La reproduction sexuée implique une différentiation extrême des gamètes qui s’accompagne de profonds remaniements des chromosomes parentaux. Au moment de la fécondation, ces chromosomes doivent être rendus compétents pour la formation du premier noyau zygotique. Au cours de ma thèse, j’ai étudié l’importance fonctionnelle de plusieurs voies moléculaires paternelles et maternelles participant à cette étape chez la drosophile. Le complexe HIRA est impliqué dans l’assemblage de nucléosomes dans le pronoyau mâle à la fécondation. J’ai décrit le rôle de HIRA et de son partenaire Yemanucléine-α dans cette voie. J’ai caractérisé plus finement ce complexe en étudiant son rôle somatique dans l’assemblage des nucléosomes et son implication dans la stabilité de l’hétérochromatine, améliorant notre compréhension des besoins biologiques qui conditionnent sa conservation et son évolution. Je me suis aussi intéressé à diverses situations affectant l’intégrité des chromosomes parentaux à la fécondation. (1) J’ai décrit les conséquences catastrophiques pour la méiose femelle de l’expression naturelle d’un transposon à travers l’étude d’un cas de dysgénésie hybride. (2) J’ai contribué à montrer que la protéine K81 est essentielle pour la protection des télomères dans les chromosomes paternels au cours de la spermatogénèse. (3) J’ai participé à caractériser les conséquences pour les chromosomes paternels de l’incompatibilité cytoplasmique induite par la bactérie Wolbachia. Ensemble, ces travaux soulignent les particularités des chromosomes parentaux à la fécondation et aident à cerner l’importance des voies maternelles et paternelles dans leur intégration dans le premier noyau du zygote / Sexual reproduction involves dramatic gamete differentiation and profound parental chromosomes remodelling. At fertilization, these chromosomes need to be rendered competent for the formation of the fist zygotic nucleus. I have studied the functional relevance of several paternal and maternal molecular pathways that participate during this process in Drosophila. The HIRA complex is required for nucleosome assembly in the male pronucleus at fertilization. I have further described the rôle of HIRA and its obligatory partner Yemanuclein-α during this step. I have characterized the somatic roles of this complex during nucleosome assembly and its involvment in heterochromatin stability, which gives us a better understanding of the biological needs that drive its conservation and evolution. I have also focused on several situations where parental chromosomes integrity at fertilization is compromised. (1) I have described a meiotic catastrophe associated with the natural expression of a transposon in the female germline during hybrid dysgenesis. (2) I have contributed to show that K81 is an essential protein for telomere protection in paternal chromosomes during spermiogenesis. (3) I have participated in the characterization of the chromosomal abnormalities associated with cytoplasmic incompatibility induced by Wolbachia. Together, these results underscore the specificities of parental chromosomes at fertilization and shed light into the importance of maternal and paternal pathways for their integration in the first zygotic nucleus

Orientadores: Maricilda Palandi de Mello, Celso Eduardo Benedetti, Fernanda Caroline Soardi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T21:27:39Z (GMT). No. of bitstreams: 1 CunhaJunior_JoseLuizRosenberis_M.pdf: 3002580 bytes, checksum: 1980aba7f72fdfd74e758a924771c055 (MD5) Previous issue date: 2010 / Resumo: A expressão do gene SRY (Sex Determining Region in chromosome Y) é responsável por desencadear a determinação testicular durante o desenvolvimento embrionário, a partir das gônadas ainda indiferenciadas. Mutações nesse gene são encontradas em muitos casos de anomalias do desenvolvimento gonadal. O projeto teve por objetivo principal a análise funcional do efeito de uma mutação na região promotora do gene SRY, sendo que essa mutação consiste em uma deleção de 3 pares de base em um dos sítios consenso de ligação do fator de transcrição Sp1 ao promotor do gene. O portador dessa mutação é um indivíduo com disgenesia gonadal pura 46,XY, sendo que outros membros da família apresentavam ambiguidade genital e o pai, também portador da mutação, possuía grave