Thesis (doctoral)--Ludwig Maximilians-Universität zu München, 1979.

Thesis (M.A.)--Rowan University, 2009. / Typescript. Includes bibliographical references.

Thesis (Ph. D.)--Ohio State University, 2005. / Title from first page of PDF file. Document formatted into pages; contains xvii, 181 p. : ill. Includes bibliographical references (p. 159-181). Available online via OhioLINK's ETD Center

AN ABSTRACT OF THE DISSERTATION OF LAN HAI, for the Doctor of Philosophy degree in Molecular Cellular and Systemic Physiology, presented on 11th December, 2015 at Southern Illinois University Carbondale. TITLE: EFFECT OF CONSTITUTIVELY ACTIVATED LUTEINIZING HORMONE RECEPTOR ON THE MOUSE REPRODUCTIVE SYSTEM MAJOR PROFESSOR: Dr. Prema Narayan The luteinizing hormone/chorionic gonadotropin receptor (LHCGR) is crucial for fertility, and genetic mutations in LHCGR cause adverse effects in reproductive development. Among the activating mutations identified in LHCGR, replacement of aspartic acid 578 by glycine (D578G) is the most common inherited mutation. Boys with this mutation undergo puberty by 2-4 years, caused by elevated testosterone in the context of prepubertal luteinizing hormone levels and present with Leydig cell hyperplasia. Clinically, these symptoms are associated with familial male-limited precocious puberty (FMPP). Our lab has published a mouse model for FMPP (KiLHRD582G) with D582G mutation equivalent to D578G in human LHCGR. We have previously demonstrated that KiLHRD582G male mice exhibited precocious puberty, Leydig cell hyperplasia and elevated testosterone and was a good model for FMPP. However, unlike women with the D578G mutation who show no abnormal phenotype, our studies revealed that female KiLHRD582G mice were infertile. KiLHRD582G female mice exhibit precocious puberty and irregular estrous cyclicity. A temporal study from 2-24 weeks of age indicated elevated steroid levels and upregulation of steroidogenic acute regulatory protein as well as several steroidogenic enzyme genes. Ovaries of KiLHRD582G mice exhibited significant pathology with the development of large hemorrhagic cysts as early as 3 weeks of age, extensive stromal cell hyperplasia with luteinization, numerous atretic follicles and granulosa cell tumors. Anovulation could not be rescued by exogenous gonadotropins. The body weights of the KiLHRD582G mice was higher that wild type counterparts, but there were no differences in the body fat composition. Hyperandrogenism and polycystic ovary phenotype was not accompanied by impaired glucose tolerance. Blocking the androgen action and estrogen synthesis indicated that reproductive phenotype was primarily due to excess estradiol. These studies demonstrate that activating LHCGR mutations do not produce the same phenotype in humans and mice and clearly illustrates species differences in the expression and regulation of LHCGR in the ovary. As we use male KiLHRD582G mice as breeders, we observed that the KiLHRD582G mice became progressive infertile, and only 8% of KiLHRD582G were fertile at 6 months of age despite normal sperm production. The infertile KiLHRD582G males were not able to form copulatory plugs in WT females, and mating studies suggested that the KiLHRD582G males were not capable of mating and/or ejaculating. Sexual behavioral testing revealed that the infertile KiLHRD582G males were capable of mounting the receptive WT females but were unable to achieve ejaculation indicating a problem with erectile and/or ejaculatory function. To address the reason for the ejaculatory dysfunction, we performed histopathological analysis of the accessory glands and penis. Hematoxylin and eosin staining showed that the normal columnar epithelium was replaced by pseudostratified columnar epithelium in the ampulla and several aggregates of chondrocyte metaplasia were apparent in the penile body of KiLHRD582G male mice. A temporal study indicated the histopathological changes in ampulla and penile body initiated at 7-8 and 12 weeks of age, respectively. Immunohistochemistry indicated that the chondrocytes stained positive for collagen type II, SOX9 and androgen receptor in the nucleus and for LHCGR in the cytoplasm. Penile fibrosis is a major cause of erectile dysfunction and is characterized by an increased collagen/smooth muscle ratio. However, our Image J analysis, hydroxyproline assay and western blot showed that KiLHRD582G penile body exhibited reduced levels of smooth muscle actin but similar total collagen content compared to WT mice. Thus, penile fibrosis was not responsible for the progressive infertility of adult KiLHRD582G mice. We also observed Leydig cell adenoma and disruption of spermatogenesis at 1 year of age. Our results suggest FMPP patients may be susceptible to infertility and testicular tumors later in their life and a follow-up study of FMPP patients is recommended.

