O diabetes melito gestacional (DMG) está relacionado ao crescimento fetal exagerado. Entender a influência de fatores relacionados ao crescimento fetal auxilia na identificação dos fetos com maior risco de desvios da normalidade. Objetivo: comparar fatores clínicos, laboratoriais e a expressão placentária de transportadores de glicose segundo o crescimento fetal em pacientes com DMG. Método: Para análise dos fatores clínicos e laboratoriais foi realizado um estudo retrospectivo com 425 gestantes com DMG do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HC FM-USP) no período de janeiro de 2003 a novembro de 2009. Para a análise da expressão placentária dos transportadores de glicose dos tipos 1 (GLUT1), 3 (GLUT3) e 4 (GLUT4) foram selecionados todos os casos de recém-nascidos grandes para idade gestacional (RNGIG) pareados com um caso controle de recém-nascido adequado para idade gestacional (RNAIG). Foram incluídas apenas gestações únicas e com DMG diagnosticado pelo teste de tolerância à glicose oral de 100 gramas, sem malformações fetais e com idade gestacional definida e confiável. Todas as gestantes realizaram dieta para diabetes, controle glicêmico diário e uso de insulina quando necessário. Os critérios de seguimento e tratamento seguiram rigorosamente as normas do Protocolo de Condutas do Setor de Endocrinopatias da Divisão de Clínica Obstétrica do HC-FMUSP. As gestantes foram divididas para análise dos dados em dois grupos: Fatores clínicos e laboratoriais com: 376 RNAIG e 49 RNGIG num total de 425 DMG. Expressão Placentária dos Transportadores de Glicose: 50 RNAIG e 44 RNGIG. Foram realizados testes de associação e médias das variáveis e relacionadas com os grupos de RNAIG e RNGIG. Resultados: Na análise univariada, dos fatores clínicos e laboratoriais, não houve diferenças entre os grupos quanto a: idade materna, antecedente familiar de diabetes, antecedente pessoal de hipertensão arterial, número de gestações, valores de glicemia de jejum e 1 hora no TTGO-100g, idade gestacional no parto, sexo do RN, tipo de parto e índice de Apgar no 1º e 5º minutos. Houve diferenças estatisticamente significativas entre os grupos quanto a: índice de massa corpórea pré-gestacional (p < 0,02); uso de insulina (p < 0,041); macrossomia anterior (p < 0,001); idade gestacional do diagnóstico do DMG (p < 0,001); glicemias de duas e três horas no TTGO-100g respectivamente com (p < 0,003) e (p < 0,026). Na análise de regressão logística foram considerados preditores independentes da ocorrência de RNGIG: o índice de massa corpórea pré - gestacional, a macrossomia anterior, aidade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas. Em relação a expressão dos transportadores de glicose não diferiram entre os grupos em relação a expressão de GLUT1 na decídua, GLUT3 na decídua e vilosidades e GLUT4 na decídua e vilosidades. Houve diferença entre os grupos quanto à: a expressão do GLUT1 nas vilosidades. Conclusões: O índice de massa corpórea pré - gestacional, a macrossomia anterior, a idade gestacional do diagnóstico do DMG e a glicemia de duas horas após sobrecarga de 100 gramas foram preditores da ocorrência de RNGIG. A expressão de GLUT1 nas vilosidades coriônicas teve relação com a ocorrência de RNGIG / Gestational diabetes mellitus (GDM) is related to excessive fetal growth. Knowing the influence of factors related to fetal growth assists in the identification of fetuses at high risk of deviations from normality. Objective: To compare clinical and laboratory tests and the placental expression of glucose transporters according to fetal growth in patients with GDM. Method: A retrospective study of clinical and laboratory factors related with large for gestational age newborns, included 425 pregnant women with GDM was carried out at Sector Endocrine Clinic of Obstetrics Hospital of the School of Medicine, University of São Paulo (HC-FMUSP), between January 2003 to November 2009. For the analysis of placental expression of glucose transporters types 1 (GLUT1), 3 (GLUT3) and 4 (GLUT4) were selected all cases of newborns large for gestational age (LGA) paired with a case control newly born appropriate for gestational age (AGA). We included only patients with singleton pregnancies and GDM diagnosed by OGTT-100g, with newborns without malformations and birth weight classified as adequate or large for gestational age. All pregnant women received diet for diabetes, daily glycemic control and insulin when necessary. The criteria for monitoring and treatment followed strictly the standards of