The airway epithelium plays a central role in respiratory physiology through its transport and immunological functions. Our previous study suggested that P2Y receptors are expressed in airway epithelia and play a significant role in regulating transepithelial ion transport. P2Y receptors belong to the family of purinergic receptors, which can be stimulated by nucleotides such as UTP and UDP. P2Y receptors are G protein-coupled receptors and classically signal through G[subscript q], resulting in an increase in intracellular Ca²⁺ concentration ([Ca²⁺]ᵢ) and thereby in the activation of Ca²⁺-dependent ion channels and downstream signalling pathway(s). Furthermore, P2Y receptors are involved in asthmatic inflammation. / Estrogen (or E₂) is an important hormone in human physiology. In addition to the classical nuclear hormone receptors ERα and ERβ, a novel estrogen receptor, G protein-coupled estrogen receptor (GPER), was recently identified and found to be involved in both rapid signalling and transcriptional regulations. The action of GPER is unclear, but it has been implicated in mediating anti-inflammatory responses. / In our experiments, both human bronchial epithelial cell line, 16HBE14o-, and primary normal human bronchial epithelial cells expressed GPER at mRNA and protein levels, as demonstrated by RT-PCR and western blotting, respectively. ERα and ERβ expression were also detected at mRNA and protein level. Expression of GPER receptors was localized in the human bronchial epithelial cells by immunofluorescence staining and western blotting of fractionated cell lysates. / [Ca²⁺]ᵢ induced by nucleotides were monitored by calcium imaging technique using MetaFluor fluorescence ratio imaging system. Stimulation of epithelial cells with E₂ or with the specific agonist of GPER, G1, rapidly attenuated a UDP-, UTP- or ATPyS- evoked increase in [Ca²⁺]ᵢ in both 16HBE14o- cell line and primary cells. This inhibitory effect of E₂ and G1 were concentration dependent, while this effect was reversed by GPER specific antagonist, G15. To examine the effect of E₂ and G1 on UDP-activated intracellular Ca²⁺ release and influx, the epithelia were exposed to nominally Ca²⁺ -free solution in the presence or absence of G1 or E₂, and then stimulated with UDP. Subsequently, Ca²⁺ was added to the perfusate. Both E₂ and G1 could inhibit UDP-induced Ca²⁺ release. However, only E₂ but not G1 could inhibit UDP-induced Ca²⁺ influx. / E₂ or G1 inhibited the secretion of two pro-inflammatory cytokines, interleukin (IL)-6 or IL-8, in cells stimulated by different nucleotides or the cationic protein, poly-L-arginie, as quantified by ELISA. CFP-Epac-YPF, an Epac-based polypeptide FRET reporter was used to monitor the real-time cAMP changes in 16HBE14o- cells. Both G1 and E₂ induced an increase in cAMP production. The transepithelial chloride (Cl⁻) secretion was measured using short circuit current technique in cells grown on permeable support. Cl⁻ secretion induced by apical UDP was partially inhibited by G1 in a concentration dependent manner. / Our results provide the first evidence that human bronchial epithelia express GPER, which interact with the P2Y receptor-mediated calcium signalling pathway and cytokine secretion. Moreover, the anti-inflammatory role of GPER may be due to its opposing effect on the pro-inflammatory pathway activated by the P2Y receptors in inflamed airway epithelia. / 气道上皮具有调节运输以及参与免疫反应等功能，在呼吸生理学研究中有着十分关键的意义。