Introdução - A puberdade precoce verdadeira ou dependente de GnRH apresenta importante morbidade: a baixa estatura, conseqüência da rápida progressão da idade óssea, além das seqüelas psico-emocionais do desenvolvimento sexual secundário precoce. Daí a importância da realização de um diagnóstico precoce e preciso, a fim de que a terapêutica adequada seja instituída o quanto antes. O uso do análogo do GnRH (aGnRH) em teste diagnóstico vem sendo utilizado com este objetivo. Neste estudo avaliou-se os valores de corte para o diagnóstico de puberdade precoce verdadeira, usando-se o teste do aGnRH. Material e métodos - Estudo prospectivo, com 44 meninas, com desenvolvimento dos caracteres sexuais secundários antes dos 8 anos de idade, atendidas no Ambulatório de Ginecologia Infanto-Puberal do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo. Realizou-se, em todos os casos, o teste do aGnRH, que consistiu na coleta de amostra sanguínea basal para dosagem de FSH e LH, seguida da aplicação subcutânea de 500µg de acetato de leuprolida (Lupron®). Novas amostras sanguíneas foram realizadas após 3 horas, para dosagem de FSH e LH, e após 24 horas da aplicação, para dosagem de estradiol Compararam-se os níveis de LH e FSH basais, de 3 horas e a relação LH/FSH obtida, além do estradiol de 24h, com a evolução clínica das pacientes. Este foi o padrão ouro utilizado para análise do teste, sendo que, após 6 meses, as pacientes foram divididas em 2 grupos: puberdade progressiva (puberdade precoce verdadeira) e não-progressiva. Para análise estatística, utilizou-se curvas ROC, estabelecendo-se sensibilidade, especificidade e melhor nível de corte para o diagnóstico de puberdade precoce verdadeira, para os diferentes critérios analisados. Além disso, avaliou-se a concordância entre os diversos tipos de análise do teste, através do coeficiente kappa. Resultados - O LH de 3 horas apresentou valor de corte > 4,5 mUI/mL, sensibilidade 59,1% e especificidade 86,4%, com área sobre a curva de 0,723. O valor de kappa foi de 0,45, com concordância de 0,73. O estradiol de 24 horas apresentou valor de corte > 40,6 pg/mL, sensibilidade 70% e especificidade 73,7%, com área sobre a curva de 0,703. O valor de kappa foi de 0,436, com concordância de 0,718. Dentre todos os critérios analisados, o melhor deles foi a relação LH/FSH de 3 horas, com valor de corte > 0,14, sensibilidade 72,7% e especificidade 77,3%, com área sobre a curva de 0,771. O valor de kappa foi de 0,5, com concordância de 0,75. Conclusões - Em nossa avaliação, a relação LH/FSH de 3 horas foi superior ao valor de LH de 3 horas ou estradiol de 24 horas, que haviam sidos os melhores critérios diagnósticos no trabalho pioneiro na utilização deste teste. / Introduction - True or GnRH-dependent precocious puberty involves important morbidity such as short stature due to the rapid progression of bone age, as well as psycho-emotional sequels of precocious secondary sexual development. Thus, it is important to make an early and precise diagnosis so that appropriate treatment can be instituted as early as possible. The GnRH analogue (aGnRH) in the diagnostic test has been used for this purpose. In the present study, the sensitivity and specificity of different laboratory criteria for the diagnosis of true precocious puberty were compared using the aGnRH test. Material and methods - This was a prospective study conducted on 44 girls with the development of secondary sexual traits before 8 years of age attended at the Childhood-Pubertal Gynecology Outpatient Clinic of the University Hospital, Faculty of Medicine of Ribeirão Preto, University of São Paulo. The aGnRH test was performed in all cases, consisting of collection of a basal blood sample for the determination of FSH and LH, followed by subcutaneous application of 500 µg leuprolide acetate (Lupron®). New blood samples were obtained after 3 hours, for the determination of FSH and LH, and after 24hours of application, for determination of estradiol. Basal LH and FSH levels and levels after 3 hours, the LH/FSH ratio obtained 3 hours after the administration of 500 µg Lupron®, and 24 hour estradiol levels were compared with the clinical course of the patients. This was the gold standard used for the analysis of the test and after 6 months the patients were divided into 2 groups: progressive puberty (true precocious puberty) and non-progressive