Conduct Protocol Endocrine Obstetric Clinic of the Clinic Hospital, School of Medicine, University of São Paulo. The pregnancies were divided for analysis into two groups: 376 cases of newborns AGA and 49 cases of newborns LGA. Data were analyzed and considered the probability value p <0.05. Results: In the univariate analysis of clinical and laboratory factors, there were no differences between the groups regarding maternal age, family history of diabetes, personal history of hypertension, number of pregnancies, blood fasting glucose and 1 hour in- OGTT 100g, gestational age at delivery, gender of the newborn, type of delivery, Apgar score at 1st and 5th minutes. There were statistically significant differences between the groups regarding: body mass index before pregnancy (p <0.02), insulin (p <0.041), previous macrosomia (p <0.001), gestational age at diagnosis of GDM (p <0.001), blood glucose levels two and three hours at 100 g OGTT, respectively, with (p <0.003) (p <0.026). In logistic regression analysis were considered independent predictors of the occurrence of LGA: body mass index before pregnancy, previous macrosomia gestational age at diagnosis of GDM and two hours after glucose overload 100 grams. Regarding the expression of glucose transporters, the groups did not differ regarding the expression of GLUT1 in the decidua, GLUT3 in the decidua and villi and GLUT4 in the decidua and villi. There were differences between the groups regarding the expression of GLUT1 in the villi. Conclusions: The body mass index before pregnancy, previous macrosomia, gestational age of diagnosis of GDM and two hours after glucose overload 100 grams were predictors of the occurrence of LGA. The expression of GLUT1 in chorionic villi was related to the occurrence of LGA newborn

Introdução: A incidência de defeitos do tubo neural é de cerca de 1/1000 nascimentos. Na mielomenngocele, o tratamento intra-uterino utilizando a técnica neurocirúrgica clássica, apresenta melhor prognóstico neurológico, do que o tratamento pós natal, mas está associado a complicações maternas e fetais. Novas técnicas de correção estão sendo estudadas para diminuir a morbidade materna e fetal. Objetivo: Comparar os efeitos sobre a medula de duas técnicas cirúrgicas de correção intra-uterina de um defeito semelhante à mielomeningocele , em fetos de ovelha. Métodos: Em 15 fetos foi criado um defeito semelhante à mielomeningocele (laminectomia e excisão de dura-máter) no 90° dia de gestação. O tipo de correção foi randomizado. No grupo 1, o defeito foi corrigido usando a técnica neurocirúrgica clássica, com a sutura de três camadas (dura-máter, músculo e pele), realizada por um neurocirurgião. No grupo 2, um especialista em Medicina Fetal, utilizou uma técnica simplificada, colocando um fragmento de celulose Biosintética sobre a medula e suturando apenas a pele sobre a celulose. Próximo ao termo da gestação (132° dias), os fetos foram sacrificados para análise anatomopatológica. Resultados: Ocorreram 1 morte materna, 3 casos de trabalho de parto precoce e 4 tardios. Um total de 10 casos foram viáveis para avaliação anatomopatológica 10 casos, 6 no grupo 1 e 4 no grupo 2. No grupo 1, todos os casos mostraram aderência da medula à cicatriz (meningoadesão) e perda da arquitetura medular por destruição do funículo posterior e 5 de 6 casos apresentaram perda da visualização da substância cinzenta. No grupo 2, observamos em todos os casos, a formação de uma neoduramater, separando o tecido nervoso do músculo adjacente, sendo que o funículo posterior e a substância cinzenta estavam preservados. Conclusão: A técnica simplificada foi superior à técnica neurocirúrgica, com maior preservação da medula e evitando as aderências do tecido nervoso. Nossos achados sugerem que a técnica utilizada atualmente na correção de mielomeningocele em fetos humanos deva ser reavaliada / Introduction: The incidence of neural tube defects is about 1/1000 births. In myelomeninocele, the intrauterine treatment with the classical neurosurgical technique suggest better neurological prognosis for the fetus, than postnatal treatment, but it is associated with maternal and fetal complications. New correction techniques are being studied to decrease maternal and fetal morbidity. Objective: To compare the effects on the medulla of two surgical techniques for intrauterine correction of myelomeningocele-like defect in sheep. Methods: In 15 pregnant sheep a myelomeningocele -like defect (laminectomy and dural excision) was created in the lumbar region in 