published_or_final_version / Zoology / Master / Master of Philosophy

Classically, testosterone (T) was considered the principal regulator of aggression. However, recent studies in birds have found aggression and T uncoupled during the non-breeding season. Circulating testosterone comes with costs such as immunosuppression and energy expenditure. Instead, the pro-hormone, dehydroepiandrosterone (DHEA), is circulated and activated within the brain via conversion into oestradiol (E2), avoiding the costs associated with T. At present the site of DHEA synthesis is unknown. My thesis investigated the existence of an analogous pathway in non-breeding red squirrels (Tamiasciurus hudsonicus) with two studies: (a) a field study investigating the effects of E2 on aggression and (b) a laboratory study that attempted to determine the site of DHEA synthesis. I conclude that E2 regulates non-breeding aggression in red squirrels and that the adrenals are not the site of DHEA synthesis. My results suggest the existence of a mammalian analogue to the regulatory pathway found in birds.

Classically, testosterone (T) was considered the principal regulator of aggression. However, recent studies in birds have found aggression and T uncoupled during the non-breeding season. Circulating testosterone comes with costs such as immunosuppression and energy expenditure. Instead, the pro-hormone, dehydroepiandrosterone (DHEA), is circulated and activated within the brain via conversion into oestradiol (E2), avoiding the costs associated with T. At present the site of DHEA synthesis is unknown. My thesis investigated the existence of an analogous pathway in non-breeding red squirrels (Tamiasciurus hudsonicus) with two studies: (a) a field study investigating the effects of E2 on aggression and (b) a laboratory study that attempted to determine the site of DHEA synthesis. I conclude that E2 regulates non-breeding aggression in red squirrels and that the adrenals are not the site of DHEA synthesis. My results suggest the existence of a mammalian analogue to the regulatory pathway found in birds.

Orientador: Rosicleire Veríssimo Silveira / Resumo: A atividade de piscicultura em tanques-rede encontra-se em expansão nas represas brasileiras, entretanto, esta prática pode alterar a biologia reprodutiva das espécies que vivem ao seu entorno. Deste modo, pretendeu-se avaliar a influência de pisciculturas em tanques-rede na reprodução de espécies de peixes, (mandi) Pimelodus cf. platicirris, (corvina) Plagioscion squamosissimus e (porquinho) Geophagus proximus, comparando dois pontos. As coletas foram realizadas em dois pontos no reservatório de Ilha Solteira, rio Grande. Um ponto denominado Área Tanque (AT), uma piscicultura em tanques-rede no munícipio de Santa Clara D’Oeste – SP, no braço Can-Can do reservatório de Ilha Solteira, rio Parana e um segundo ponto, Área Controle (AC) a aproximadamente 10 km, a montante. Os espécimes foram coletados bimestralmente com auxílio de dois lotes de redes com malhas 3, 4, 5, 6 e 7 cm e um lote de redes com malhas 7, 8, 10, 12 e 14 cm, no período de Dez/2014 a Dez/2016). Realizou-se a caracterização morfológica da gametogênese e do ciclo reprodutivo das espécies, além do uso do Ìndice gonadossomático e medida do diâmetro dos oócitos maduros nas diferentes áreas. A frequencia sexual foi similar em P. squamosissimus espécie sedentária e G. proximus territorialista e um pouco discrepante para P. cf. platicirris, espécie migratória, mas se mostrou um dado constante não havendo interferência da piscicultura. A proporção de femeas na fase de apto a desova foi similar para as três espécies, r... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The fish farming activity in net cages is in expansion in Brazilian dams, however, this practice may modify the biology of the species which live around it. This way, this work aimed to evaluate the influence of fish farming in net cages in the reproductive biology of fish species such as (mandi) Pimelodus cf. platicirris, (corvina) Plagioscion squamosissimus and (porquinho) Geophagus proximus, comparing the Net Cage Areas (NCA) to Control Areas (CA). The samples were collected at two points at Ilha Solteira reservoir, Rio Grande. One of them named Net Cage Area (NCA), a fish farm with Net Cages in the County of Santa Clara D’Oeste – SP, at the Can-Can arm of Ilha Solteira reservoir, Parana River and the other one, Control Area, (CA) at