puberty. ROC curves were used for statistical analysis, with the determination of the sensitivity, specificity and best cut-off value for the diagnosis of true precocious puberty of the different criteria analyzed. In addition, the agreement of the various types of test analysis was evaluated using the kappa coefficient. Results - Three hour LH presented a cut-off value of > 4.5 mIU/mL, 59.1% sensitivity and 86.4% specificity, with an area under the curve of 0.723. The kappa value was 0.45, with 0.73 agreement. Twenty-four hour estradiol presented a cut-off value of > 40.6 pg/mL, 70% sensitivity and 73.7% specificity, with an area under the curve of 0.703. The kappa value was 0.436, with 0.718 agreement. The best of all criteria used was the 3 hour LH/FSH ratio, with a cut-off value of > 0.14, 72.7% sensitivity and 77.3% specificity, with an area under the curve of 0.771. The kappa value was 0.5, with 0.75 agreement. Conclusions - In the present evaluation, the 3 hour LH/FSH ratio was superior to the 3 hour LH value and the 24 hour estradiol value, which had been the best diagnostic criteria in the pioneering study using this test.

INTRODUÇÃO: Os fatores genéticos que influenciam o início da puberdade precoce ainda não são totalmente conhecidos. Assim, investigar os mecanismos gênicos que estariam envolvidos na sua gênese é muito importante, pois, além de possibilitar o diagnóstico em fases iniciais, pode contribuir para o desenvolvimento de novas terapias, com melhora do prognóstico. Para alguns investigadores, o estradiol também seria um fator contribuinte no determinismo da puberdade. OBJETIVOS: Estudar três genes que codificam enzimas relacionadas à esteroidogênese (CYP1A1, CYP1B1 e CYP17) em meninas com puberdade precoce central. Avaliar a associação entre variações na sequência desses genes e a puberdade precoce central. MÉTODOS: Foram incluídas 177 pacientes, divididas em dois grupos: Grupo Controle - formado por 104 meninas sem puberdade precoce, acompanhadas no Setor de Ginecologia da Infância e da Adolescência da Divisão de Clínica Ginecológica do HC-FMUSP por outros diagnósticos; Grupo Caso - composto por 73 meninas com diagnóstico de puberdade precoce central, acompanhadas no mesmo setor. Foi avaliada a presença de mutação em genes envolvidos no metabolismo do estrogênio (CYP1A1, CYP1B1 e CYP17) pela técnica de RFLP (Restriction Fragment Length Polymorphism), utilizando DNA obtido a partir de sangue periférico. RESULTADOS: A distribuição dos genótipos de CYP1A1 MspI (p=0,86) e CYP17 (p=0,12) não apresentou diferença significante entre os grupos. Para o CYP1B1 Eco571, o genótipo mutado C/C foi mais frequente no Grupo Controle que no Grupo Caso (p=0,03). CONCLUSÃO: Nossos dados sugerem que a variação do gene CYP1B1 Eco571 poderia estar associada ao determinismo da puberdade / INTRODUCTION: The genetic factors influencing onset of precocious puberty are not as yet fully known. Therefore, it is very important to investigate the genetic mechanisms involved in its genesis, for the resulting knowledge would not only enable diagnosis in the early stages but also contribute to the development of new therapies for improvement in prognosis. According to some researchers, estradiol would also be a contributory factor in puberty timing. OBJECTIVES: To investigate three genes which codify enzymes associated with steroidogenesis (CYP1A1, CYP1B1, and CYP17) in girls with central precocious puberty by focusing on the association between the sequence variation of these genes and central precocious puberty. METHODS: A total of 177 patients was included and divided into two groups: Control Group with 104 girls without precocious puberty who were being treated for other diagnoses at the Sector of Gynecology of Childhood and Adolescence, Division of Gynecology Clinic, HC-FMUSP; Case Group with 73 girls diagnosed with central precocious puberty. Mutations in genes involved in estrogen metabolism (CYP1A1, CYP1B1, and CYP17) were assessed by the RFLP (restriction fragment length polymorphism) technique using DNA obtained from peripheral blood. RESULTS: No significant difference in the distribution of the CYP1A1 MspI (p=0.86) and CYP17 (p=0.12) genotypes was detected between the two study groups. As for CYP1B1 Eco571, the mutated C/C genotype was found to be more frequent in the Control Group than in the Case Group (p=0.03). CONCLUSION: Our data suggest the CYP1B1 Eco571 gene variation is associated with puberty timing