90o day gestation. The type of correction was randomized. In group 1 the defect was corrected using the classic neurosurgical technique of three layers suture (dura-mater, muscle and skin closure) performed by a neurosurgeon. In group 2, a fetal medicine specialist used a biosynthetic cellulose patch to protect the medulla and only the skin was sutured above it. Near term (132o day gestation) fetuses were sacrificed for pathological analysis. Results: One maternal death occurred, early preterm labour, and late preterm labour occurred in 3 and 4 cases, respectively. A total of 10 cases were available for pathological analysis, 6 in group 1 and 4 in group 2. In group 1, all the cases showed a adherence of the medulla to the scar (meningoneural adhesion) and a destruction of the normal architecture of nervous tissue, whithout view of posterior funiculus and in 5 of 6 cases without view of grey matter. In group 2, in all the cases were observed a formation of an organized tissue involving the cellulose patch, a neoduramater, separating the nervous tissue of adjacent muscle, preserving the posterior funicullus and grey matter. Conclusion: The simplified new technique was better than the classical neurosurgical technique. It preserved the nervous tissued and avoided the adherence of the medulla to the scar. This suggests the current technique used for the correction of spina bifida in humans may need to be reassessed

Ceratite ulcerativa é uma das enfermidades oculares mais comuns em cães e requer tratamento clínico-cirúrgico urgente. Neste contexto, o tratamento cirúrgico com implante de membrana biológica é opção benéfica. Estudou-se o implante de membrana amniótica (MA) e membrana alantoamniótica (MAAM) alógenas preservadas em glicerina a 98% em vinte córneas de cães submetidas previamente à ceratectomia lamelar superficial. Dividiram-se os cães em dois grupos para o estudo aos 2, 7, 15, 30 e 60 dias de pós-operatório. Avaliaram-se, segundo parâmetros clínico-oftalmológicos e histopatológicos. Clinicamente detectou-se fotofobia e blefarospasmo, tipos e graus de secreção, edema e neovascularização corneana e transparência da córnea no local do implante. Nos períodos pré-determinados as córneas foram submetidas à avaliação histológica. Foram constatados, com base no exame ocular, graus leves a moderado de fotofobia e blefarospasmo; predominou o tipo de secreção mucoso com grau moderado e o edema corneano permaneceu leve no grupo que recebeu MA, e moderada a leve no grupo que recebeu MAAM. A neovascularização intensificou-se no 15º dia de pós-operatório em ambos os grupos. Com base nos achados histopatológicos os grupos evoluíram de forma semelhante; constatou-se o desenvolvimento do processo de cicatrização dentro dos parâmetros normais para a córnea. Concluiu-se que a utilização da MA comparada com a MAAM de cães preservadas em glicerina a 98% em ceratoplastias não diferem quanto sua evolução segundo os parâmetros adotados para análise. Ambas as membranas são igualmente efetivas como membrana biológica alógena na reparação de defeito corneano. / Ulcerative ceratitis is one of the most frequent ocular diseases in dogs and it requires prompt surgical and clinical therapy. In this context, surgical treatment using biological membrane implants is a benefic option. In this study alogenous 98% glycerin-preserved amniotic (MA) and alantoamniotic (MAAM) membranes were implanted on twenty corneas from dogs previously submitted to superficial lamellar keratectomy. Clinical, ophthalmologic and histological parameters were investigated. The animals were divided in two groups and pos-operative corneal histological evaluation was taken on days 2, 7, 15, 30 and 60. Photophobia, blefarospasm, different types and severity of ocular secretion, corneal edema and neovascularization and corneal transparency at the implant site. Ocular examination revealed mild to severe photophobia and blefarospasm, moderate mucous secretion was the most frequent. Corneal edema was mild in the MA group and mild to moderate in the MAAM group. From the 15th pos-operative day neovascularization was more pronounced for both groups. Based on the histological findings the groups showed similar evolution and the healing process was found to be within normal parameters for the corneal tissue. We concluded that in dogs 98% glycerin-preserved MA and MAAM applied to keratoplasty do not differ among the parameters evaluated. Both membranes are equally effective as allogenous biological membrane for the repair of corneal defects.

