveau des centrosomes. D’autre part, Cep76 peut être acétylée au site K279 en phase G2, ce qui régule négativement son activité et sa phosphorylation sur le site S83. Ces études permettent d'améliorer notre compréhension de la biologie des centrosomes et des cils et pourraient conduire au développement de nouvelles applications diagnostiques et thérapeutiques.<br>Centrosomes are small organelles that regulate diverse cellular processes such as polarity or mitosis in mammalian cells. They are composed of two centrioles surrounded by a pericentriolar matrix. These centrosomes are the major microtubule organizing centers. Moreover, they promote the formation of cilia, protrusions on the surface of quiescent cells that are critical for signal transduction. A wide variety of human diseases such as cancers or ciliopathies are linked to a malfunction of centrosomes and cilia. Therefore the aim of my research is to understand the mechanisms necessary for the biogenesis and function of centrosomes and cilia. First, I have characterized a novel centrosomal protein called nephrocystin - 5 (NPHP5). This protein is localized, in interphase cells, in the distal region of centrioles. Its depletion inhibits the migration of centrosomes to the cell surface during the early stage of cilia formation. NPHP5 interacts with CEP290 via its C-terminal region that is essential for ciliogenesis. It also interacts with calmodulin, which prevents its self-aggregation. I have demonstrated that the Cep290- and CaM-binding domains as well as the centrosomal localization domain of NPHP5 are separable. Moreover, I have shown that NPHP5 proteins with pathogenic mutations can no longer interact with CEP290 and are not localized to centrosomes, rendering these proteins non-functional. Finally, using a pharmacological approach to modulate the downstream events in the ciliogenic pathway, I showed that cilia formation can be restored even without NPHP5. On the other hand, I studied the role of NPHP5 in the assembly and trafficking of the BBSome into the cilium. The BBSome consists of eight different subunits that assemble into a functional complex of which little is known about the spatiotemporal regulation of its assembly process. I have previously shown that NPHP5 favored the formation of cilia and its dysfunction contributes to the development of nephronophthisis (NPHP). Although the NPHP and BBS syndrome (BBS) are ciliopathies that share common clinical features, molecular basis of these phenotypic similarities is not understood. I found that NPHP5, located at the base of the cilium, contains two separate binding sites for BBSome. Furthermore, I demonstrated that NPHP5 and his partner CEP290 interact dynamically with the BBSome during the transition from quiescence to proliferation. Depletion NPHP5 or CEP290 leads to the dissociation of at least two subunits of BBSome forming a sub-complex that can still traffic into the cilium. I have shown that the transport of cargo to the ciliary compartment through this sub-complex is only partially altered. Finally, I have also focused my research on another centrosomal protein poorly characterized. The centrosomal protein of 76 kDa (Cep76) was previously involved in the maintenance of a single duplication of centrioles per cell cycle, and interacts with the cyclindependent kinase 2 (CDK2). Cep76 is preferentially phosphorylated by cyclin A/CDK2 on the single site S83. This event is essential to suppress centrioles amplification in S phase. I have demonstrated that Cep76 inhibits amplification by blocking the phosphorylation of Plk1 at the centrosome. Moreover, Cep76 can be acetylated at the K279 site in G2 phase, which negatively regulates its activity and phosphorylation on the site S83. These studies will improve our understanding of the biology of centrosomes and cilia and could lead to development of new diagnostic and therapeutic applications.

碩士<br>國立臺北大學<br>國際財務金融碩士在職專班<br>106<br>This study collects the data of Small and Medium Enterprise (SME) managers’ credit card credit status at the " Joint Credit Information Center " between 2009 and 2016 from a domestic commercial bank. There are 300 samples in total containing 150 non-performing loans and 150 normal credit loans. The methodology of this study is using Logistic Regression Model to find out the relationship between SME managers’ credit card credit status and corporate credit default risk. The results show that: I. A significant correlation between occurrences of non-performing loans of SME and three factors, which are SME managers whom have“credit card overdue payment records”,“none credit card overdue payment records, but pay with minimum required amount”and“pay by installment payments”. II. The rate of forecast accuracy of non-performing loans possibility is higher, when the model uses three foresaid variables with comparison one factors–credit card overdue payment records of SME managers. Due to there is only one lagging indicator, which is credit card overdue payment records of SME managers, has been applied in the existing SME credit risk assessment model of population when it rates the default risk of SME. It is suggested that there are two more leading indicators, which are “none credit card overdue payment records of SME managers, but pay with minimum required amount” and “pay by installment payments”, should be added into the model as these two factors may also imply the risk of credit expansion. It not only optimizes the SME credit risk assessment model, but also upgrades the quality of credit assets. Meanwhile, it is also reducing the possibility of non-performing loans as well as strengthening the Capital Adequacy Ratio of financial institutions and its operation structure. Consequently, it helps to built up a robust financial market body of abstract starts here.

