A idade normal para início da puberdade em meninas varia bastante, de 8 a 13 anos, e os genes envolvidos nesse controle são parcialmente conhecidos. Fatores ambientais, como alimentação e exposição a disruptores endócrinos, contribuem para essa variabilidade, de modo que genes modulados epigeneticamente podem justificar parte da complexidade desse processo. O termo epigenética se refere às modificações na expressão gênica que não são causadas por alterações na sequência do DNA. A metilação do DNA é o mecanismo epigenético mais bem estudado. Na última década surgiram evidências demonstrando a relação entre metilação do DNA e desenvolvimento puberal. Em fêmeas de roedores, a hipermetilação do DNA levou à puberdade precoce. Em humanos, a puberdade precoce central (PPC) familial causada por mutações nos genes MKRN3 e DLK1 é considerada um defeito do imprinting, fenômeno epigenético no qual apenas um dos alelos parentais é expresso, estando o outro metilado e inativo. Além disso, um conceito atual propõe que o início da puberdade requer a repressão epigenética de fatores inibidores do eixo gonadotrófico. Recentemente, genes zinc finger (ZNF) foram relacionados ao processo puberal, e muitos deles codificam repressores transcricionais. Neste trabalho, estudamos a metilação do DNA do sangue periférico de 10 pacientes do sexo feminino com PPC familial (casos índices) e 33 meninas com desenvolvimento puberal normal (15 pré-púberes e 18 púberes), usando a plataforma Human Methylation 450 BeadChip. Duas pacientes tinham PPC de causa genética (uma com mutação no MKRN3 e outra com deleção no DLK1) e oito tinham PPC idiopática, sem mutações identificadas pelo sequenciamento exômico global. Cento e vinte regiões diferencialmente metiladas foram identificadas entre as meninas saudáveis pré-púberes e púberes, estando 74% delas no cromossomo X. Apenas uma região mostrou-se hipometilada no grupo púbere e, de maneira importante, contém a região promotora do ZFP57, fator necessário para manutenção do imprinting. Uma vez que a hipermetilação nas regiões promotoras dos genes é relacionada à inibição transcricional, o achado de hipermetilação global do DNA na puberdade sugere que haja inibição de fatores inibidores do eixo gonadotrófico, o que resultaria no início do processo puberal. O receptor estrogênico destacou-se como um fator transcricional que se liga a sete genes diferencialmente metilados entre os controles pré-púberes e púberes. As pacientes com PPC apresentaram mais sítios CpG hipermetilados tanto na comparação com as meninas pré-púberes (81%) quanto púberes (89%). Há doze genes ZNF contendo sítios CpG hipermetilados na PPC. Não foram encontradas anormalidades de metilação nos genes MKRN3 e DLK1 nem em suas regiões regulatórias. Em conclusão, este estudo evidenciou hipermetilação global do DNA em meninas com puberdade normal e precoce, sugerindo que esse padrão é uma marca epigenética da puberdade. Pela primeira vez, mudanças no metiloma de pacientes com PPC foram descritas. Modificações na metilação de vários genes ZNF parecem compor a complexa rede de mecanismos que leva ao início da puberdade humana / Normal puberty initiation varies greatly among girls, from 8 to 13 years, and the genetic basis for its control is partially known. Environmental factors, such as nutrition and exposure to endocrine disruptors, contribute to this variance, and epigenetically modulated genes may justify some of the complexity observed in this process. Epigenetics refers to alterations in gene expression that are not caused by changes in DNA sequence itself. DNA methylation is the best studied epigenetic mechanism. In the last decade, evidence has emerged showing the relationship between DNA methylation and pubertal development. In female mice, DNA hypermethylation led to precocious puberty. In humans, familial central precocious puberty (CPP) caused by mutations in the MKRN3 and DLK1 genes is considered a disorder of imprinting, an epigenetic phenomenon in which only one parental allele is expressed, and the other allele is methylated and inactive. In addition, animal studies indicated that pubertal timing requires epigenetic repression of inhibitory factors of the gonadotrophic axis. Recently, zinc finger genes (ZNF) were related to pubertal development, many of which encode transcriptional repressors. In the present study, we analyzed the DNA methylation of peripheral blood samples from 10 female patients with familial CPP (index cases) and 33 girls with normal pubertal development (15 pre-pubertal and 18 pubertal), using the Human Methylation 450 BeadChip assay. Genetic CPP was diagnosed in two patients (one with a MKRN3 mutation and the other with a DLK1 deletion). The remaining eight cases with idiopathic CPP were previously evaluated by whole exome sequencing and no causative mutations were identified so far. We evidenced 120 differentially methylated regions between pre-pubertal and pubertal healthy girls, and 74% of them were located at the X chromosome. Only one genomic region was hypomethylated in the pubertal group. Of note, it contains the promoter region of ZFP57, an important factor for imprinting maintenance. As DNA hypermethylation in gene promoters is related to gene silencing, the finding of global DNA hypermethylation in puberty suggests inhibition of inhibitory factors of the hypothalamic-pituitary-gonadal axis that results in puberty onset. Importantly, the estrogen receptor was identified as a transcriptional factor that binds to seven differentially methylated genes associated with pubertal process. Patients with CPP exhibited more hypermethylated CpG sites compared to both pre-pubertal (81%) and pubertal (89%) controls. Twelve ZNF genes were recognized as having hypermethylated CpG sites in CPP. The methylation analyses of MKRN3 and DLK1 genes showed no abnormalities. In conclusion, this study revealed a widespread DNA hypermethylation in girls with normal and precocious puberty, suggesting that this pattern can be an epigenetic signature of puberty. For the first time, changes in the methylome of patients with CPP were described. We highlight that alterations in methylation levels of several ZNF genes may impact the onset of human puberty