Tse On Ki. / Thesis submitted in: June, 1997. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (leaves 97-113). / Abstract also in Chinese. / List of Tables --- p.xiii / List of Figures --- p.xiv / List of Abbreviations --- p.xv / Chapter Chapter 1. --- Introduction to thesis --- p.2 / Chapter 1.1 --- Misoprostol --- p.2 / Chapter 1.1.1 --- Description and History of Drug --- p.2 / Chapter 1.1.2 --- Current Use in Obstetrics and Gynaecology --- p.3 / Chapter 1.1.2.1 --- Termination of Pregnancy (TOP) --- p.3 / Chapter 1.1.2.2 --- Cervix Priming prior to Surgical Treatment of Pregnancy Failure --- p.4 / Chapter 1.1.2.3 --- Medical management of spontaneous abortion --- p.4 / Chapter 1.2 --- Gemeprost --- p.4 / Chapter 1.3 --- Doppler Sonography and Assessment of Blood Velocity --- p.5 / Chapter 1.4 --- Overview of Thesis --- p.5 / Chapter 1.5 --- Aim of this Study --- p.8 / Chapter Chapter 2. --- Physiological and Anatomical Features of Pregnancy --- p.10 / Chapter 2.1 --- Cardiovascular System Changes in Pregnancy --- p.10 / Chapter 2.1.1 --- Changes in the Blood --- p.10 / Chapter 2.1.2 --- Changes in Circulation --- p.11 / Chapter 2.1.3 --- The Distribution of Blood Flow in Uterus --- p.12 / Chapter 2.2 --- Blood Supply to Uterus --- p.13 / Chapter 2.3 --- Pelvic Anatomy in Early Pregnancy via Transvaginal Sonography --- p.15 / Chapter 2.3.1 --- Uterus --- p.15 / Chapter 2.3.2 --- The Adnexa --- p.17 / Chapter 2.3.3 --- Other Pelvic Structures --- p.17 / Chapter Chapter 3. --- Prostaglandins and Analogues --- p.19 / Chapter 3.1 --- Natural Prostaglandins --- p.19 / Chapter 3.2 --- The Source of PGs in Reproductive Organs of Women --- p.19 / Chapter 3.3 --- PGs Synthesis and Metabolism --- p.20 / Chapter 3.4 --- PGE1 Analogue: Misoprostol --- p.21 / Chapter 3.4.1 --- Misoprostol --- p.22 / Chapter 3.4.1.1 --- Pharmacology --- p.22 / Chapter 3.4.1.2 --- Adverse Side Effects --- p.23 / Chapter 3.4.1.3 --- Toxicology --- p.23 / Chapter 3.4.1.4 --- Misoprostol in Obstetrics & Gynaecology --- p.24 / Chapter 3.4.1.4.1 --- To Induce Abortion in First and Second Trimesters --- p.24 / Chapter 3.4.1.4.2 --- Cervix Priming prior to Surgical Evacuation of Uterus --- p.27 / Chapter 3.4.1.4.3 --- Medical Management of Miscarriage --- p.28 / Chapter 3.4.1.4.4 --- Induction of Labour with Dead Fetus --- p.29 / Chapter 3.4.1.4.5 --- Induction of labour --- p.29 / Chapter 3.4.2 --- The Potential Dangers of PGE1 analogues --- p.30 / Chapter Chapter 4. --- Doppler Sonography and Parameter Measurements --- p.34 / Chapter 4.1 --- The Principles of Ultrasound and Doppler Sonography --- p.34 / Chapter 4.1.1 --- The Basic Principles of Ultrasound --- p.34 / Chapter 4.1.2 --- Principles of Doppler Sonography --- p.36 / Chapter 4.2 --- Doppler Mode --- p.39 / Chapter 4.2.1 --- Continuous Wave Doppler Imaging --- p.39 / Chapter 4.2.2 --- Pulsed Wave Doppler Systems --- p.39 / Chapter 4.2.3 --- Colour Doppler Sonography (CDS) --- p.40 / Chapter 4.3 --- The Instrument of Doppler Sonography --- p.40 / Chapter 4.4 --- "Resistance Indices - S/D, PI and RI" --- p.42 / Chapter 4.5 --- Flow Measurement of Uterine artery --- p.44 / Chapter 4.5.1 --- Sampling Sites and Waveforms --- p.44 / Chapter 4.5.2 --- Waveform Components --- p.45 / Chapter 4.5.3 --- Identification of the Main Uterine Arteries --- p.45 / Chapter 4.5.4 --- UA Waveform Changes in Normal Pregnancy --- p.46 / Chapter 4.5.5 --- Factors Affecting the UA Waveforms --- p.48 / Chapter 4.5.6 --- Uterine Artery Resistance in Normal Pregnancy and Labour --- p.49 / Chapter 4.5.6.1 --- Uterine Artery Resistance in Normal Pregnancy --- p.49 / Chapter 4.5.6.2 --- Uterine Artery Resistance during normal labour --- p.51 / Chapter 4.5.7 --- Doppler Measure of Fetal Heart Rate --- p.52 / Chapter 4.6 --- Sonography in Estimation of Gestational Age --- p.53 / Chapter Chapter 5. --- Research Protocol --- p.56 / Chapter 5.1 --- The Ethics --- p.56 / Chapter 5.2 --- Apparatus --- p.58 / Chapter 5.3 --- Drug and Dosage --- p.59 / Chapter 5.4 --- Research Protocol --- p.59 / Chapter 5.4.1 --- Subjects --- p.59 / Chapter 5.4.2 --- Transvaginal