about 10 km upstream. The specimens were collected bimonthly with the use of two lots of nets with mesh of 3, 4, 5, 6 e 7 cm and one lot of net with mesh of 7, 8, 10, 12 e 14 cm, between December/2014 and December/2016. Besides the morphological characterization of gametogenesis and the reproductive cycle of the species, the Gonadosomatic index and the measurement of mature oocytes diameter at different areas were made. The sexual frequency was similar in P. squamosissimus secondary species and G. proximus territorialist and with a little discrepancy to P. cf. platicirris, migratory species, but it showed constant data not having interference from the fish farm. The proportion of females, which were suitable for spawning, was similar for the t... (Complete abstract click electronic access below) / Doutor

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The seminal plasma is particularly important in sperm protection against damage caused by reactive oxygen species (ROS). The paraoxonase type 1 (PON1) is a multi-enzymatic complex with antioxidant properties, preventing the increase of ROS quantities, thus protects cell membranes and neutralizes the effects of lipid oxidation. The objective of this study was to correlate the influence of PON1 activity with the CASA system parameters and sperm viability (membrane integrity acrosome integrity, functionality of mitochondria and DNA fragmentation) of swine semen in natura, and investigate the PON1 expression (mRNA) in testicle and epididymis (head, body and tail) tissue of boars. The sperm fraction (FE) presented the major PON1 activity and males 81, 80 and 79 (P<0.05) showed the highest enzyme activity. But, when comparing the differences between the ejaculate samples, our study showed differences (P<0.05) between males 81 and 92 in FE and sperm post fraction (PF). However, when the ejaculated was diluted (1:1), it has presented 44% average reduction in the activity of PON1. The activity of PON1 in FE showed positive correlation with sperm concentration, curvaceous distance, curvy velocity and DNA integrity (P<0.05), and also showed negative correlation with straightness and linearity parameters advised by the CASA system. The mRNA expression was observed only in the body portion of the epididymis. The FE is the part of the ejaculate that more PON1 activity and presented the parameters DCL, VCL, STR and LIN, together, with concentration and sperm viability (integrity of DNA) are those who showed the greatest association with the enzyme. Already the PON1 was only expressed (Mrna) in the body of epididymis pig breeder. / O plasma seminal é particularmente importante na proteção do espermatozoide contra os danos causados pelas espécies reativas ao oxigênio (EROs). A paraoxonase tipo 1 (PON1) é um complexo multienzimático com propriedades antioxidantes, impedindo o aumento da quantidade de EROs, o que confere proteção às membranas celulares e neutraliza os efeitos da oxidação lipídica. O estudo objetivou correlacionar a influência da atividade de PON1 com os parâmetros de avaliação do sistema Computer Assisted Semen Analysis (CASA) e a viabilidade espermática (integridade de membrana, integridade de acrossoma, funcionalidade de mitocôndria e fragmentação do DNA) do sêmen suíno in natura e investigar a expressão da PON1 (RNAm) no tecido do testículo e epidídimo (cabeça, corpo e cauda) do reprodutor suíno adulto. A fração espermática (FE) apresentou a maior atividade de PON1 e os machos 81, 80 e 79 (P<0,05) apresentaram a maior atividade de PON1. Já em relação a comparação entre as diferentes amostras do ejaculado, nosso estudo demonstrou diferenças (P<0,05) nos machos 81 e 92 em relação a FE e a fração pós-espermática (FP). Entretanto, quando o ejaculado foi diluído (1:1) apresentou redução média de 44% na atividade da PON1. A atividade de PON1 na FE apresentou uma correlação positiva com a concentração espermática, DCL, VCL e IDNA (P<0,05) e apresentou correlação negativa para os parâmetros de STR e LIN assessorados pelo sistema CASA. A expressão de RNAm somente foi observada para a porção do corpo do epidídimo. A FE é a parte do ejaculado que mais apresentou atividade da PON1 e os parâmetros DCL, VCL, STR e LIN, juntamente, com concentração e viabilidade espermática (integridade de DNA) são os que apresentaram a maior associação com a enzima. Já a PON1 foi somente expressa (RNAm) no corpo de epidídimo do reprodutor suíno.