Dissertação de Mestrado em Genética Molecular, Comparativa e Tecnológica<br>A distribuição equitativa dos cromossomas durante a divisão celular é essencial à viabilidade do organismo. Erros na segregação dos cromossomas estão na origem da aneuploidia associada a doenças do recém-nascido e tumorigénese. Deste modo, é fundamental a compreensão dos mecanismos moleculares envolvidos na fidelidade da segregação cromossómica. A separação dos cromossomas durante a mitose requer a formação de uma estrutura simétrica bipolar, designada fuso mitótico, que é formada por microtúbulos nucleados a partir dos centrossomas em células animais. Os cromossomas são ligados ao fuso através da interacção dos cinetocoros com os microtúbulos. Existe um elevado número de proteínas associadas ao fuso, centrossomas e cinetocoros que regulam a bipolaridade, os movimentos cromossómicos e a fidelidade da segregação. A proteína xCep57 foi previamente caracterizada em Xenopus como uma proteína adaptadora das ligações dos microtúbulos aos centrossomas e cinetocoros. A imunodepleção desta proteína num sistema in vitro de extractos de oócitos compromete a estrutura do fuso, o alinhamento dos cromossomas e a estabilidade das ligações cinetocoro-microtúbulo e centrosoma-microtúbulo. Consistentemente, esta proteína interactua com proteínas cinetocorianas reguladoras das ligações ao fuso bem como a γ-tubulina que faz parte dos anéis de nucleação de microtúbulos nos centrossomas. Tem sido questionada a possibilidade da xCep57 ser o homólogo funcional da proteína Dam1 de levedura, para a qual foi demonstrado um papel fulcral na acoplagem da ligação do cinetocoro aos microtúbulos dinâmicos. Existe assim grande expectativa quanto à identificação de um homólogo em eucariotas superiores de função determinante na regulação dos movimentos cromossómicos e fidelidade de segregação. Neste trabalho, partiu-se para um estudo funcional da proteína Cep57 humana com recurso à metodologia de RNA de interferência (RNAi) e microscopia de fluorescência confocal, com o objectivo de se caracterizar a função desta proteína num sistema in vivo através da sua repressão específica por RNAi. Verificou-se por imunofluorescência que a proteína Cep57 se localiza nos centrossomas em interfase e mitose, mas não se detectou nos cinetocoros. Também se demonstrou que a proteína Cep57 se liga directamente a microtúbulos in vitro. A análise do fenótipo mitótico de células depletadas de Cep57 por RNAi revelou a existência de problemas no alinhamento dos cromossomas na placa metafásica e na organização do fuso mitótico. As ligações cinetocoro-microtúbulos estão significativamente comprometidas e a distância interpolar está diminuída. No entanto, ao contrário dos resultados anteriormente descritos que apontam para a interacção de xCep57 com as proteínas cinetocorianas Zwint e Mis12, neste estudo não se observou a diminuição destas proteínas no cinetocoro de células RNAi-depletadas. Deste modo, o fenótipo mitótico observado parece resultar de uma função da proteína Cep57 no centrossoma e não no cinetocoro, possivelmente, e em particular, na ancoragem dos microtúbulos aos centrossomas. Curiosamente, através de um estudo in silico, descobriu-se a existência de um segundo homólogo de xCep57 em humano e em ratinho. Deixa-se assim em aberto a questão de, em humano, poderem existir duas proteínas homólogas de xCep57 com função acopladora aos microtúbulos, actuando uma nos centrossomas (provavelmente a Cep57 aqui descrita) e outra nos cinetocoros. Estudos futuros centrar-se-ão no esclarecimento desta questão.<br>The equal distribution of chromosomes during cell division is essential to the viability of the organism. Errors in chromosome segregation are the cause of aneuploidy associated with diseases of the newborn and tumorigenesis. Thus, it is essential the understanding of the molecular mechanisms involved in the fidelity of chromosome segregation. The separation of chromosomes during mitosis requires the formation of a symmetric bipolar structure, called spindle, which is formed by microtubules nucleated from centrosomes in animal cells. Chromosomes are attached to the spindle through interaction of the kinetochores with microtubules. There are a large number of proteins associated with the spindle, centrosomes and kinetochores governing spindle bipolarity, chromosome movement and the fidelity of chromosome segregation. xCep57 protein was previously characterized in Xenopus as an adapter of the connections of microtubules to the centrosomes and kinetochores. Immunodepletion