A Síndrome de Moebius é caracterizada principalmente pela falta de mobilidade dos músculos da face e abdução dos olhos, conferindo ao paciente um aspecto de face em máscara devido a um comprometimento na formação das estruturas neuromusculares. A sua causa primária permanece desconhecida e várias teorias foram propostas, entre elas a teoria genética. Nesse sentido, o gene HOXB1 localizado no cromossomo 17 é um possível candidato à alteração, uma vez que em modelo animal, quando esse gene está mutado, os animais exibem o fenótipo bastante semelhante aos pacientes portadores da Síndrome de Moebius, pois perdem a mobilidade da musculatura facial. Assim sendo, este projeto propõe analisar, utilizando técnicas de amplificação e seqüenciamento de DNA, possíveis mutações no gene HOXB1 e em seu parálogo HOXA1 em pacientes portadores da Síndrome de Moebius. Palavras-chave: Síndrome de Moebius; HOXA1; HOXB1. / The Moebius Syndrome is characterized by the absence of facial and eyes mobility due to the underdevelopment of facial nerves and muscles causing a face-like mask in the compromised patients. The primary cause still need to be identified, however, different hypothesis have been established including a possible genetic alteration. The HOXB1 gene, located in the chromosome 17 is a possible candidate, since when it was mutated in animals; the phenotype found closely resembles features of clinical profile associated with humans suffering from Moebius Syndrome. Therefore, the aim of this study was to analyze possible mutations in the HOXB1 and in its paralogue HOXA1 in patients with the Moebius Syndrome.

A Síndrome de Moebius é caracterizada principalmente pela falta de mobilidade dos músculos da face e abdução dos olhos, conferindo ao paciente um aspecto de face em máscara devido a um comprometimento na formação das estruturas neuromusculares. A sua causa primária permanece desconhecida e várias teorias foram propostas, entre elas a teoria genética. Nesse sentido, o gene HOXB1 localizado no cromossomo 17 é um possível candidato à alteração, uma vez que em modelo animal, quando esse gene está mutado, os animais exibem o fenótipo bastante semelhante aos pacientes portadores da Síndrome de Moebius, pois perdem a mobilidade da musculatura facial. Assim sendo, este projeto propõe analisar, utilizando técnicas de amplificação e seqüenciamento de DNA, possíveis mutações no gene HOXB1 e em seu parálogo HOXA1 em pacientes portadores da Síndrome de Moebius. Palavras-chave: Síndrome de Moebius; HOXA1; HOXB1. / The Moebius Syndrome is characterized by the absence of facial and eyes mobility due to the underdevelopment of facial nerves and muscles causing a face-like mask in the compromised patients. The primary cause still need to be identified, however, different hypothesis have been established including a possible genetic alteration. The HOXB1 gene, located in the chromosome 17 is a possible candidate, since when it was mutated in animals; the phenotype found closely resembles features of clinical profile associated with humans suffering from Moebius Syndrome. Therefore, the aim of this study was to analyze possible mutations in the HOXB1 and in its paralogue HOXA1 in patients with the Moebius Syndrome.

All-trans retinoic acid (atRA) mediated growth inhibition results in the arrest of the cell cycle during the G1 phase in CAOV3 cells but not SKOV3 ovarian carcinoma cells. The G1 checkpoint is regulated by a multitude of molecules such as the retinoblastoma family of proteins, cyclins, cyclin dependent kinases (CDKs) and cyclin dependent kinase inhibitors (CDKis). CAOV3 cells, which are atRA sensitive, have been shown to express p16INK4a (p16), a cyclin dependent kinase inhibitor regulating the G1 checkpoint. However, atRA resistant SKOV3 cells do not express p16. In these studies, we investigated the role of p16 in mediating atRA induced growth arrest. Our results show that overexpression of p16 in SKOV3 cells leads to growth inhibition following atRA treatment. However, the inhibition is short-term due to the loss of p16 expression. Nevertheless, these results show that p16 plays a role in atRA mediated growth inhibition in ovarian carcinoma cells and that modulation of p16 expression can determine the growth response to atRA. Additionally, we also examined the effect of atRA treatment on the expression of homeobox genes in the CAOV3 cells and SKOV3 cells model system. Homeobox genes comprise a family of transcription factors which function during embryonic development to control pattern formation, differentiation and proliferation. Besides their dominant role during embryogenesis, they are also expressed in adults. In human tumors, an association between the deregulation of the expression of homeobox genes and oncogenic transformation has been reported. It is known that some homeobox genes are atRA targets due to the presence of retinoic acid response element (RARE) either in their promoter region or in their 3' region. In these studies we examined the expression of 13 homeobox genes in CAOV3 cells and SKOV3 cells following ethanol or atRA treatment. The 13 homeobox genes were analyzed because previous studies done by our laboratory observed differences in expression of these homeobox genes when comparing atRA sensitive oral squamous carcinoma cells (SCC) to atRA resistant oral squamous cell carcinoma cells. Of the 13 homeobox genes analyzed in the ovarian carcinoma cell model system, we found HOXA1 and HOXB4 to be upregulated by atRA in CAOV3 cells but not in SKOV3 cells. We also found that the induction of HOXA1 and HOXB4 mRNA expression in CAOV3 cells occurred as a respond to atRA treatment and is not due to a generalized response because of overall growth reduction. Interestingly, HOXA1 has two alternatively spliced forms. The mRNA expression of the truncated form of HOXA1 is highly induced by atRA when compared to its full length form. HOXB1, which is HOXA1 target gene, was not upregulated following atRA treatment. These results suggest that: 1) expression of p16 plays a role in mediating atRA growth inhibition; 2) HOXA1 and HOXB4 also play a role in mediating growth suppression by atRA; and 3) the truncated form of HOXA1 is induced by atRA treatment and may play a role in mediating growth inhibition by atRA, perhaps by acting in a dominant negative fashion. / Microbiology and Immunology