Submitted by Natalia de Souza Gonçalves (natalia.goncalves@ufpe.br) on 2016-09-19T13:15:03Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) FABRICYA ROBERTA DA SILVA_PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE HUMANA E MEIO AMBIENTE _CENTRO ACADÊMICO DE VITÓRIA_2016.pdf: 2637722 bytes, checksum: 92b453d850e6c8353a4a077e3f5b4a8d (MD5) / Made available in DSpace on 2016-09-19T13:15:03Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) FABRICYA ROBERTA DA SILVA_PROGRAMA DE PÓS-GRADUAÇÃO EM SAÚDE HUMANA E MEIO AMBIENTE _CENTRO ACADÊMICO DE VITÓRIA_2016.pdf: 2637722 bytes, checksum: 92b453d850e6c8353a4a077e3f5b4a8d (MD5) Previous issue date: 2016-02-24 / CAPES / Em regiões tropicais os morcegos apresentam diferentes estratégias reprodutivas, conforme as variações climáticas e os recursos alimentares disponíveis em seus habitats. Assim, para compreender as peculiaridades da biologia reprodutiva desse grupo, são necessários estudos que abordem análises histomorfométricas das suas gônadas ao longo do ano. Em vista disso, o objetivo desse estudo foi analisar através da morfologia e histomorfometria, as gônadas masculina e feminina do morcego Artibeus planirostris, ao longo do ciclo reprodutivo, em um fragmento de Mata Atlântica no Nordeste do Brasil. Para verificar a condição reprodutiva, os espécimes foram classificados em: machos (testículos descendentes e não descendentes) e fêmeas (inativas, grávidas, lactantes e pós-lactantes). As coletas foram realizadas durante 18 meses (junho/2014 a novembro/2015) e os animais foram agrupados em seis períodos de três meses cada: períodos I, II, III, IV, V e VI. Para a análise histomorfométrica, os animais foram eutanasiados e tiverem seus órgãos removidos. As lâminas produzidas foram coradas por Hematoxilina e Eosina. Nos testículos, foram mensurados: a área de ocupação do compartimento tubular e intertubular; número de células de Leydig, de Sertoli, de espermatócitos e de espermátides alongadas. No epidídimo foi medido: altura do epitélio, diâmetro tubular, do lúmen e do epitélio. Nos ovários foi considerada a área de ocupação e o número de folículos. Os resultados mostraram a presença de machos com testículos descendentes e aumento significativo dos compartimentos testiculares, bem como de seus tipos celulares ao longo de todos os períodos. Tais parâmetros, apresentaram-se estatisticamente pronunciados em dois períodos de baixa precipitação. Foi observado um aumento gradativo da área de ocupação dos ovários, junto ao número de folículos antrais e corpo lúteo, à medida que aproximava-se dos meses mais secos, além disso fêmeas lactantes foram capturadas em meses chuvosos. No epidídimo, os diâmetros luminal, epitelial e tubular apresentaram significâncias na cauda, porém com expressividade acentuada em dois períodos. A altura do epitélio aumentou gradativamente, no entanto uma redução foi verificada em um dos períodos. Em vista dos resultados, é possível concluir que machos de A. planirostris estejam ativos reprodutivamente ao longo do ano, porém podem apresentar picos espermatogênicos em meses secos, os quais seriam para sincronizar com o padrão de poliestria bimodal observado nas fêmeas. / In tropical regions the bats have different reproductive strategies, according climatic variations and food resources available in their habitats. Thus, to understand the peculiarities of the reproductive biology of this group, are necessary studies that address histomorphometric analysis their gonads throughout the year. In view of this, the aim of this study was to analyze through morphology and histomorphometry, the male and female gonads bat Artibeus planirostris along the reproductive cycle, in an Atlantic Forest fragment, in the northeast of Brazil. To check the reproductive condition, the specimens were classified as: male (descended and not descended testes) and female (inactive, nursing mothers, pregnant women and post-lactating). Samples were collected for 18 months (June / 2014 to November / 2015) and the animals were grouped into six periods of three months each: periods I, II, III, IV, V and VI. For histomorphometric analysis, the animals were euthanized and had their organs removed. The produced slides were stained with hematoxylin and eosin. Testicles were measured: the footprint of the tubular and intertubular compartment; number of Leydig cells, Sertoli cells, spermatocytes and spermatids elongated. In the epididymis was measured: epithelial height, diameter tubular, lumen and epithelium. In the ovaries was considered Ovarian footprint and the number of ovarian follicles. The results show the presence males with testes and descendants significant increase in testicular compartments, as well as their cellular types throughout all periods. These parameters were statistically pronounced in two periods of low rainfall. Was observed a gradual increase of ovarian footprint, with the number of antral follicles and corpus luteum, as they approached of the driest months, beyond of this lactating they were captured in rainy months. In the epididymis, the luminal diameters, epithelial and tubular they have the significances tail, but expressively sharp in two periods. The height of the epithelium gradually increased, but a reduction was observed in one of the periods. In view of the results, we conclude that males A. planirostris are reproductively active throughout the year, but may present spermatogenic peaks in dry months, which would be to synchronize with bimodal polyestry observed in females.