of this protein in an in vitro system of oocyte extracts compromises the structure of the spindle, the alignment of chromosomes and stability of kinetochore-microtubule as well as centrosome-microtuble connections. Consistently, this protein interacts with regulatory kinetochore proteins linked to the spindle and γ-tubulin which is a main component of the nucleation rings of microtubules at centrosomes. It has been called into question the possibility of xCep57 be the functional homolog of the Dam1 yeast protein, for which a central role in coupling the attachment of kinetochore to dynamic microtubules has been shown. Thus, there are great expectations to the identification of a homolog with decisive role in regulating the movement and fidelity of chromosome segregation in higher eukaryotes. In this work, a functional study of the human protein Cep57 has been carried out in an in vivo system using the RNA interference (RNAi) methodology and confocal microscopy analysis. It was found by immunofluorescence that the Cep57 protein is located in centrosomes during both interphase and mitosis, but could not be detected in kinetochores. It was also demonstrated that the protein Cep57 binds directly to microtubules in vitro. Phenotypic analysis of Cep57 RNAi-depleted mitotic cells revealed the existence of chromosome alignment and spindle defects. The kinetochore-microtubule connections are significantly compromised and the interpolar distance is decreased. Futhermore, the kinetochore localization of known interactors of xCep57 is not altered in Cep57 RNAi-depleted cells consistently to the fact that human Cep57 could not be detected at kinetochores. Thus, overall, the mitotic phenotype observed seems to result from a centrosomal function of Cep57, possibly in microtubule anchorage to centrosomes. Interestingly, through an in silico study, we found the existence of a second homolog of xCep57 in human and mouse. Therefore, an open question remains whether both xCep57 protein homologues in human and mouse have a function in microtubule attachment, with Cep57 described here acting at the centrosome level and the other Cep57-related protein perhaps acting at the kinetochore level. Future studies will be focused on addressing this question.

Le syndrome de Joubert est une maladie récessive caractérisée par une malformation congénitale distincte du tronc cérébral et du cervelet, associée à une anomalie des mouvements oculaires (apraxie oculomotrice), une respiration irrégulière, un retard de développement, et une ataxie à la démarche. Au cours de la dernière décennie, plus de 20 gènes responsables ont été identifiés, tous ayant un rôle important dans la structure et la fonction des cils primaires. Ainsi, le syndrome de Joubert est considéré une ciliopathie. Bien que le Syndrome de Joubert ait été décrit pour la première fois dans une famille canadienne-française en 1969, le(s) gène(s) causal demeurait inconnu dans presque tous les cas de syndrome de Joubert recensés en 2010 dans la population canadienne-française, soit début de mon projet doctoral. Nous avons identifié un total de 43 individus canadiens-français (35 familles) atteints du syndrome de Joubert. Il y avait un regroupement de familles dans la région du Bas-Saint-Laurent de la province de Québec, suggérant la présence d'un effet fondateur. L’objectif de ce projet était de caractériser la génétique du syndrome de Joubert dans la population canadienne-française. Notre hypothèse était qu’il existait un effet fondateur impliquant au moins un nouveau gène JBTS. Ainsi, dans un premier temps, nous avons utilisé une approche de cartographie par homozygotie. Cependant, nous n’avons pas identifié de région d’homozygotie partagée parmi les individus atteints, suggérant la présence d’une hétérogénéité génétique ou allélique. Nous avons donc utilisé le séquençage exomique chez nos patients, ce qui représente une approche plus puissante pour l’étude de conditions génétiquement hétérogènes. Nos travaux ont permis l’identification de deux nouveaux gènes responsables du syndrome de Joubert: C5orf42 et TMEM231. Bien que la localisation cellulaire et la fonction de C5orf42 soient inconnus au moment de cette découverte, nos résultats génétiques combinés avec des études ultérieures ont établi un rôle important de C5orf42 dans la structure et la fonction ciliaire, en particulier dans la zone de transition, qui est une zone de transition entre le cil et le reste de la cellule. TMEM231 avait déjà un rôle établi dans la zone de transition ciliaire et son interaction avec