hipospadia ao nascimento. Para tentar esclarecer os efeitos desta mutação nos mecanismos moleculares de regulação da expressão do gene SRY, este trabalho primeiramente analisou a interação da proteína Sp1 com os sítios localizados na região promotora de SRY e o efeito da mutação nesta interação, através de ensaios de EMSA (Electrophoretic Mobility Shift Assay). Concluiu-se que os sítios Sp1A e Sp1B se ligam a duas moléculas de Sp1 e que a mutação no Sp1A praticamente abole esta ligação. Possivelmente a ausência dessa ligação impediu a formação de um complexo de transcrição, causando uma diminuição na expressão do gene SRY e levando à ausência de formação dos testículos e reversão sexual completa na paciente. Complementando, foi analisado o efeito dessa mutação na expressão através de ensaio de expressão com gene repórter, no qual o promotor normal se mostrou em média duas vezes mais eficiente na ativação da expressão da luciferase que o promotor mutante. Entretanto, mais experimentos de transfecção, inclusive com outras linhagens celulares, devem ser realizados para confirmação desse resultado. Além disso, foi analisado o efeito de uma nova mutação (localizada na região codificante do gene SRY) na ligação da proteína SRY com o DNA, através de ensaios de EMSA. Concluiu-se que a mutação E89K, associada com disgenesia gonadal pura 46,XY, reduziu em alto nível a atividade de ligação in vitro da proteína SRY mutante ao DNA, o que representa um forte indício de que atividade reduzida da proteína mutante não foi suficiente para desencadear o processo de determinação testicular. Paralelamente, foi feito o rastreamento de mutações no gene SRY e sua região promotora em novos casos de disgenesia gonadal, não tendo sido encontradas, porém, alterações nos pacientes analisados / Abstract: The SRY (Sex Determining Region in chromosome Y) gene expression is responsible for testicular determination during embrionary development. Mutations in SRY are found in many cases of anomalies of gonadal development. This project analyzed the functional effect of a mutation in SRY promoter region; the mutation is a 3-bp deletion in a consensus binding site for Sp1 transcription factor. The patient presented 46,XY pure gonadal dysgenesis, and a family history of relatives with different levels of genital ambiguity. Her father shares the same mutation in the SRY promoter region. In order to investigate the effects of the mutation upon the molecular mechanisms that regulate SRY gene expression, the interaction of the Sp1 transcription factor with normal and mutant binding sites was analyzed by EMSA (Electrophoretic Mobility Shift Assay). Each of Sp1A and Sp1B normal sites binds to a single Sp1 molecule, whereas the 3-bp deletion in Sp1A abolishs the binding to this site. Probably, the lack of Sp1 binding to the Sp1A site prevented the formation of a stable transcription complex, reducing the level of SRY expression and leading to the absence of testicles and complete sex reversal in the patient. Parallely, the effect of the mutation was analyzed by a reporter gene assay, indicating that the normal promoter is almost two times more efficient than the mutant promoter in the activation of luciferase gene expression using HeLa cells. However, further transfection experiments with other cell lineages must be performed to confirm this result. In addition, the effect of a new mutation (E89K, located in the SRY gene coding region) was analyzed by testing the ability of the SRY mutant protein to bind its DNA consensus sequence. EMSA assays revealed that the E89K mutation, which is associated with 46,XY pure gonadal dysgenesis, strongtly reduced the SRY protein binding activity in vitro. This result is a strong evidence that the reduced activity of SRY mutant protein was not sufficient to trigger the testicular determination in the patient, leading to the pure gonadal dysgenesis phenotype. Screening of mutations in the SRY gene coding and promoter regions was also performed in six diferent patients with 46,XY gonadal dysgenesis. However, other mutations have not been identified / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Glaucoma is a heterogeneous group of diseases that is currently considered to be the leading cause of irreversible blindness worldwide. Of the identified risk