Submitted by Geandra Rodrigues (geandrar@gmail.com) on 2018-06-18T15:07:51Z No. of bitstreams: 1 fernandapereirasilva.pdf: 1453424 bytes, checksum: b2a5a43450827f6489877e9e4ea29cca (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2018-06-27T11:38:25Z (GMT) No. of bitstreams: 1 fernandapereirasilva.pdf: 1453424 bytes, checksum: b2a5a43450827f6489877e9e4ea29cca (MD5) / Made available in DSpace on 2018-06-27T11:38:25Z (GMT). No. of bitstreams: 1 fernandapereirasilva.pdf: 1453424 bytes, checksum: b2a5a43450827f6489877e9e4ea29cca (MD5) Previous issue date: 2018-02-26 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A forma e tamanho do corpo são características que diferem entre machos e fêmeas adultos de crocodilianos, entretanto, não existe relato do dimorfismo sexual para filhotes de Melanosuchus niger. O presente estudo teve como objetivo analisar a existência de dimorfismo sexual em filhotes de M. niger com base em dados morfológicos. Os espécimes analisados foram oriundos de um experimento de incubação artificial. Os ovos foram coletados no Lago Tracajá da Reserva de Desenvolvimento Sustentável Mamirauá. As gônadas foram analisadas quanto aos parâmetros macroscópicos (forma, textura, coloração e presença/ausência de ducto de Müller) para verificar se havia dimorfismo sexual. A identificação sexual dos filhotes foi confirmada a partir da análise histológica do complexo-Gônada-Adrenal-Mesonefro (GAM) conforme procedimentos de rotina. Para a análise da variação da forma utilizou-se a morfologia quantificada das placas epidérmicas da cabeça dos filhotes a partir de 13 landmarks submetidos a uma análise de morfometria geométrica. Para isto, foram fotografadas em vista dorsal, a cabeça dos exemplares e utilizados os softwares TPSUtil, TPSDig2 e MorphoJ para análises. A análise histológica serviu para confirmar o sexo dos indivíduos analisados. Foram identificados 38 fêmeas e 12 machos. Nos testículos foi possível observar a presença da túnica albugínea associada a medula pouco diferenciada, onde não foi observada a presença de túbulos seminíferos, apenas a presença de células vacuolizadas. Associado ao mesonefro observou-se a presença de ducto de Müller remanescente. Nos ovários observou-se a presença de um epitélio germinativo evidente, formado por tecido epitelial cuboide simples. Na região medular observou-se a presença de folículos ovarianos e lacunas medulares. O Ducto de Müller apresentou-se ligado ao mesonefro pelo mesossalpinge. Foi observado que as características macroscópicas das gônadas diferem entre os sexos. Os testículos apresentaram um formato elíptico com textura lisa e coloração amarela. Os ovários apresentaram textura granulosa, coloração bege clara e associada a ele observou-se a presença de Ducto de Müller. A análise de componentes principais (PCA) mostrou variação na forma das placas epidérmicas da cabeça de filhotes de M. niger, indicando a presença de dimorfismo sexual. O Componente Principal 1 (PC1) explicou 52,6 % da variação da forma, enquanto o Componente Principal 2 (PC2) explicou 14,4%, e a variação geral explicada por esses dois eixos principais foi de 67,0%. A análise de função discriminante confirmou a presença de dimorfismo sexual (p = 0,01). Foi observado que as fêmeas possuem a região posterior da cabeça mais longa e larga, e a mandíbula mais curta e estreita. Enquanto a região posterior da cabeça dos machos é ligeiramente mais curta e estreita, e a mandíbula mais longa e larga. As características macroscópicas e histológicas das gônadas foram semelhantes às encontradas em outras espécies de crocodilianos. A análise macroscópica demonstrou-se suficiente para identificar o sexo de filhotes de M. niger. Em crocodilianos adultos, o dimorfismo sexual pode estar presente na região do crânio relacionado evolutivamente ao reconhecimento visual do gênero. Entretanto, a seleção sexual não seria tão relevante para os jovens quanto para os indivíduos sexualmente maduros. A variação da forma tanto no crânio quanto na mandíbula encontrada em filhotes de M. niger, sugere que machos e fêmeas possivelmente passaram por diferentes processos ontogênicos. A falta de trabalhos envolvendo o estudo de dimorfismo sexual por meio da técnica de morfometria geométrica em filhotes de outras populações de M. niger e/ou de outras espécies de crocodilianos, dificulta a comparação direta. No entanto, fica claro a importância do estudo de dimorfismo sexual em crias de crocodilianos para estimativa da proporção sexual, principalmente