INTRODUÇÃO: Os fatores genéticos que influenciam o início da puberdade precoce ainda não são totalmente conhecidos. Assim, investigar os mecanismos gênicos que estariam envolvidos na sua gênese é muito importante, pois, além de possibilitar o diagnóstico em fases iniciais, pode contribuir para o desenvolvimento de novas terapias, com melhora do prognóstico. Para alguns investigadores, o estradiol também seria um fator contribuinte no determinismo da puberdade. OBJETIVOS: Estudar três genes que codificam enzimas relacionadas à esteroidogênese (CYP1A1, CYP1B1 e CYP17) em meninas com puberdade precoce central. Avaliar a associação entre variações na sequência desses genes e a puberdade precoce central. MÉTODOS: Foram incluídas 177 pacientes, divididas em dois grupos: Grupo Controle - formado por 104 meninas sem puberdade precoce, acompanhadas no Setor de Ginecologia da Infância e da Adolescência da Divisão de Clínica Ginecológica do HC-FMUSP por outros diagnósticos; Grupo Caso - composto por 73 meninas com diagnóstico de puberdade precoce central, acompanhadas no mesmo setor. Foi avaliada a presença de mutação em genes envolvidos no metabolismo do estrogênio (CYP1A1, CYP1B1 e CYP17) pela técnica de RFLP (Restriction Fragment Length Polymorphism), utilizando DNA obtido a partir de sangue periférico. RESULTADOS: A distribuição dos genótipos de CYP1A1 MspI (p=0,86) e CYP17 (p=0,12) não apresentou diferença significante entre os grupos. Para o CYP1B1 Eco571, o genótipo mutado C/C foi mais frequente no Grupo Controle que no Grupo Caso (p=0,03). CONCLUSÃO: Nossos dados sugerem que a variação do gene CYP1B1 Eco571 poderia estar associada ao determinismo da puberdade / INTRODUCTION: The genetic factors influencing onset of precocious puberty are not as yet fully known. Therefore, it is very important to investigate the genetic mechanisms involved in its genesis, for the resulting knowledge would not only enable diagnosis in the early stages but also contribute to the development of new therapies for improvement in prognosis. According to some researchers, estradiol would also be a contributory factor in puberty timing. OBJECTIVES: To investigate three genes which codify enzymes associated with steroidogenesis (CYP1A1, CYP1B1, and CYP17) in girls with central precocious puberty by focusing on the association between the sequence variation of these genes and central precocious puberty. METHODS: A total of 177 patients was included and divided into two groups: Control Group with 104 girls without precocious puberty who were being treated for other diagnoses at the Sector of Gynecology of Childhood and Adolescence, Division of Gynecology Clinic, HC-FMUSP; Case Group with 73 girls diagnosed with central precocious puberty. Mutations in genes involved in estrogen metabolism (CYP1A1, CYP1B1, and CYP17) were assessed by the RFLP (restriction fragment length polymorphism) technique using DNA obtained from peripheral blood. RESULTS: No significant difference in the distribution of the CYP1A1 MspI (p=0.86) and CYP17 (p=0.12) genotypes was detected between the two study groups. As for CYP1B1 Eco571, the mutated C/C genotype was found to be more frequent in the Control Group than in the Case Group (p=0.03). CONCLUSION: Our data suggest the CYP1B1 Eco571 gene variation is associated with puberty timing

Co-variation in the genetic architecture, and co-localization of quantitative trait loci (QTL), for embryonic developmental rate and age at maturation in male rainbow trout (Oncorhynchus mykiss) was examined. Two lots (September and December spawning) of 2 x 5 (male x female) diallel crosses were performed and the resulting progeny were raised to maturity. Higher incidences of precocious maturation were observed in the September lot fish compared to December lot fish, which was uncoupled to developmental rate. However, precocious maturation and body size was higher in early hatching December lot fish. A genome scan was also completed across 4 linkage groups previously reported to possess embryonic developmental rate QTL and/or age at maturation QTL. No evidence of co-localization of QTL for the two traits was detected. However, the presence of QTL for embryonic developmental rate on RT-8 and RT-24 and one for age at maturation on RT-17 supports previous results.