我们曾经的研究发现P2Y受体在气道上皮中表达并调节上皮细胞离子运输过程。P2Y受体属于嘌呤受体，可被三磷酸尿苷（UTP），二磷酸尿苷（UDP）等核苷酸激活。同时，P2Y受体也是一类G蛋白偶联受体，可通过活化G[subscript q]蛋白调控细胞内钙离子浓度而激活钙依赖性离子通道及其他下游信号通路。此外P2Y受体还参与哮喘炎症的调控。 / 雌激素（或雌二醇，E₂）是人体一类十分重要的激素。除传统的核受体ERα与ERβ外，一类新型雌激素受体GPER已被发现和鉴定。GPER属于G蛋白偶联受体，可同时参与转录调控和非基因依赖的快速信号调节。尽管具体机理尚不明确，但研究发现GPER可介导抗炎症反应。 / 实验结果显示，在支气管上皮细胞株16HBE14o-和原代人支气管上皮细胞中GPER都被检测到基因和蛋白水平的表达。GPER在人支气管上皮细胞中的定位也通过免疫荧光染色（immunofluorescence）和亚细胞组分蛋白质印迹（western blot of fractionated cells）得到鉴定。 / 本研究中，荧光显微技术（fluorescence microscopy）被用于测定核苷酸介导的细胞内钙离子浓度（[Ca²⁺]ᵢ）。在16HBE14o- 和原代培养人支气管上皮细胞中，E₂和GPER特异性激动剂G1都可抑制核苷酸介导的 [Ca²⁺]ᵢ增加，且这种抑制作用呈浓度依赖。GPER特异性拮抗剂G15可抵消G1的抑制作用。进一步研究表明，E₂和G1都可抑制UTP诱导的胞内钙库释放，然而只有E₂抑制UTP诱导的胞外钙离子内流。 / 除钙离子调节外，E₂和G1还可抑制支气管上皮细胞中核苷酸或聚精氨酸（poly-L-arginine）刺激介导的两种促炎症细胞因子，白介素6（IL-6）和白介素8（IL-8）的分泌。酶联免疫法（ELISA）被用于细胞因子的定量。同时，CFP-Epac-YPF作为一类多肽荧光共振能量转移（FRET）探针被转染入16HBE14o- ，探测细胞内腺苷-3',5'-环化一磷酸（cAMP）的实时变化。结果显示在人支气管上皮细胞中E₂和G1都可引导cAMP生成。此外，我们使用短路电流（short-circuit current, Isc）技术测定单层上皮细胞的氯离子（Cl⁻）分泌，并发现人支气管上皮顶膜面UDP诱导的Cl⁻ 分泌可被G1部分抑制，且抑制效果呈浓度依赖。 / 本研究首次证明GPER表达于人支气管上皮, 且激活GPER对P2Y受体介导的钙离子信号通路以及细胞因子生成起到抑制作用。这些结果表明在气道炎症反应中，GPER可通过反向调节P2Y受体激活的促炎症作用，达到抗炎症的效果。 / Hao, Yuan. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 187-211). / Abstracts also in Chinese. / Title from PDF title page (viewed on 03, November, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Lin Sze-Wah. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 111-127). / Abstracts in English and Chinese. / Abstract (in English) --- p.i / Abstract (in Chinese) --- p.iii / Acknowledgements --- p.iv / Table of Contents --- p.v / Abbreviations --- p.x / Scientific Names --- p.xii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Gonadotropins --- p.1 / Chapter 1.1.1 --- Structure --- p.1 / Chapter 1.1.2 --- Function --- p.1 / Chapter 1.1.3 --- Regulation --- p.2 / Chapter 1.1.3.1 --- Gonadotropin-releasing hormone (GnRH) --- p.3 / Chapter 1.1.3.2 --- Dopamine --- p.4 / Chapter 1.1.3.3 --- Sex steroids --- p.5 / Chapter 1.1.3.3.1 --- Functions --- p.5 / Chapter 1.1.3.3.2 --- Working mechanism´ؤEstrogen signaling pathway --- p.7 / Chapter 1.1.3.4 --- Gonadal peptides --- p.9 / Chapter 1.1.3.4.1 --- Functions --- p.9 / Chapter 1.1.3.4.2 --- Working mechanism一Activin signaling pathway --- p.11 / Chapter 1.2 --- Transcriptional regulation of pituitary gonadotropin subunit genes at the promoter level --- p.13 / Chapter 1.2.1 --- Transcriptional regulation of mammalian glycoprotein a subunits --- p.13 / Chapter 1.2.1.1 --- GnRH --- p.14 / Chapter 1.2.1.2 --- Activin --- p.15 / Chapter 1.2.1.3 --- Steroids --- p.15 / Chapter 1.2.2 --- Transcriptional regulation of mammalian FSHβ and LHβ subunits --- p.16 / Chapter 1.2.2.1 --- Regulation of LHβ expression by GnRH --- p.17 / Chapter 1.2.2.1.1 --- Roles of SP-1 binding sites on LHβ promoter --- p.17 / Chapter 1.2.2.1.2 --- Effect of SF-1 on LHp expression --- p.17 / Chapter 1.2.2.1.3 --- Effect of Egr-1 on LHp expression --- p.18 / Chapter 1.2.2.1.4 --- "Synergistic effect ofSP-1, SF-1 and Egr-1 on LHp expression." --- p.18 / Chapter 1.2.2.1.5 --- Effect of Pitx-1 on LHβ expression --- p.19 / Chapter 1.2.2.1.6 --- "Effect of SF-1, Egr-1 and Pitx-1 on LHβ expression of other mammalian counterparts" --- p.19 / Chapter 1.2.2.1.7 --- Effect of other transcription factors on mammalian LHβ expression --- p.19 / Chapter 1.2.2.2 --- Regulation of LHβ expression by steroids and activin --- p.20 / Chapter 1.2.2.3 --- Regulation of FSHβ expression by activin and GnRH --- p.20 / Chapter 1.2.2.4 --- Regulation of FSHβ expression by steroids --- p.21 / Chapter 1.2.2.5 --- Regulation of FSHβ expression by other transcription factors --- p.22 / Chapter 1.2.3 --- Transcriptional regulation of fish FSHβ and LHβ subunits --- p.22 / Chapter 1.3 --- The project objectives and long-term significance --- p.24 / Chapter CHAPTER 2 --- CLONING OF ZEBRAFISH FSHB AND LHB PROMOTERS. --- p.26 / Chapter 2.1 --- Introduction --- p.26 / Chapter 2.2 --- Materials and Methods --- p.27 / Chapter 2.2.1 --- Chemicals --- p.27 / Chapter 2.2.2 --- Animals --- p.27 / Chapter 2.2.3 --- Isolation of genomic DNA --- p.28 / Chapter 2.2.4 --- Cloning of promoters of zebrafish FSHβ and LHβ from the genomic DNA --- p.28 / Chapter 2.2.5 --- Construction of the reporter plasmids containing zebrafish FSHβ and LHβ promoters --- p.30 / Chapter 2.2.6 --- Cell culture and transient transfection --- p.31 / Chapter 2.2.7 --- SEAP reporter gene assay --- p.32 / Chapter 2.2.8 --- β-galactosidase reporter gene assay --- p.32 / Chapter 2.2.9 --- Data analysis --- p.33 / Chapter 2.3 --- Results --- p.33 / Chapter 2.3.1 --- Cloning of zebrafish FSHβ and LHβ promoters --- p.33 / Chapter 2.3.2 --- Sequence characterization of zebrafish FSHβ and LHβ promoters --- p.34 / Chapter 2.3.3 --- Basal FSHp and LHβ promoter activities in LβT2 cells --- p.35 / Chapter 2.4 --- Discussion --- p.36 / Chapter CHAPTER 3 --- ROLES OF ACTIVIN/SMADS AND ESTROGEN/ERS IN THE REGULATION OF ZEBRAFISH FSHB AND LHB PROMOTER ACTIVITY --- p.51 / Chapter 3.1 --- Introduction --- p.52 / Chapter 3.2 --- Materials and Methods --- p.56 / Chapter 3.2.1 --- Chemicals --- p.56 / Chapter 3.2.2 --- Animals --- p.56 / Chapter 3.2.3 --- Isolation of total RNA --- p.57 / Chapter 3.2.4 --- Rapid amplification of full-length cDNA (RACE) --- p.57 / Chapter 3.2.5 --- Construction of expression plasmids --- p.57 / Chapter 3.2.6 --- cell culture and transient transfection --- p.59 / Chapter 3.2.7 --- SEAP reporter gene assay --- p.59 / Chapter 3.2.8 --- p-galactosidase reporter gene assay --- p.59 / Chapter 3.2.9 --- Data analysis --- p.59 / Chapter 3.3 --- Results --- p.60 / Chapter 3.3.1 --- Cloning and sequence characterization of zebrafish Smad 4 (zfSmad 4) --- p.60 / Chapter 3.3.2 --- Smads regulate FSHβ transcription in LβT2 cells --- p.61 / Chapter 3.3.3 --- Smads regulate LHβ transcription in LPβT2 cells --- p.61 / Chapter 3.3.4 --- Functionality of the two forms of Smad 4 cloned --- p.62 / Chapter 3.3.5 --- Estrogen and ERs regulate zJFSHβ transcription in LβT2 cells --- p.63 / Chapter 3.3.6 --- Estrogen and ERs regulate zfLHβ transcription in LβT2 cells --- p.63 / Chapter 3.4 --- Discussion --- p.64 / Chapter CHAPTER 4 --- PROMOTER ANALYSIS FOR SMAD RESPONSIVE ELEMENT AND ESTROGEN RESPONSIVE ELEMENT IN ZEBRAFISH FSHB AND LHB PROMOTERS --- p.82 / Chapter 4.1 --- Introduction --- p.83 / Chapter 4.2 --- Materials and Methods --- p.85 / Chapter 4.2.1 --- Chemicals and animals --- p.85 / Chapter 4.2.2 --- Construction of SEAP reporter plasmids containing different lengths of zfFSHβ promoter --- p.85 / Chapter 4.2.3 --- Construction of SEAP reporter plasmids containing different lengths of zfLHβ promoter --- p.85 / Chapter 4.2.4 --- Site-directed mutagenesis --- p.86 / Chapter 4.2.5 --- cell culture and transient transfection --- p.87 / Chapter 4.2.6 --- SEAP reporter gene assay --- p.87 / Chapter 4.2.7 --- P-galactosidase reporter gene assay --- p.87 / Chapter 4.2.8 --- Data analysis --- p.88 / Chapter 4.3 --- Results --- p.88 / Chapter 4.3.1 --- Localization of Smad-responsive element (SRE) on zfFSHβ promoter --- p.88 / Chapter 4.3.2 --- Localization of estrogen-responsive element (ERE) on zfLHβ promoter --- p.89 / Chapter 4.3.3 --- Localization of estrogen-responsive element (ERE) on zfFSHβ promoter --- p.90 / Chapter 4.3.4 --- Confirmation of SRE by site-directed mutagenesis --- p.91 / Chapter 4.3.5 --- Confirmation of ERE by site-directed mutagenesis --- p.92 / Chapter 4.4 --- Discussion --- p.92 / Chapter CHAPTER 5 --- GENERAL DISCUSSION --- p.106 / Chapter 5.1 --- Overview --- p.106 / Chapter 5.2 --- Contribution of the present research --- p.107 / Chapter 5.3 --- Future research direction --- p.108 / REFERENCE: --- p.111

Orientador: Lúcia Helena Simões da Costa Paiva / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T13:42:39Z (GMT). No. of bitstreams: 1 Pinheiro_Anderson_M.pdf: 3122120 bytes, checksum: dced5efa50ee11a7d5554366020908bb (MD5) Previous issue date: 2012 / Resumo: Introdução: A prevalência de obesidade tem aumentado em todo o mundo e hoje já representa um problema de saúde pública. Na população feminina, seu aumento ocorre principalmente nos anos próximos da transição para menopausa. O aumento de peso representa um risco para diversas comorbidades, dentre elas um importante fator de risco para patologia endometrial. A etiologia e a patogênese dos pólipos não estão completamente esclarecidas. Estuda-se se o desenvolvimento dos pólipos endometriais está diretamente relacionado à presença de receptores hormonais, além de estar relacionado a mecanismos envolvidos à proliferação e à apoptose celular. Objetivos: Avaliar a imunoexpressão dos receptores de estrógeno (RE), progesterona (RP), Cox-2, Ki67 e Bcl-2 em pólipos endometriais benignos na pré e pós-menopausa e associação com a obesidade. Materiais e métodos: Dentre 1050 mulheres submetidas à histeroscopia cirúrgica no Hospital da Mulher Prof. Dr. Aristodemo Pinotti - CAISM/UNICAMP, de janeiro de 1998 a dezembro de 2008, 800 foram casos de polipectomia endometrial confirmados com exame anatomopatológico. Deste total, foram excluídas as usuárias de Tamoxifeno, as que faziam uso de terapia hormonal e os casos de pólipos malignos ou pré-malignos. Obteve-se uma amostra de 515 pólipos endometriais benignos em mulheres na pré e pós-menopausa. Foram avaliadas as expressões de RE, RP, Bcl-2, Ki67 e Cox-2, através de imuno-histoquímica, segundo a porcentagem de células coradas, intensidade da coloração e escore final. Os escores finais de RE, RP, Bcl-2, Cox-2 variam de 0 a 8 e o de Ki67 de 0 a 3. A mediana dos escores finais de RE, RP, Bcl-2, Cox-2 e Ki67 no epitélio glandular e no estroma dos pólipos foi comparada entre mulheres obesas e não obesas na pré e pós-menopausa, utilizando os testes qui-quadrado, exato de Fisher ou não paramétrico de Mann-Whitney. Resultados: A mediana do escore final de receptores hormonais mostrou maior expressão de RP no estroma e no epitélio glandular das mulheres obesas na pós-menopausa, sem diferença em relação à expressão dos RE. Em mulheres na pré-menopausa não houve diferença na expressão de RE e RP entre obesas e não obesas. Nos pólipos endometriais de mulheres pós-menopausadas houve maior expressão de Cox-2 e Bcl-2 no