se aplicada em populações naturais. Os modelos morfométricos geométricos podem ajudar a desenvolver representações gráficas do dimorfismo sexual da cabeça nos crocodilianos, o que torna estes resultados importantes para programas de conservação em condições naturais, pois representa uma alternativa não-letal às técnicas utilizadas para identificação sexual em M. niger. / The shape and size of the body are characteristics that differ between adult males and females of crocodilians; however, there is no report about sexual dimorphism for Melanosuchus niger hatchling. The present study had as objective to analyze the existence of sexual dimorphism in of M. niger hatchling based on morphological data. The specimens analyzed in this study came from an artificial incubation experimente. The eggs were collected at Lake Tracajá on Mamirauá Sustainable Development Reserve. The gonads were analyzed with macroscopic parameters (shape, texture, color and presence / absence of Müller duct) to verify if there was sexual dimorphism. The sexual identification of the hatchling was confirmed by the histological analysis of Gonad-Adrenal-Mesonefro (GAM) complex according to routine procedures. For the analysis of the variation of the form the quantified morphology of the epidermal plates of the hatchling' head was used from 13 landmarks submitted to a geometric morphometry analysis. For this, the heads of the specimens were photographed in dorsal view and the TPSUtil, TPSDig2 and MorphoJ software were used for analysis. Histological analysis served to confirm the animal´s sex of this study. There were identified, 38 females and 12 males. In the testes it was possible to observe the presence of the tunica albuginea associated with a little differentiated marrow, where the presence of seminiferous tubules was not observed, only the presence of vacuolated cells. Associated with the mesonephro we observed the presence of the remaining Müller duct. In the ovaries, the presence of an evident germinative epithelium, formed by simple cuboid epithelial tissue was observed. In the medullary region the presence of ovarian follicles and marrow gaps were observed. The Müller Duct was connected to the mesonephro by the mesosalpinge. It was observed that the macroscopic characteristics of the gonads differ between the sexes. They showed elliptical, smoothly, yellow testes. The ovary presents granular structure with light beige coloration and associated with the presence of Müller's Duct. Principal component analysis (PCA) showed variation in the shape of epidermal scales of M. niger hatchling, indicating the presence of sexual dimorphism. The Principal Component 1 (PC1) explained 52.6% of the shape variation, as the Principal Component 2 (PC2) explained for 14.4%, and the overall variation explained by these two main axes was 67.0%. The analysis of discriminant function confirmed the presence of sexual dimorphism (p = 0.01). It was observed that the females have the posterior region of the head longer and wider, and the jaw shorter and narrower. While the posterior region of the males head is slightly shorter and narrower, and the jaw longer and wider. The macroscopic and histological characteristics of the gonads were similar to those found in other species of crocodilians. Macroscopic analysis was sufficient to identify the sex of M. niger hatchling. In adult crocodilians, sexual dimorphism may be present in the region of the skull related evolutionarily to visual recognition of the genus. However, sexual selection would not be as relevant to young people as to sexually mature individuals. The variation of the shape in both the skull and mandible found in M. niger hatchling suggests that males and females may have undergone different ontogenic processes. The lack of work involving the study of sexual dimorphism using a geometric morphometry technique in hatchling of other populations of M. niger and/or other species of crocodilians makes difficult direct comparison. However, it is clear the importance of the study of sexual dimorphism in gilts of crocodilians to estimate the sexual proportion, especially if applied in natural populations. Geometric morphometric models can help to develop graphic representations of sexual dimorphism of the head in crocodilians, which makes these results important for conservation programs under natural conditions, since it represents a non-lethal alternative to the techniques used for sexual identification in M. niger.