factors, elevated intraocular pressure remains the only modifiable risk factor that can be targeted clinically. Ocular hypertension is often a result of dysregulation of aqueous humour fluid dynamics in the anterior eye segment. Aqueous humour drainage is regulated by structures located in the anterior chamber of the eye. In some circumstances dysregulation occurs due to developmental abnormalities of these structures. The malformation of structures in the anterior segment is thought to be due to a defect in the differentiation and/or migration of the periocular mesenchyme during development. Unique to vertebrates, the neural crest cell (NCC) population contributes to the periocular mesenchyme and is instrumental to the proper development of structures in the anterior segment. For many years our laboratory has examined the role of the Activating Protein-2 (AP-2) transcription factors that are expressed in the neural crest and vital during the development of the eye. The purpose of this research project is to investigate the role of AP-2β in the NCC population during the development of the anterior segment of the eye. Conditional deletion of AP-2β expression in the NCC population demonstrated that mutants have dysgenesis of structures in the anterior segment including defects of the corneal endothelium, corneal stroma, ciliary body and a closed iridocorneal angle. Loss of retinal ganglion cells and their axons was also observed, likely due to the disruption of aqueous outflow, suggesting the development of glaucoma. The data generated from this research project will be critical in elucidating the role of AP-2β in the genetic cascade dictating the development of the anterior eye segment in addition to providing scientific research with a novel model of glaucomatous optic neuropathy. / Thesis / Master of Science (MSc)

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

O Pseudohermafroditismo masculino disgenético (Anomalia da diferenciação sexual 46,XY ADS 46,XY) é definido como ambigüidade genital num paciente com testículos e/ou cariótipo 46,XY com uma das seguintes características: alteração histológica testicular, ausência ou hipoplasia das células de Leydig em tecido previamente estimulado com gonadotrofina coriônica humana(hCG), falta de resposta de testosterona ao estímulo com hCG sem acúmulo de precursores, ausência de células germinativas, presença de derivados müllerianos indicando inadequada produção do hormônio antiMülleriano (HAM) ou resistência de seus receptores. Esse estudo apresenta uma avaliação clínica, laboratorial, anátomopatológica, morfométrica e molecular de 10 pacientes com ADS 46,XY; dois pacientes apresentaram mutação no SF1 (fator esteroidogênico 1), duas mutações no domínio hingee uma terceira produziu um stop códon na posição 404; três pacientes com deleção da cópia do DAZ2. A morfometria testicular mostrou todos os diâmetros tubulares médios (DTM) moderado a gravemente diminuídos e os índices de fertilidade tubular leve a moderadamente diminuídos. Devido à dificuldade do diagnóstico diferencial e etiológico, o estudo morfométrico e molecular deve sempre acompanhar esses casos de ADS 46,XY. / The dysgenetic male pseudohermaphroditism 46,XY ; disorders of sex development (DSD 46,XY) is defined as sexual ambiguity in patients with testis and/or 46,XY karyotype and one of the characteristics: hystologic alteration of the testis; absence or hypoplasia of Leydig cells; a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors; absent germ cells; presence of müllerian duct derivatives showing inappropriate production of antimüllerian hormone (AMH) or resistance to its receptors. This study shows the clinic, laboratory, histologic, morphometric and molecular evaluation of 10 patients with DSD 46,XY; two patients showed mutations in the SF1 gene (steroidogenic factor-1); two in the hinge domain and one stop codon at the position 404 of the protein; three patients exhibited deletion of DAZ2. The testis morphometry showed reduction: marked to severe of all mean tubular diameter (MTD) while the reduction of the tubular fertility index (TFI) were slight to marked. Due to difficulties establishing the differential diagnosis and the etiology, the morphometric and molecular evaluation must be always done in the patients with DSD 46,XY.