Puberty is the process of becoming sexually mature and it has fundamental somatic and psychosocial implications. The focus of this dissertation was the short and long term developmental significance, concerning both soma et psyche, of female pubertal timing. Four studies were designed to accomplish these aims. Six samples of different ages from different countries and from different time points, comprising several thousand females some of which were followed longitudinally, were used. Age at menarche was used as the primary measure of pubertal maturation. The first main aim of this dissertation was to explore the mechanisms that might explain the well-established link between female pubertal timing and problem behavior, and to identify contextual conditions at which associations are stronger or weaker. Existing explanations are unsatisfactory and little is known about conditions that might affect the strength of the associations. In Paper I, we tested and confirmed a peer socialization hypothesis as a satisfactory explanation for the link between early puberty and problematic adjustment. In short, this hypothesis posits that early developing girls associate with older peers and boyfriends because they feel more mature than their same age peers, and through these peers and boyfriends the early developed girls are channeled into more socially advanced behaviors, including normbreaking. This should be particularly true in contexts where heterosexual relationships are sanctioned and where there is an abundance of deviant youth. In Paper II, I used a biopsychosocial approach and studied pubertal timing along with self-perceptions of maturity and early romantic relationships. The findings revealed that early puberty had very different implications depending on the psychological and social contexts in which it was embedded. For instance, when early puberty was coupled with feeling mature and having early romantic relationships, it was associated with adjustment problems. When early puberty was coupled with neither, it was not linked to particularly high levels of problem behavior. In stark contrast to the vast literature on the role of female pubertal timing in adolescence, the literature on long-term implications is remarkably limited. For this reason, the second main aim of this dissertation was to study the adult implications of female pubertal timing. In Papers III and IV, we examined long term implications of pubertal timing, particularly as it relates to somatic development. The findings suggested that pubertal timing does have future implications for women’s body perception and composition, with early developing females having higher body mass indexes in adulthood, but only under certain circumstances. The findings of this dissertation help further understanding of the soma et psyche implications of female pubertal timing. They indicate that pubertal timing has concurrent and future implications. It seems, however, that timing is not everything. The developmental significance of female pubertal timing appears to be very different under different contextual conditions. Thus, it is only when girls’ psychological and social contexts are considered that fruitful predictions can be made. As such, the findings have important implications for prevention, policy, and practice.