Abstract Endocrine disorders are common in adults with epilepsy on antiepileptic drugs (AEDs). This study aimed to evaluate endocrine function, lipid metabolism and growth in patients with childhood- or adolescence-onset epilepsy. Altogether 148 patients with epilepsy on carbamazepine (CBZ), oxcarbazepine (OXC), valproate (VPA) or lamotrigine monotherapy during pubertal maturation and 124 healthy controls participated in this population-based cohort study. Boys and young men (n = 140) underwent cross-sectional evaluation once, and girls and young women (n = 132) twice at an approximate interval of 6 years. Gonadal structure and serum reproductive and thyroid hormone and lipid concentrations were evaluated and growth data were gathered. Elevated serum testosterone and androstenedione levels and polycystic ovary syndrome (PCOS) were common in female subjects whose medication, especially VPA, continued into young adulthood. Serum reproductive hormone concentrations and ovarian structure were similar in patients off medication and controls in young adulthood. CBZ was associated with elevated serum sex hormone binding globulin and decreased dehydroepiandrosterone sulfate levels and VPA with elevated serum androstenedione concentrations in male patients. Testicular structure was similar in patients and controls. CBZ, OXC and VPA were associated with changes in serum thyroid hormone, thyrotropin and lipid levels during pubertal maturation in female patients, but these levels returned to normal after withdrawal of medication. Linear growth and final height were normal in female patients, but overweight was common if they had been obese and on VPA medication during pubertal maturation. Elevated serum androgen levels, PCOS and overweight are common if epilepsy and AED use, especially VPA, continue into young adulthood. These untoward changes or alterations in serum thyroid hormone or lipid concentrations are not present in young women with medication withdrawn. CBZ and VPA are associated with changes in serum sex hormone levels in boys and young men with epilepsy. Epilepsy and AED use during pubertal maturation does not seem to affect growth.

FOXL2 constitue un gène majeur de la différenciation et de la fonction ovarienne. Chez l’Homme, l’haploinsuffisance de ce gène induit des malformations palpébrales qui peuvent être associées ou non à une insuffisance ovarienne prématurée (BPES de type I ou II). Dans certains cas, des anomalies de l’expression de FOXL2 sont liées à des délétions de régions situées très en amont du gène, témoignant de la présence d’activateurs distaux. L’existence de cette régulation à longue distance a aussi été mise en évidence dans l’espèce caprine par notre équipe. La mutation naturelle PIS (Polled Intersex Syndrome) induit à l’état homozygote l’absence d’expression du gène FOXL2 dans les gonades XX, conduisant au développement de testicules à la place d’ovaires. Ainsi, au cours de mon doctorat j’ai été amenée à travailler sur deux aspects différents : (i) l’analyse des cibles/voies activées par FOXL2 dans la gonade, et (ii) l’étude de la régulation à distance de l’expression de FOXL2.En ce qui concerne le premier point, l’analyse du rôle d’un gène candidat, Dmxl2, chez la souris a nécessité la mise en place d’une invalidation conditionnelle de ce gène (Knock-out ou KO) dans les gonades (le KO total étant létal à la naissance). Chez les mâles, une diminution de 60% de la production de spermatozoïdes a été observée à la puberté pendant la première vague de spermatogénèse.En ce qui concerne le second point, l’étude d’une région régulatrice potentielle de l’expression de FOXL2 a permis de définir des éléments très conservés présentant un profil épigénétique caractéristique de régions de type « enhancers ». J’ai ensuite établi un modèle in vitro « d’édition de l’épigénome » du locus FOXL2, en utilisant la technologie CRISPR/dCas9-p300 pour modifier la marque épigénétique H3K27ac et activer l’expression de ce gène. A long terme, ces travaux pourraient aboutir à la création de « médicaments épigénétiques » pour soutenir l’expression de FOXL2 et rétablir la fertilité des patientes atteintes d’une mutation de ce gène. / FOXL2 is a major gene for ovarian differentiation and functions. In humans, FOXL2 haploinsufficiency induces eyelid malformations with or without premature ovarian failure (BPES type I or II). In some cases, abnormalities of FOXL2 expression are related to deletions of regions located far upstream of this gene, indicating