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis

O Pseudohermafroditismo masculino disgenético (Anomalia da diferenciação sexual 46,XY ADS 46,XY) é definido como ambigüidade genital num paciente com testículos e/ou cariótipo 46,XY com uma das seguintes características: alteração histológica testicular, ausência ou hipoplasia das células de Leydig em tecido previamente estimulado com gonadotrofina coriônica humana(hCG), falta de resposta de testosterona ao estímulo com hCG sem acúmulo de precursores, ausência de células germinativas, presença de derivados müllerianos indicando inadequada produção do hormônio antiMülleriano (HAM) ou resistência de seus receptores. Esse estudo apresenta uma avaliação clínica, laboratorial, anátomopatológica, morfométrica e molecular de 10 pacientes com ADS 46,XY; dois pacientes apresentaram mutação no SF1 (fator esteroidogênico 1), duas mutações no domínio hingee uma terceira produziu um stop códon na posição 404; três pacientes com deleção da cópia do DAZ2. A morfometria testicular mostrou todos os diâmetros tubulares médios (DTM) moderado a gravemente diminuídos e os índices de fertilidade tubular leve a moderadamente diminuídos. Devido à dificuldade do diagnóstico diferencial e etiológico, o estudo morfométrico e molecular deve sempre acompanhar esses casos de ADS 46,XY. / The dysgenetic male pseudohermaphroditism 46,XY ; disorders of sex development (DSD 46,XY) is defined as sexual ambiguity in patients with testis and/or 46,XY karyotype and one of the characteristics: hystologic alteration of the testis; absence or hypoplasia of Leydig cells; a decreased testosterone response to human chorionic gonadotropin stimulation without accumulation of testosterone precursors; absent germ cells; presence of müllerian duct derivatives showing inappropriate production of antimüllerian hormone (AMH) or resistance to its receptors. This study shows the clinic, laboratory, histologic, morphometric and molecular evaluation of 10 patients with DSD 46,XY; two patients showed mutations in the SF1 gene (steroidogenic factor-1); two in the hinge domain and one stop codon at the position 404 of the protein; three patients exhibited deletion of DAZ2. The testis morphometry showed reduction: marked to severe of all mean tubular diameter (MTD) while the reduction of the tubular fertility index (TFI) were slight to marked. Due to difficulties establishing the differential diagnosis and the etiology, the morphometric and molecular evaluation must be always done in the patients with DSD 46,XY.

Comme tous les ciliés, P. tetraurelia réarrange son génome à chaque génération sexuelle pendant le développement de son macronoyau somatique ¿ partir du micronoyau germinal. Les réarrangements incluent l’excision précise de courtes séquences dérivant de transposons et appelés IES (Internal Eliminated Sequences) dont la majorité sont intragéniques. L’excision d’une fraction d’entre elles dépend de petits ARN maternels (appelés scnARN) qui sont produits à partir de tout le génome germinal pendant la méiose. Ce mécanisme pose un problème lors d’une conjugaison entre deux souches présentant des polymorphismes d’insertion : une cellule sera théoriquement incapable d’exciser une IES portée par l’allèle paternel reçu si cette IES est absente de l’allèle maternel ou si la séquence est trop divergente. Mes résultats montrent cependant que les allèles paternels divergents sont correctement excisés en utilisant les scnARN produit par la cellule paternelle. Dans le cas d’un polymorphisme absence/présence, l’IES que j’ai étudié est excisée chez 70 % des hétérozygotes F1, également via les scnARN paternels. Nous avons exploré deux hypothèses pour expliquer comment ils pouvaient agir. Il pourrait s’agir d’une programmation précoce des noyaux gamétiques ou alors d’un échange cytoplasmique des scnARN. Finalement, j’ai montré qu’un défaut de scnARN maternels n’est pas une cause possible de dysgénésie hybride. Cependant, 30 % des hétérozygotes F1 présentent une rétention variable de l’IES étudié via un mécanisme inconnu. Si cela est généralisable à toutes les IES homozygotes, alors ce mécanisme aurait un effet délétère sérieux sur les F1 et pourrait contribuer à l’isolement reproductif. / Like all ciliates, P. tetraurelia entirely rearranges its genome during development of the somatic macronucleus from the germline micronucleus, in each sexual generation. Rearrangements include the precise excision of IESs (Internal Eliminated Sequences), single-copy intervening sequences likely derived from transposon insertions. At least for a fraction of IESs, correct excision, which is required to reconstitute