Pubertal timing is a human life history variable representing a trade-off between early reproduction and continued growth. Dominance is an important feature to male mate value. These two variables should have far-reaching effects on adult male life. Chapter 1 reviews evolution, r/K selection and life history theory to derive hypotheses concerning variation in male mating strategy. Chapters 2-4 investigate the effects of pubertal timing and dominance on mating strategy using sociosexual orientation and preferences for faces and mate characteristics. Both early puberty and high dominance associate with unrestricted sociosexuality (increased interest in casual sex) as predicted. Dominance is shown to relate to preferences for cues of sociosexuality but not femininity, while pubertal timing relates to neither facial characteristic. Earlier and later developing men do not differ in their mate characteristic preferences, while dominant men exhibit enhanced female-typical mate preferences counter to predictions. A dominance-dependent, dual, male mating strategy is proposed to account for results. Chapter 5 introduces sensitivity to putative human pheromones as an indicator of mating strategy. Dominant men are found to be more sensitive to and more averse to a putative female pheromone. Pubertal timing has no effect on sensitivity. Results are interpreted in terms of dominant male avoidance of infertile matings. Chapter 6 finds that early puberty associates with facial masculinity, attractiveness and apparent age. Chapter 7 offers a hormonal underpinning of effects related to pubertal timing, showing that early development associates with higher levels of testosterone in men. Chapter 8 uses digit length ratios to show that early developing men may have been exposed to greater levels of uterine testosterone, suggesting prenatal influences on male pubertal timing. Chapter 9 shows dominance associates with bodily, vocal and general attractiveness but not facial attractiveness. Chapter 10 reports that dominance associates with high levels of the stress hormone cortisol, suggesting costs of high dominance. Chapter 11 shows early pubertal timing relates to the visual appearance of skin, perhaps because of lower sebum production among early developing men, leading to them having darker, less reflective skin. This may reflect accelerated ageing of early developing males, potentially representing a cost to longevity.

published_or_final_version / Community Medicine / Doctoral / Doctor of Philosophy

Adolescent substance abuse represents a complex, difficult challenge in the United States. Substance addiction research requires rich contextualization that takes into account individual, familial, and community experiences. This project focuses on how adolescent girls' substance use interacts with risk and vulnerability. More specifically, how the social and biological body influences substance initiation and how local contexts and constraints effects recovery from addiction. The sample includes adolescent girls who are enrolled in substance abuse treatment programs. The methodological approach encompasses a mixture of quantitative and qualitative, including analysis of a nation-wide dataset, narrative interviews, participant observation, and case following. While the quantitative analysis was with the nation-wide dataset, the qualitative data are derived from a sample of adolescent girls in Tucson, Arizona. Risk is assessed along three axes, biocultural, familial, and structural. Biocultural risk examines the influence that an early pubertal developmental trajectory has on substance initiation. Familial risk analyzes how the culture and habitus of the family affects youth initiation of substance use. Structural risk highlights the continued vulnerability that youth who are raised in the `system' face and specifically their challenges to recovery after substance abuse treatment. Findings from the nation-wide sample include a significant relationship between pubertal timing and age of onset of substance use. In the Tucson sample, familial immersion in substance use was so extensive that girls were not only expected to begin using but also initiation of use became a 'rite of passage' within the family. Finally this research documents the unintended role the child welfare system plays as a structural impediment to girls' recovery from substance abuse.

A descriptive study concerning the Navajo Puberty Ceremony for girls, the Kinaalda', examined the extent of the practice of the ceremony, and the frequency in which the girls who have had the ceremony and the girls who have not had the ceremony differ in traditional characteristics. Fifty-four percent of the girls questioned have had the Kinaalda'. Significant differences between the girls who had the ceremony and those who had not had the ceremony were found, using a chi square test of significance at an alpha level of .05, in the frequency of a set of traditional characteristics. The Kinaalda' girl possessed the set of traditional qualities more frequently than the non-Kinaalda' girl.

Relationships between body dissatisfaction, dieting methods, father absence, and puberty timing were investigated in this study. Participants included adolescent females from Wave 1 of the National Longitudinal Survey of Adolescent Health. Logistic regression results indicated that girls without a biological father in the home were significantly more likely to have an early onset of puberty than on-time or late. Girls who experienced early puberty exhibited higher levels of body dissatisfaction, but didn't use more dieting methods. Early onset girls more likely used dieting methods if their biological father was present than absent; however, no significant difference in body dissatisfaction was shown. A negative relationship with fathers indicated more body dissatisfaction. None of the attained findings were found when the biological mother was absent, and having a stepfather did not seem to matter. Evidence was revealed that fathers play a role in their daughters' view of their own bodies.