Scan --- p.60 / Chapter 5.4.3 --- Parameters Measured --- p.61 / Chapter 5.4.4 --- Misoprostol --- p.65 / Chapter 5.5 --- Data analysis --- p.66 / Chapter Chapter 6. --- Results --- p.68 / Chapter 6.1 --- The Patients' Characteristics --- p.68 / Chapter 6.2 --- The Intra-observer Error --- p.70 / Chapter 6.3 --- Results of Study --- p.70 / Chapter 6.3.1 --- Effect of Misoprostol on the S/D ratio of Both Uterine Arteries --- p.70 / Chapter 6.3.2 --- Effect of Misoprostol on the PI of Both Uterine Arteries --- p.73 / Chapter 6.3.3 --- Effect of Misoprostol on Fetal Heart Rate (FHR) --- p.76 / Chapter 6.3.4 --- Left and Right UA-S/D --- p.78 / Chapter 6.3.5 --- Left and Right UA-PI --- p.81 / Chapter 6.3.6 --- The relationship Between Subgroups --- p.84 / Chapter 6.3.7 --- Side Effects of Misoprostol --- p.85 / Chapter 6.3.8 --- The Changes of UA-S/D and UA-PI According to Gestation --- p.86 / Chapter Chapter 7. --- Discussions and Conclusions --- p.89 / Chapter 7.1 --- Difficulties Encountered during Study --- p.89 / Chapter 7.2 --- Results of Study --- p.90 / Chapter 7.3 --- Implications --- p.94 / Chapter 7.4 --- Summary of Thesis --- p.95 / Chapter 7.5 --- Conclusions --- p.96 / Chapter 8. --- References and Appendix --- p.97

Orientador: Marcus Antonio Rossi Feliciano / Coorientador: Wilter Ricardo Russiano Vicente / Coorientador: Ricardo Andres Ramirez Uscategui / Banca: Felipe Farias Pereira da Câmara Barros / Banca: Eliandra Antonia Pires Buttler / Resumo: O objetivo deste estudo foi avaliar a rigidez de tecidos materno fetais em ovinos a partir da décima semana de gestação, por meio da elastografia ARFI (Acoustic Radiation Force Impulse), determinando padrões qualitativos e quantitativos, com o intuito de verificar a aplicabilidade da técnica elastográfica no estudo do desenvolvimento desses tecidos. Foram avaliadas 24 ovelhas multíparas, com peso entre 40 e 60 quilos e idade entre 2 e 5 anos, da raça Santa Inês. Após a realização de exames prévios e verificada a higidez dos animais, estes foram submetidos a um protocolo de sincronização de estro seguido da monta natural. Quando confirmada a prenhez, foram submetidos aos exames ultrassonográfico convencional e elastografia (método ARFI quantitativo e qualitativo) do tecido pulmonar, hepático, renal e placentônios, com a utilização do aparelho ultrassonográfico ACUSON S2000/SIEMENS e softwares específicos. Os exames foram realizados semanalmente, a partir da décima semana gestacional (64 a 70 dias) até a semana da parição, sendo verificados os achados sonográficos destes tecidos, velocidade de cisalhamento (avaliação elastográfica quantitativa) e suas características de rigidez (avaliação elastográfica qualitativa). O delineamento experimental foi inteiramente casualizado e se utilizou nível de significância de 5% para todos os testes realizados. Os animais não apresentaram quaisquer alterações clínicas e obstétricas durante o período gestacional e parto. Os neonatos apresentar... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the stiffness of maternal fetal tissues of ovines from the tenth week of gestation, using elastography ARFI (Acoustic Radiation Force Impulse), with the purpose of verifying the applicability of the elastographic technique in the study of the development of these tissues throughout the gestational period. Twenty - four multiparous ewes, weighing between 40 and 60 kilos and age between 2 and 5 years, of the Santa Inês breed were evaluated. After conducting preliminary exams and checked the healthiness of the animals, these were submitted to an estrus synchronization protocol and natural mating. When the pregnancy was confirmed, they were submitted to the conventional ultrasound examinations and elastography (quantitative and qualitative method ARFI) with the use of the ultrasound equipment ACUSON S2000 / SIEMENS and specific software. The exams were performed weekly, from the 10th gestational week (64 to 70 days) until the week of parturition and the shear wave velocity (quantitative elastographic evaluation) and stiffness characteristics were verified (Qualitative elastographic evaluation).The experimental lineation was completely randomized and used 5% significance level for all tests. The animals did not present any clinical and obstetric alterations during pregnancy and birth. The newborns presented themselves healthy and normal. ARFI elastography was performed consistently and without any difficulty. Qualitative elastography did not appear deformable, demonstrating homogeneous image. For qu... (Complete abstract click electronic access below) / Mestre