the presence of distal activators. The existence of this long-range regulation has also been demonstrated in the goat species by our laboratory. The natural mutation PIS (Polled Intersex Syndrome) when homozygous induces the silencing of FOXL2 expression in XX gonads, leading to the development of testes instead of ovaries. Thus, during my PhD, I worked on two different aspects: (i) the analysis of FOXL2-activated targets/pathways in the gonad, and (ii) the study of the long-range regulation of FOXL2 expression.Regarding the first point, gene function analysis of the candidate gene Dmx12 required the establishment of a conditional knock-out in the mouse gonad (Dmxl2 total KO is lethal at birth). In males, a 60% decrease in sperm production was observed at puberty during the first wave of spermatogenesis.Regarding the second point, the study of a putative regulatory region of FOXL2 expression allowed to define highly conserved elements harbouring typical enhancer epigenetic profile. Then, I established an in vitro model of FOXL2 locus “epigenome editing”, using the CRISPR/dCas9-p300 technology to modify the epigenetic mark H3K27ac. In the long term, this work may lead to the development of "epigenetic drugs" to support the expression of FOXL2 and restore the fertility of patients with a mutation of this gene.

Organisms are comprised of many cells with multiple distinct cell types, each of which must be decided precisely to ensure proper formation of a functional organism. In C. elegans, the basic helix-loop helix transcription factor HLH-2 is required for the specification of the anchor cell, or AC. The AC arises from a group of four somatic gonad cells, all of which initially express HLH-2. Two of the four cells, which we call β cells, lose AC competence early and instead become ventral uterine precursor cells, or VUs. We call the remaining two cells α cells. One α cell becomes the AC, while the other becomes a VU. Which α cell becomes the AC is random—50% of the time one α cell becomes the AC, while the other 50% of the time the other α cell becomes the AC. The choice of which cell becomes the AC and which becomes the VU is called the AC/VU decision, and occurs through reciprocal signaling by LIN-12/Notch and its ligand LAG-2/DSL. At first, both α cells express similar levels of lin-12 and lag-2. As the AC/VU decision progresses, the AC expresses higher levels of lag-2, and the VU expresses higher levels of lin-12. By this time, HLH-2 is only present in the specified AC, while it is post-translationally degraded in VUs. The mechanism by which HLH-2 is degraded and the consequences of disrupting its degradation on AC specification are unknown. In this work, we studied the function and regulation of HLH-2 during two stages of somatic gonad development. First, we used long-term fluorescence microscopy to visualize HLH-2 over the course of somatic gonad development. We found that HLH-2 expression begins in the parents of the α and β cells a consistent amount of time after their birth, and that the parent cell that first expresses HLH-2 almost always gives rise to the α cell that becomes the VU, while the second cell to express HLH-2 gives rise to the AC. This led us to study the effect of a loss of hlh-2 activity in the α and β cells. We generated an α and β cell-specific hlh-2(0) allele using genome editing tools and found that LIN-12 protein is not present in the absence of hlh-2 activity. Based on this discovery, we conceived a model where HLH-2 expression biases the first-expressing cell towards the VU fate by endowing it with an edge in lin-12 activity. Next, we focused on restriction of HLH-2 to the AC. Typically, HLH-2 protein is degraded in VUs, which we hypothesized was a crucial step in restriction of the AC fate to a single cell. We found that in a lin-12(0) background, HLH-2 is stabilized in VUs even when the resulting cell does not become an AC, indicating that lin-12 directly promotes HLH-2 degradation. This led us to search for a lin-12-regulated factor that targets HLH-2 for degradation in VUs. We identified seven ubiquitin-related genes whose depletion resulted in stabilized HLH-2 in VUs, but surprisingly did not cause an AC/VU defect. We suspect that HLH-2 degradation in VUs is one of multiple negative regulatory mechanisms that ensure the robustness of the AC/VU decision. The following research contributes new insights into how stochastic cell fate decisions amplify noise to ensure a consistent and reproducible outcome.