epitélio glandular das mulheres obesas do que em relação às mulheres não obesas. Não houve diferenças em relação ao estroma endometrial. Na pré-menopausa, houve maior expressão de Bcl-2 apenas no epitélio glandular das mulheres obesas. Não houve diferenças na expressão de Ki67 entre obesas e não obesas tanto na pós-menopausa quanto na pré-menopausa. Conclusões: Os pólipos de mulheres obesas apresentam, na pós-menopausa, maior expressão de RP glandular e estromal, Cox-2 glandular e Bcl-2 glandular, sem diferenças na expressão de Ki67. Estes dados sugerem que sua etiopatogênese dos pólipos em obesas parece estar mais relacionada aos receptores de progesterona, à inibição da apoptose e aos mecanismos relacionados à inflamação celular / Abstract: Introduction: The prevalence of obesity has increased worldwide and represents a public health problem nowadays. The female population, considerably presents its increase in the coming years of the transition to menopause. Weight gaining represents a risk for various comorbidities, but among them all, it is an important risk factor for the endometrial pathology. The polyps etiology and pathogenesis have not been completely clarified so far. It has been studied whether the endometrial polyps development is directly related to the presence of hormone receptors, besides being associated with mechanisms involved in the proliferation and cellular apoptosis.Objectives: To evaluate the immunoexpression of estrogen and progesterone receptors, Ki67, Bcl-2 and Cox-2 in benign endometrial polyps in pre and postmenopausal women and their association with obesity. Methods: It was observed that among 1050 women who underwent hysteroscopic surgery at the "Prof. Dr. José Aristodemo Pinotti" Women's Hospital-CAISM-UNICAMP from January 1998 to December 2008, 800 were confirmed with endometrial polyp anatomopathological diagnosis. Of this total amount, it was excluded tamoxifen users, those who used hormone therapy and cases of malignant or pre-malignant polyps. It was obtained a sample of 515 benign endometrial polyps in women before and after menopause. It was also assessed the expression of ER, PR, Bcl-2, COX-2 and Ki67 through immunohistochemistry according to stained cells percentage, staining intensity, and the final score. The ER, PR, Bcl-2, Cox-2 final score ranges from 0 to 8 and the Ki67 from 0 to 3). The ER, PR, Bcl-2, Cox-2 and Ki67 median final scores in the glandular epithelium and stroma of the polyps were compared among obese and nonobese women, in pre and postmenopausal condition, using the Chi-square Fisher's exact test or nonparametric Mann-Whitney test. Results: The hormonal receptors median final score has showed an increased expression of progesterone receptors in the stroma and glandular epithelium of postmenopausal obese women but there was no difference in expression of ER estrogen receptors. In premenopausal women, there was no difference in expression of ER and PR among obese and nonobese women. The endometrial polyps in postmenopausal women have showed a higher expression of Cox-2 and Bcl-2 in glandular epithelium in obese women rather than in nonobese women. There were no differences in the endometrial stroma. In premenopausal women, there was a higher expression of Bcl-2 only in the obese women glandular epithelium. There were no differences in Ki67 expression among obese and nonobese both postmenopausal and premenopausal women. Conclusions: Obese women polyps, in postmenopausal condition, have increased expression of glandular and stromal PR, Cox-2 and Bcl-2 glandular. However, there are no differences in the Ki67 expression . These data suggest that its etiopathogenesis in obese women polyps, seem to be related to progesterone receptors, apoptosis inhibition and also to mechanisms associated with cellular inflammation / Mestrado / Fisiopatologia Ginecológica / Mestre em Ciências da Saúde