Ethanol exposure during pregnancy is one of the leading causes of preventable birth defects, leading to a range of symptoms collectively known as fetal alcohol spectrum disorder (FASD). More moderate levels of prenatal ethanol exposure (PNE) lead to a range of behavioural deficits including aggression, poor social interaction, poor cognitive performance and increased likelihood of addiction in later life. Current theories suggest that adaptation in the hypothalamic-pituitaryadrenal (HPA) axis and neuroendocrine systems contributes to mood alterations underlying behavioural deficits and vulnerability to addiction. This has led to the suggestion that corticotrophin-releasing factor (CRF) antagonists and glucocorticoid (steroid) inhibitors may be potential therapeutics to address the deficits of PNE and for the treatment of addiction. The zebrafish (Danio rerio) has several advantages over mammalian models, such as low cost of maintenance, short life cycle, easy embryological manipulation and the possibility of large-scale genetic screening. By using this model, our aim is to determine whether developmental ethanol exposure provokes changes in the HPA axis (HPI axis in fish), as it does in mammalian models, therefore opening the possibilities of using zebrafish to elucidate the mechanisms involved, and to test novel therapeutics to alleviate deleterious symptoms. Thus this thesis focuses solely on the effect of developmental ethanol exposure on the functioning of the HPI axis in zebrafish. Stress-reactivity in zebrafish larvae ethanol-treated 1-9 days post 4 fertilisation (dpf) was assessed using thigmotaxis and thigmotaxis following airstress. In both tests, lower stress-related responses were obtained with ethanol treated animals, in that they spent less time at the edges of the apparatus (P<0.01, n=3). They also showed lower total body cortisol (P=0.04, n=14). Larvae also showed the same behaviour pattern two weeks after ethanol exposure, (23dpf) (P=0.04, n=3), again with reduced total cortisol (P=0.03, n=4). HPI-related gene transcription was also assessed in 9dpf ethanol treated zebrafish larvae, by qRT-PCR. Revealing up-regulation of CRH, CRHBP and CRHR2, normalized against β-Actin, Elav1 and Gap43 housekeeping genes. In situ hybridization revealed no spatial changes in CRH, CRH-BP and POMC with animals at the same stage. Behavioural stress-reactivity differences in 6-months old adults that had been exposed developmentally to ethanol were assessed using novel tank diving and thigmotaxis. Both assays indicated a decrease in stress-like behaviour due to early ethanol exposure compared to controls (P<0.05, n=5 both). Finally, cortisol levels were assayed from 9dpf larvae and 6-month-old adults that had been treated with ethanol during early development showed a significant reduction in cortisol output when air-exposed stressed compared to controls (P=0.04, n=5). Conclusion: Early ethanol exposure produced significant changes in cortisol, HPI gene mRNA expression and stress-reactive behaviour in 9dpf animals. Changes in cortisol and behaviour were still detected in 6-months old adults, developmentally treated with ethanol, indicating that early ethanol exposure has permanent effects on the HPI axis. 5 As our data contradicts the findings in mammalian literature where early ethanol exposure increases stress-like behaviour in later life, it is also possible that more permanent effects of PNE in mammals may arise through maternal-offspring interactions, during and post gestation, such as breastfeeding and maternal grooming of the offspring, which are absent in the zebrafish model.