Orientador: Urara Kawazoe / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T09:56:42Z (GMT). No. of bitstreams: 1 Pinola_TalineBueno_M.pdf: 812699 bytes, checksum: 060ac3635a386505de2cf212ec4c9a59 (MD5) Previous issue date: 2009 / Resumo: Este estudo teve como objetivos obter o isolado precoce de Eimeria maxima "Ca" e posterior estudo comparativo da patogenicidade entre as aves (Gallus gallus) inoculadas com isolados: parental e precoce, com os seguintes critérios de avaliação: ganho de peso corporal, escore de lesão da mucosa intestinal e contagem de oocistos produzidos pelas aves infectadas. Para a obtenção do isolado precoce foram inoculados sucessivamente em aves sadias, os primeiros oocistos eliminados, a partir da geração parental. Os oocistos contidos nas fezes foram isolados e cultivados em solução de dicromato de potássio 2% para a esporulação. Através deste processo foram realizadas 25 passagens dos primeiros oocistos nas aves até a estabilização do período prépatente. Foi obtida uma redução de 36 horas na eliminação dos primeiros oocistos, de 124h30min para 88h30min. Os isolados parental e precoce foram comparados quanto a patogenicidade, através da inoculação de doses específicas em aves sadias. Nesse estudo foram utilizadas 75 aves distribuídas em cinco grupos: I e II para a produção de oocistos, III e IV para ganho de peso e escore de lesão e grupo V como controle negativo (aves sadias). Houve uma diminuição significativa da patogenicidade em aves infectadas com o isolado precoce quando comparadas a aves inoculadas com a geração parental. As aves infectadas com o isolado precoce obtiveram uma média de escore de lesão da mucosa do intestino estatisticamente inferior à parental. O valor médio do escore de lesão obtido pelo isolado precoce foi de 0,77 enquanto que a geração parental 1,67. As aves infectadas com o isolado precoce apresentaram uma redução significativa na produção de oocistos, quando comparadas à respectiva geração parental. Apresentaram, também, ganho de peso estatisticamente superior em relação à geração parental. Este isolado precoce é um excelente candidato à produção e comercialização de uma vacina viva atenuada. No entanto, testes em bateria e no campo sobre imunidade protetora desse isolado precoce devem ser realizados no futuro que poderá caracterizá-lo confiável para o uso comercial como vacina viva atenuada no controle da coccidiose aviária. / Abstract: The aim of this study was to obtain a precocious line of Eimeria maxima, "Ca" strain and the comparative pathogenicity study between precocious line and parental strain. The criteria used for this evaluation was: body weight gain, lesion score of intestinal mucosa and oocyst counting dropped by infected chickens. The precocious line was obtained by successive oral inoculation of parental E. maxima oocysts in chickens. First oocysts eliminated after inoculation were collected, isolated and cultivated in 2% dichromate potassium solution for their sporulation. The pre-patent period of first oocysts elimination diminished and stabilized after 25 passages in chickens. It was obtained a time reduction of 36 hours between precocious line and parental strain: 124h30' for parental strain and 88h30' for precocious line. The comparative evaluation of pathogenicity between precocious line and parental strain were performed using body weight gain, lesion score and oocyst production compared to non - infected chickens in groups of five chickens per cage with three replications. Parental strain infected in chickens showed loss of body weight while in chickens inoculated with precocious line showed weight gain of 75% when compared to non-infected control birds. The lesion score of intestine mucosa was 0.8 for chickens with precocious line and 1.7 for parental strain showing statistically significant difference. The precocious line showed also significant less oocyst production than parental strain in infected chickens. The precocious line of E. maxima obtained in the present study seems a good candidate for live attenuated vaccine against avian coccidiosis although there is a need for complementary study about protective immunity in battery and field tests. / Mestrado / Mestre em Parasitologia