Orientador: José Luis Moraes Vasconcelos / Banca: Ciro Moraes Barros / Banca: Roberto Sartori Filho / Resumo: O objetivo desse experimento foi avaliar o efeito de tratamentos com progesterona (P4) na indução de estro, concepção e prenhez em novilhas Nelore prépuberes. Novilhas Nelore (n = 935) com idade média de 24,0 ± 1,13 meses, peso de 298,0 ± 1,89 Kg e ECC 3,2 ± 0,26, foram submetidas a dois exames ultrassonográficos com intervalo de sete dias (d-19 e d-12) para determinar a presença ou ausência de CL, e aquelas com presença de CL em uma ou ambas avaliações foram consideradas púberes (Grupo PGF; n = 346). No dia -12 as novilhas consideradas pré-púberes foram divididas aleatoriamente para não receberem tratamento (Grupo CIDR0; n = 113), para receberem um dispositivo intravaginal novo contendo 1,9 g de P4 (Grupo CIDR1; n = 237), ou para receberem um dispositivo utilizado previamente por 27 dias (Grupo CIDR4; n = 239). No dia zero foi retirado o CIDR das novilhas dos tratamentos CIDR1 e CIDR4, e as novilhas do tratamento PGF receberam aplicação de prostaglandina F2 . Todas as novilhas foram submetidas à palpação retal para avaliação do escore uterino (EU) e amostras de sangue foram colhidas para dosagens de P4. O diâmetro do maior folículo (ØFOL) foi mensurado em todas as novilhas no dia zero. A partir do dia um (d1) todas as novilhas foram submetidas à observação de estro durante 45 dias e inseminadas seguindo o esquema: cio manhã / IA tarde do mesmo dia e cio tarde / IA no dia seguinte de manhã e depois foram expostas a touros por mais 45 dias de estação de monta (EM). As variáveis contínuas foram avaliadas pelo PROC GLM e as binomiais pelo PROC LOGISTIC do SAS. Nas novilhas prépúberes, houve influência (P < 0,05) do tratamento nas concentrações de P4 no dia 0 (CIDR0: 0,43 ± 0,16; CIDR1: 2,26 ± 0,11; CIDR4: 1,22 ± 0,11 ng/ml), ØFOL (CIDR0: 9,41 ± 0,24; CIDR1: 9,73 ± 0,17; CIDR4: 11,37 ± 0,16 mm), EU no dia... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this trial was to evaluate the effects of treatments with progesterone (P4) on the rates of induced estrus, conception and pregnancy in pre-pubertal Nelore heifers. Nelore heifers (n = 935) with 24.0 ± 1.13 months, body weight of 298.0 ± 1.89 Kg and body conditional score (BCS) of 3.2 ± 0.26 were submitted to two ultrasound examinations 7 d apart (d -19 and -12) to determine the presence or absence of CL, and those with presence of CL in at least one examination were considered pubertal (PGF; n = 346). On d -12, the pre-pubertal heifers were randomly assigned to receive no treatments (group CIDR0; n = 113), a new intravaginal insert containing 1.9 g of P4 (group CIDR1; n = 237) or a P4 insert used previously for 27 d (group CIDR4; n = 239). On d 0, heifers from treatments CIDR1 and CIDR4 had inserts removed, and heifers from treatment PGF received a prostaglandin F2 treatment. Also, heifers were rectally palpated for uterine score (US) evaluation, blood samples were taken for P4 analysis and follicular diameter (ØFOL) was measured in all heifers on d 0. Beginning on d 1, animals were observed for estrus and inseminated during 45 d [estrus morning / artificial insemination (AI) afternoon of same day and estrus afternoon / AI on next day morning] and further exposed to natural breeding from d 46 to 90 of breeding season (BS). Continuous variables were evaluated by PROC GLM and binomial variables by PROC LOGISTIC of SAS. In pre-pubertal heifers, there were effects of treatment (P < 0.05) on serum concentrations of P4 at d 0 (CIDR0: 0.43 ± 0.16; CIDR1: 2.26 ± 0.11; CIDR4: 1.22 ± 0.11 ng/mL), ØFOL (CIDR0: 9.41 ± 0.24; CIDR1: 9.73 ± 0.17; CIDR4: 11.37 ± 0.16 mm), US at d 0 (CIDR0: 1.46 ± 0.06; CIDR1: 1.86 ± 0.04; CIDR4: 2.20 ± 0.04), means days to show estrus (CIDR0: 4.45 ± 0.28; CIDR1: 3.52 ± 0.13; CIDR4: 3.19 ± 0.14 days), estrus detection rate in 7 d of BS... (Complete abstract click electronic access below) / Mestre