d’autres protéines impliquées dans le syndrome de Joubert était connu. Nos études ont également identifié des variants rares délétères chez un patient JBTS dans le gène ciliaire CEP104. Nous proposons donc CEP104 comme un gène candidat JBTS. Nous avons identifié des mutations causales dans 10 gènes, y compris des mutations dans CC2D2A dans 9 familles et NPHP1 dans 3 familles. Au total, nous avons identifié les mutations causales définitives chez 32 des 35 familles étudiées (91% des cas). Nous avons documenté un effet fondateur complexe dans la population canadienne-française avec de multiples mutations récurrentes dans quatre gènes différents (C5orf42, CC2D2A, TMEM231, NPHP1). Au début de ce projet de recherche, l’étiologie génétique était inconnue chez les 35 familles touchées du syndrome de Joubert. Maintenant, un diagnostique moléculaire définitif est identifié chez 32 familles, et probable chez les 3 autres. Nos travaux ont abouti à la caractérisation génétique du syndrome de Joubert dans la population canadienne-française grâce au séquençage exomique, et révèlent la présence d'un effet fondateur complexe avec une l'hétérogénéité allélique et intralocus importante. Ces découvertes ont éclairé la physiologie de cette maladie. Finalement, l’identification des gènes responsables ouvre de nouvelles perspectives diagnostiques ante-natales, et de conseils génétique, très précieuses pour les familles.<br>Joubert syndrome (JBTS) is a primarily autosomal recessive disorder characterized by a distinctive mid-hindbrain/cerebellum malformation, eye movement abnormalities (oculomotor apraxia), irregular breathing, developmental delay, and ataxia. Over the past decade, over 20 causal genes have been identified, all of which have an important role in the structure and function of the primary cilia. Thus, JBTS joins an expanding category of diseases termed “ciliopathies”. Though JBTS was first described in affected siblings of a French Canadian (FC) family in 1969, the underlying genesis basis of the disorder was unknown in the overwhelming majority of FC cases at the onset of this doctoral project in 2010. We identified a total of 43 FC individuals with JBTS from 35 families. We observed a clustering of the affected families in the Lower Saint-Lawrence region of the province of Quebec, suggesting the presence of a founder effect. The aim of this doctoral project was to characterize the genetic landscape of JBTS in the FC population, and we hypothesized the presence of a founder effect in novel JBTS gene(s). Therefore, we initially used a homozygosity mapping approach. However, we did not identify any shared regions of homozygosity amongst affected individuals, suggesting the presence of genetic and/or allelic heterogeneity. We therefore primarily used a whole exome sequencing approach in our JBTS patients, a strategy that is better suited for the study of genetically heterogeneous conditions. Our work has resulted in the identification of two novel JBTS genes: C5orf42 and TMEM231. In total, we have identified causal mutations in C5orf42 in 14 families (including the original JBTS family described in 1969), and TMEM231 in 2 families. Though the function and cellular localization of C5orf42 was not known at the time of the publication of our manuscript, our genetic findings combined with subsequent animal and cellular work establish the important role of C5orf42 in ciliary structure and function, particularly at the ciliary transition zone. TMEM231 had been previously shown to localize to the ciliary transition zone and interact with several JBTS gene products. We also identified deleterious rare variants in one JBTS patient in the ciliary gene CEP104, implicating CEP104 as a strong candidate JBTS gene. We identified causal mutations in 10 JBTS genes, including CC2D2A in 9 families and NPHP1 in 3 families. Definite causal mutations were identified in 32 of 35 families (91% of cases). We documented a complex founder effect in the FC population with multiple recurrent mutations in 4 different genes (C5orf42, CC2D2A, TMEM231, NPHP1). Prior to the start of this research endeavor, the underlying genetic etiology of Joubert syndrome was unknown in all 35 families. Now, a definite molecular diagnosis has been identified in 32 families, and a probable molecular diagnosis in the remaining 3. Therefore, our work has resulted in the unraveling of the genetic basis of JBTS in the French-Canadian population using WES, and reveals the presence of a complex founder effect with substantial locus and allelic heterogeneity.