IntroduÃÃo: A Puberdade Precoce (PP) à problema cada vez mais freqÃente em todo o mundo. CrianÃas em PP estÃo em risco de iniciaÃÃo sexual mais cedo, abuso sexual, baixa estatura na vida adulta, risco aumentado de obesidade, hiperinsulinemia e hipertensÃo arterial. O estudo trata da freqÃÃncia e condiÃÃes associadas à PP, visando propor medidas de SaÃde PÃblica para reduÃÃo deste problema e de suas possÃveis seqÃelas. MÃtodos: O estudo transversal, desenvolve abordagem analÃtica das crianÃas acometidas de PP, atendidas no ambulatÃrio de endocrinologia do Hospital UniversitÃrio Walter CantÃdio (HUWC) no perÃodo 1994 a 2010, contando com uma amostra de 342 crianÃas de 1 a 11 anos. As variÃveis analisadas incluÃram as caracterÃsticas biolÃgicas, sÃcioeconÃmicas, familiares e nutricionais. A anÃlise dos dados foi realizada atravÃs do programa SPSS,utilizando-se o teste do qui-quadrado para medir associaÃÃes, com o valor alfa de 5%. Resultados: Encontrou-se uma razÃo feminino: masculino de 37:1. As caracterÃsticas mais freqÃentes das crianÃas com PP foram: cor da pele parda, 64,1%; adotadas, 7,3%; sobrepeso e obesidade, 27,1 e 25,1%, respectivamente; inÃcio da puberdade abaixo dos 5 anos, 27,1%; altura dos pais abaixo da mÃdia populacional, 80%. CrianÃas com PP atendidas tardiamente pelo serviÃo especializado tenderam a apresentar mais cedo sinais da puberdade (p<0,001) e a pertencerem a famÃlias de menor renda (p<0,002). ConclusÃes: Encontraram-se elevadas proporÃÃes de crianÃas com PP que haviam sido adotadas e que apresentavam sobrepeso/obesidade, dois fatores de risco importantes na gÃnese do problema. O retardo na atenÃÃo especializada esteve associado ao aparecimento muito precoce dos sinais da puberdade e à baixa renda familiar, sugerindo a necessidade de aÃÃes de saÃde pÃblica que promovam a detecÃÃo e a atenÃÃo ao problema nas camadas mais pobres da populaÃÃo / Introduction: Precocious puberty (PP) is an increasingly common problem worldwide. PP children are at risk of early sexual initiation, sexual abuse, short stature in adult life, increased risk of obesity, hyperinsulinemia and hypertension. The study addresses the frequency and conditions associated with PP in order to propose measures to reduce this public health problem and its possible sequels. Methods: A cross-sectional study develops analytical approach to children affected by PP, who were given assistance at the clinic of Endocrinology, in the University Hospital Walter CantÃdio (HUWC) in the period from 1994 to 2010, with a sample of 342 children aged 1 to 11 years. The variables looked over included the biological, socio-economic, family and nutrition features. Data analysis was performed by using the SPSS program by means of the chi-square test to evaluate any association with the alpha value of 5%. Results: A ratio female-male of 37:1 was found. The most frequent features of children with PP were: dark skin, 64.1%; adopted, 7.3%; overweight and obesity, 27.1 and 25.1%, respectively; onset of puberty under 5 years, 27.1%; parental height below the average population, 80%. Children with late PP assistance by specialized service tended to show early signs of puberty (p <0.001) and belong to households with lower income (p <0.002). Conclusions: It was found a high proportion of children with PP which had been adopted and that had overweight/obesity, two important risk factors in the genesis of the problem. The delay in specialized care was associated with signs of very early onset of puberty and low household income, which suggests that public health actions are required to encourage detection and attention to the problem among the poorest strata of the population.