Orientador: Guilherme de Paula Nogueira / Resumo: Comumente o biotipo é o principal critério utilizado para predicão da puberdade precoce em novilhas Nelore. Este experimento comparou os possíveis efeitos do peso a desmama (PeD), peso ao sobreano (PeS), espessura de gordura subcutânea na garupa (EGSG), concentrações séricas de IGF-I (IGF-I) e insulina ao sobreano (Ins), ganho diário de peso: até a desmama (GD), entre a desmama ao sobreano (GS) e do nascimento até o sobreano (GT), além de escores visuais de conformação (C), precocidade (P) e musculatura (M) avaliados tanto na desmama como no sobreano, sobre a idade à primeira prenhez e desempenho na primeira estação de monta (EM) de novilhas Nelore. Para isso foram utilizados dados de 241 novilhas, produzidos pela P@RM agropecuária, estas nascidas entre maio e setembro de 2016. As mesmas permaneceram à pasto num único lote a partir da desmama. Avaliações de CPM e mensuração do peso corporal em jejum alimentar e hídrico após12h foram realizadas na desmama e repetidas após ganho mínimo de 50kg de peso. Foi iniciada a EM no dia seguinte à avaliação de sobreano, quando as novilhas foram submetidas ao protocolo de indução de puberdade e duas inseminações artificiais em tempo fixo, e posterior repasse com touro. Para a idade em dias à primeira prenhes houve efeito individual de C na desmama, PeD, GD, P e M no sobreano, GS, GT e IGF-I (p<0,05), no entanto, P e M na desmama não se mostraram importantes (p>0,05). Todas as avaliações de CPM mostraram correlações positivas com o GD, GS ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lately frame has being used to predict the occurrence of precocious puberty in Nellore heifers. This experiment compared the possible effects of weaning weight (PeD), yearling weight (PeS), rump backfat thickness (EGSG), serum concentrations of IGF-I (IGF-I) and insulin to yearling (INS), daily weight gain until weaning (GD), between weaning to yearling (GS) and from birth to yearling (GT), further the visual scores of conformation (C), precocity (P) and musculature (M) on weaning and yearling, on age at first pregnancy and the performance in the first breeding season (EM) of Nellore heifers. For that, data from 241 animals, produced by P@RM farming, born between May to September 2016 was used. After weaning all heifers were kept on pasture in a single lot and CPM score and body weight gain, under restriction of food and water for 12 h, were evaluated at weaning and repeated after a minimum weight gain of 50 kg. Breeding season started a day after evaluation of yearling, where heifers were subjected to a puberty induction protocol and two fixed time artificial inseminations, followed by a clean-up bull. There was an individual effect of C at weaning, PeD, GD, P and M on the yearling, GS, GT and IGF-I on age at days at first pregnancy (p<0.05), however, the date of birth, P and M at weaning had no significant effect (p>0.05). All CPM evaluations showed positive correlations with GD, GS and GT both at weaning (r= 0.61, 0.38, 0.43, p<0.05) and yearling (0.46, 0.50, 0, 41, p<0.05... (Complete abstract click electronic access below) / Doutor

Na puberdade ocorrem mudanças significativas na organização de circuitos neurais com ajuda da ação dos hormônios esteroidais, levando a dimorfismos sexuais relevantes para a promoção de comportamentos adultos. A porção ventrolateral do núcleo ventromedial do hipotálamo (VMHvl) é o sítio neural que mais exerce controle do comportamento sexual feminino a partir da ação dos hormônios ovarianos. Dentre os núcleos que o aferentam, destaca-se o núcleo medial da amígdala (MEA), transmitindo informações essenciais para iniciar tal comportamento. Investigamos a densidade das projeções do núcleo MEA para o VMHvl, em ratas ooforectomizadas pré-ppuberes e em ratas controle. Foi feita a ooforectomia em ratas Sprague-Dawley com 35 dias de idade. Ao atingirem idade de 90 dias, injetamos por iontoforese o traçador anterógrado Phaseolus vulgaris no MEA. Sugerimos uma diferença na densidade de projeções e varicosidades do MEA para o VMHvl entre os grupos. Aparentemente, os hormônios ovarianos contribuem na organização das conexões do MEA com estruturas do circuito do comportamento sexual feminino. / During puberty, significant changes occur to organize neural circuits, with steroid hormones action, leading to significant sexual dimorphism to promote adults behaviors. The ventrolateral portion of the ventromedial nucleus of the hypothalamus (VMHvl) is the major neural site controlling this behavior with the ovarian hormones action. One of outputs to VMHvl, the medial amygdala nucleus (MEA) transmit essential information to start such behavior. We investigate the projections density of MEA to VMHvl, in ovariectomized rats at early puberty. We performed ovariectomy Sprague-Dawley rats with approximately 35 days of age, when they reach the age of 90 days, we perform unilateral iontophoretic injections of anterograde tracer Phaseolus vulgaris in MEA. Our results suggest a possible difference in projections density of the MEA to VMHvl, between ovariectomized rats the onset of puberty and control. Apparently, ovarian hormones are important factors that contribute to the organization of MEA connections with structures of the circuit of female sexual behavior during puberty.