Wang, Xiao. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 32-37). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Fetal germ cell development is a key stage of female reproductive life. The DAZ family proteins (DAZ, DAZL and BOLL) are RNA-binding proteins with critical roles in murine germ cell development but their expression and potential targets in the human are largely unknown. The studies in this Thesis investigated the expression and function of DAZL and BOLL in human fetal ovary. Both DAZL and BOLL mRNA are increased dramatically at the time of entry into meiosis. Immunohistochemical analysis with specific meiotic markers suggested that DAZL and BOLL have distinct spatial-temporal expression patterns, with minimal co-expression – BOLL expression was transient prior to follicle formation. This pattern was shown not to be present in the mouse fetal ovary, where Dazl and Boll are co-expressed, indicating a limitation of the mouse for exploring the function of Boll. Two human cell lines, embryonic kidney derived HEK293 cells and germ cell tumour derived TCam-2 cells were used as models to identify the mRNA targets of DAZL and BOLL after transfection of DAZL or BOLL vectors. In HEK293 cells, TEX19 and TEX14 were confirmed as potential targets of both DAZL and BOLL, and CDC25A as a potential DAZL target. Further experiments indicated that DAZL and BOLL did not increase target mRNA transcription but increased stabilisation. A DAZL/GFP co-transfection-FACS system for TCam-2 cells was established as this cell line has very low transfection efficiency. TEX14 and SYCP3 significantly increased in GFP+ve-DAZL+ve cells when compare to the GFP-ve-DAZL-ve cells, whilst SOX17 and DNMT3L significantly decreased in the GFP+ve-DAZL+ve cells. A 3'-UTR luciferase assay confirmed regulation of TEX14 and SOX17 by DAZL through their 3'-UTR. RNA immunoprecipitation further demonstrated direct binding between human TEX14, TEX19, SYCP3, SOX17 mRNA and DAZL protein, and that TEX14 binding is through its 3'-UTR. Dual fluorescence immunohistochemistry showed that SOX17 and DMNT3L are expressed in early germ cells with DAZL, and are later down-regulated co-incident with that of DAZL, consistent with the novel repressive effect of human DAZL on these two potential targets. These studies indicate that DAZL and BOLL are associated with different key meiotic stages of germ cell development in human fetal ovary. Several potential mRNA targets of DAZL and BOLL, and a novel repression function of human DAZL on its mRNA targets were identified giving further insight into the role of these factors in human ovarian development.

Orientador: Cesar Roberto Esper / Banca: Vênicio José de Andrade / Banca: Marcos Silva / Banca: Joaquim Mansano Garcia / Banca: Vera Fernanda Martins Hossipian de Lima / Resumo: Pouco se sabe sobre os eventos morfológicos que ocorrem durante o desenvolvimento pré-natal das gônadas nas raças zebuinas. O objetivo deste estudo foi descrever os eventos morfológicos relacionados ao desenvolvimento pré-natal da gônada, incluindo a sua formação, identificação de células germinativas primordiais, surgimento de oogônios, oócitos e folículos em embriões e fetos da raça Nelore. Oitenta e um embriões e fetos bovinos, com idade variando de 26 a 240 dias após fecundação, foram coletados em frigoríficos. A idade dos fetos foi estimada a partir de medidas tomadas no sentido crânio-caudal e aplicadas à fórmula proposta por Rexroad et. al. (1974). O sexo foi identificado a partir de observações macroscópicas e usando a técnica do PCR (Polymerase Chain Reaction) somente quando as diferenças sexuais morfológicas não foram evidentes. Para histologia, as gônadas foram fixadas em líquido de Bouin por 24 horas. Após processamento histológico, cortes de tecido de 5mm, foram corados com hematoxilina-eosina. Os resultados mostraram que a crista gonádica se formou a partir de 29 dias após fecundação. No 34º dia, células germinativas primordiais foram identificadas. As oogônias surgiram em grande quantidade entre 50 e 100 dias e seu número reduziu drasticamente, atingindo números finais aos 140 dias. Os folículos primordiais, folículos em crescimento e antrais apareceram em média aos 95, 140 e 180 dias, respectivamente. Oogônias e folículos primordiais, de forma diferente dos folículos em crescimento, apresentaram diferenças significativas no seu diâmetro nos vários períodos estudados. Folículos antrais mostraram diâmetro médio de 96,92 l 31,07mm aos 180 dias , chegando atingir médias de1331,43 l 567,43mm aos 240 dias... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Little is known about morphological events occurring during the prenatal development of gonads in the Zebu breeds. The objective of this study was to describe the morphologic events related to the prenatal development of the gonad, including its formation, identification of primordial germinative cells, appearance of oogonia, oocytes and follicles in Nelore breed embryos and fetuses. Eighty-one bovine embryos and fetuses, with age range from 26 to 240 days following fecundation, were gathered in a local slaughter-house. The age of fetuses was estimated from measures taken in the cranium-caudal direction and applied to the formula proposed by Rexroad et. al. (1974). The sex was identified from macroscopic observations and using PCR (Polymerase Chain Reaction) technique only when the morphologic sexual differences were not evident. For histology, gonads were