functional genes in the macronucleus, is thought to depend on their recognition by Piwi-bound small RNAs (called scnRNAs) produced from the maternal germline genome during meiosis. This raises a problem during conjugation between strains presenting insertion polymorphisms: a cell will be theoretically unable to excise an IES from the incoming (paternal) allele if that IES is absent from the maternal allele, or if its sequence is too divergent. Our results, however, indicate that divergent paternal alleles are correctly rearranged, using scnRNAs produced by the paternal cell. In the case of an absence/presence polymorphism, the IES we studied is excised in 70% of heterozygotes, also using paternal scnRNAs. We explored two hypotheses to explain how they can act. It could be either an early programming of the gametic nuclei or through cytoplasmic exchange of scnRNAs. My results seem to favor the latter. Overall, I showed that the lack of maternal scnRNAs is not a possible cause of hybrid dysgenesis. However, 30% of heterozygous F1 display a variable retention of the IES through an unknown mechanism. If this is true for all hemizygous IESs then it will have a strong deleterious effect on hybrid F1s and may contribute to reproductive isolation.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Introdução: O gene DMRT1 é um fator muito importante, o qual induz a determinação sexual masculina. Estudos mais recentes têm demonstrado que o Dmrt1 possui um papel significante no desenvolvimento ovariano. Deleções restritas ao gene DMRT1 têm sido raramente identificadas em pacientes com disgenesia gonadal (DG) sem outras características sindrômicas. Objetivo: Pesquisar a presença de haploinsuficiência do gene DMRT1 (deleções e/ou mutações inativadoras) em um grupo grande de pacientes não sindrômicos com distúrbios do desenvolvimento sexual (DDS) por anormalidades gonadais. Polimorfismos do DMRT1, como fatores potenciais pelas anormalidades gonadais, foram também identificados. Pacientes e Métodos: Foram avaliados cerca de 39 pacientes portadores de DDS por anormalidades do desenvolvimento gonadal 46,XY: 24 com disgenesia gonadal parcial e 15 pacientes com disgenesia gonadal completa. As regiões codificadoras do DMRT1 e o domínio DM (exon 1) foram amplificados e sequenciados. A análise de Multiplex ligation probe amplification (MLPA) do DMRT1 foi realizada usando um kit comercial. Resultados: Deleção parcial ou total do DMRT1 não foi identificada pela técnica de MLPA. Oito variantes alélicas do DMRT1 foram identificados. Uma nova variante c.968-15insTTCTCTCT foi identificada em 6,4% e em 14,3% dos alelos dos pacientes 46,XY e indivíduos controles, respectivamente. Conclusão: Este estudo sugere que deleções parciais ou completas no DMRT1 e mutações inativadoras não são frequentemente encontradas em pacientes com anormalidades do desenvolvimento gonadal. Além disso, nenhuma das variantes alélicas identificadas neste grupo de pacientes poderia ser considerada como um marcador potencial polimórfico para disgenesia gonadal / Introduction Dmrt1 gene is a very important factor in inducing male sex determination, and more recently it has been demonstrated that Dmrt1 plays a significant role in ovary development. DMRT1 deletions have rarely been identified in patients with 46,XY gonadal dysgenesis (GD) without syndromic features. Objective- To screen for the presence of DMRT1 haploinsufficiency (deletions and/or inactivating mutations) in a large cohort of non-syndromic patients with disorder of sex development (DSD) due to abnormalities of gonadal development. DMRT1 polymorphisms, as potential susceptibility factors for gonadal abnormalities, were also investigated. Subjects and Methods- We evaluated 39 patients with 46,XY GD: 24 patients with the partial, and 15 with the complete form. The entire coding region (éxons 2-5) of DMRT1 and the DM domain (exon 1) were PCR-amplified and direct sequenced. Multiplex ligation probe amplification (MLPA) analysis of DMRT1 was carried out using a commercial kit. Results- Partial or total deletion of DMRT1 was not identified by MLPA technique. Eight allelic variants of DMRT1 were identified. The novel variant c.968-15insTTCTCTCT was identified in 6.4% and in 14.3% of the alleles of 46,XY patients and control subjects, respectively Conclusion- This study suggest that complete or partial DMRT1 deletions and inactivating mutations are not frequently found in patients with abnormalities of gonadal development. Additionally, none of the allelic variants identified in this cohort of patients could be considered a potential polymorphic susceptibility marker for gonadal dysgenesis