Le syndrome de Marfan (MFS) est une maladie génétique rare. Les signes cliniques sont principalement squelettiques, oculaires et cardiovasculaires. Le premier gène identifié est FBN1. Une LSDB UMD créée en 1995 a permis de colliger les mutations identifiées chez les patients. D’autres gènes ont été identifiés comme causant des syndromes apparentés : FBN2, TGFBR1, TGFBR2, ACTA2, SMAD3, MYH11 et MYLK. Ma thèse avait pour but de mettre à jour ces différentes bases et de créer celles des nouveaux gènes. Les techniques de séquençage nouvelle génération dans la pratique clinique amènent des médecins non spécialistes à rapporter des variations secondaires dans ces gènes « actionable ». Il s’agit de la ressource la plus complète pour les cliniciens et les généticiens pour interpréter les variants associés au MFS et ses pathologies associées pour les variants primaires et secondaires.La bicuspidie de la valve aortique (2 feuillets au lieu de 3) est la malformation cardiovasculaire la plus fréquente touchant 0,6 à 2 % de la population. Ma thèse avait pour but d’identifier de nouveaux gènes impliqués dans la BAV. Grâce à une cohorte de 200 patients, le gène HOXA1, un facteur de transcription, a pu être examiné de manière plus approfondie. Alors qu’il contient une répétition de 10 histidines, les individus hétérozygotes mutés en présentent 11. Mes études ont permis de démontrer l’imputabilité de cette mutation. Dans un second temps, un séquençage d’exomes entiers a permis de mettre en évidence de nouveaux variants prédits pathogènes. Ces données permettront de mieux comprendre le rôle physiologique d’HOXA1 et des nouveaux gènes candidats dans la pathogénèse de la BAV chez l’humain. / Marfan Syndrome is a rare genetic disorder. Clinical signs are mainly skeletal, ocular and cardiovascular. The first gene identified is FBN1. A LSDB UMD was created in 1995 to colligate all the mutations identified in. Thereafter, several others genes were identified involved in related disorders : FBN2, TGFBR1 and TGFBR2, ACTA2, SMAD3, MYH11 and MYLK. The objectives of my PhD were to update these databases and to create new ones for genes. Next generation sequencing in clinical practice leads non-specialized doctors to report pathogenic secondary variants in “actionable” genes. Our databases are the only resources providing access to the full spectrum of known pathogenic mutations with checked and interpreted data from many reference diagnostic laboratories and research centers worldwide, and the most comprehensive resources for clinicians and geneticists to interpret variations linked to Marfan syndrome and related disorders not only primary but also secondary variants.Bicuspid Aortic Valve is the most common cardiovascular malformation affecting 0.6-2% of the population (2 leaflets instead of 3). Thanks to a cohort of 200 BAV patients, HOXA1 gene, a transcription factor, has been screened. While HOXA1 contains a string of 10 histidine repeats, these individuals are heterozygous for an 11 histidine repeat variant. My studies showed the imputability of this mutation. In a second phase, whole exome sequencing allow us to highlight new variants predicted pathogenic. Studies are still ongoing to confirm their imputability. These data contribute to our better understanding of the physiological of HOXA1 and new candidate genes in the pathogenesis of BAV disease in humans.