fixed into Bouin fluid for 24 hours. Then, 5mm-tissue cuts were stained with hematoxylin-eosin. The results showed that the gonadal ridge was developed from 29 days following fecundation. At the 34th day, primordial germ cells were identified. Oogonia arose in great quantity between 50 and 100 days and its number reduced dramatically, attaining final numbers at 140 days. The primordial follicles, growing follicles and antral follicles appeared on the average at 95, 140 and 180 days, respectively. Oogonia and primordial follicles, in a different way from growing follicles, presented significant differences in its diameter in the several periods studied. Antral follicles showed 96.92 l 31.07mm in diameter at 180 days, achieving means of 1331.43 l 567.43 mm at 240 days. The statistical analysis showed a positive and highly significant correlation (P< 0.01), between the oogonia diameter and its nucleus, as well as between the primordial and growing follicles with its oocytes and respective nuclei... (Complete abstract, access undermentioned eletronic address) / Doutor

O consumo desenfreado de etanol traz consequências negativas, que envolvem problemas fisiopatológicos e de socialização do indivíduo, além de questões de saúde pública. Seu consumo por mulheres grávidas acarreta em danos ao desenvolvimento e formação cerebral do feto, causando a Síndrome Alcoólica Fetal (SAF). Uma forma mais abrangente deste distúrbio é a FASD (Fetal Alcohol Spectrum Disorder), que inclui formas brandas de SAF, bem como qualquer desordem neurológica ou congênita em decorrência do etanol. O objetivo do estudo é avaliar as alterações comportamentais e neuroquímicas em peixezebra adulto após a exposição a diferentes concentrações de etanol na fase inicial do seu desenvolvimento. Para a indução desta desordem, utilizamos ovos de peixe-zebra com 24h pós-fertilização, expostos a diferentes concentrações de etanol: 0%, 0,1%, 0,25%, 0,5% e 1%, durante duas horas. Acompanhamos o desenvolvimento destes animais até a fase adulta (4 meses), onde foram realizadas diferentes tarefas comportamentais: exploração do aparato novel tank e interação social, além de captação de glutamato, como medida neuroquímica. Como resultados mais relevantes, destacam-se a exploração de todo o aparato pelo grupo controle e uma diminuição concentração-dependente da exploração pelos grupos expostos previamente ao etanol; maior permanência no fundo do novel tank nas concentrações elevadas de etanol; uma diminuição concentração-dependente do tempo de permanência próximo ao cardume de acordo com o aumento da concentração de etanol. Quanto às análises bioquímicas, a captação de glutamato mostrou uma diminuição significativa na função de transporte deste neurotransmissor nos animais tratados com concentrações mais elevadas de etanol. A posterior intervenção farmacológica com buspirona reverteu o perfil comportamental previamente observado pelo efeito do etanol. Considerando as análises bioquímicas, a captação de glutamato mostrou uma diminuição significativa na função de transporte deste neurotransmissor nos animais tratados com concentrações intermediárias (0.25 e 0,5%)de etanol. Concluímos que a exposição prévia a diferentes concentrações de etanol na fase embrionária leva a alterações comportamentais na fase adulta, tais como diminuição de exploração frente a novidades, ansiedade e diminuição de interação social. / The excessive consumption of ethanol has negative consequences, which involve pathophysiological and individual socialization issues, and public health issues. Its consumption by pregnant women causes damage to brain development and formation of the fetus, causing fetal alcohol syndrome (FAS). A broader form of this disorder is the FASD (Fetal Alcohol Spectrum Disorder), which includes milder forms of FAS, as well as any neurological disorder or congenital due to ethanol. This project proposes to assess the behavioral and neurochemical alterations in adult zebrafish after exposure to different concentrations of ethanol in the initial phase of its development. To induce this disorder, we use zebrafish eggs and 24h post-fertilization exposed to different concentrations of ethanol, 0%, 0.1%, 0.25%, 0.5% and 1%, for two hours. We observed the development of these animals to juvenile and adulthood, which will be performed different behavioral tasks: exploration of novel apparatus tank and social interaction, as well as glutamate uptake, as measured neurochemistry. Among the most relevant results, we highlight the exploration of the whole apparatus for the control group and a concentration-dependent decrease of exploration by groups previously exposed to ethanol. It was observed high time spent in the bottom of the novel tank in high concentrations of ethanol. Furthermore, elevated spend time close to social interaction chamber was observed, with a dose-dependent decrease of this time in accordance with the increase of ethanol concentration. Further pharmacological intervention with buspirone reversed the behavioral profile previously observed the effect of ethanol. Considering biochemical analysis, glutamate uptake showed a significant decrease in the transport function of this neurotransmitter in animals treated with intermediated doses of ethanol. We conclude that prior exposure to different concentrations of ethanol in infancy leads to behavioral changes in adulthood, such as decreased operating front the news, anxiety and decreased social interaction.