Introdução: Receptores hormonais modulam a resposta dos tecidos ao estímulo humoral. Evidências epidemiológicas mostram que as variações dos genes que regulam a biossíntese e metabolização dos receptores estrogênicos (RE) causam alterações no efeito dos estrógenos no tecido mamário, podendo explicar as variações individuais da densidade mamográfica. A alta densidade mamográfica é um fator de risco importante para o câncer de mama.Objetivo: Avaliar a associação das características clínicas e dos polimorfismos do gene do REalfa (XBal, Pvull e (GT)n) com a densidade mamográfica em mulheres após a menopausa. Casuística e métodos: Avaliaram-se prospectivamente 463 mulheres após a menopausa, sendo que 308 mulheres com mamas densas e 155 com mamas não densas, segundo os critérios do ACR-BIRADS (2003) por avaliação objetiva computadorizada, com idade de 45 a 60 anos, não usuárias de terapia hormonal nos últimos 12 meses e sem antecedentes pessoais de câncer de mama. Amostras de sangue periférico foram obtidas para extração de DNA e análise dos polimorfismos presente no íntron 1 e região promotora (Xbal, Pvull e (GT)n) do gene do RE?. Resutados: Dos fatores considerados de risco para o câncer de mama, houve associação com a densidade mamográfica: idade (p=0,005); medida da cintura abdominal (p=0,001); número de gravidezes (p=0,007); idade ao ter o 1º filho (p=0,035); antecedentes familiares (p=0,035); tempo decorrido deste a data da última menstruação (p=0,007) e índice de massa corpórea (p=0,022). Foi verificada diferença significante entre os grupos de mama densa e não densa para distribuição do genótipo do polimorfismo Pvull (p=0,024). Xbal (p=0,362) e, para o polimorfismo de repetição (GT)n (p=0,151) a associação não foi significante. Conclusão: Apenas o polimorfismo Pvull e os fatores clínicos: idade, cintura abdominal, número de gravidezes, idade ao ter o primeiro filho, antecedentes familiares de câncer de mama, tempo decorrido desde a data da última menstruação e o índice de massa corpórea mostraram-se associados com a densidade mamográfica após a menopausa / Introduction: The hormone receptors modulate the tissue response to hormonal stimulation. Epidemiological evidences show that the variations in genes that regulate the biosynthesis and metabolism of estrogen receptors (ER) cause changes in the effect of estrogen in breast tissue, which may explain individual variations of the mammographic density. High mammographic density is an important risk factor for the breast cancer. Objective: Evaluating the association of clinical characteristics and polymorphisms of the gene ERalfa [XbaI, PvuII and (GT)n] and mammographic density in postmenopausal women. Methods: It was prospectively evaluated 463 postmenopausal women - 308 women with high mammographic density (HMD) and 155 controls (to 50% density or less), according to the criteria of the ACR - BIRADS (2003 ) computed objective assessment , aged 45 to 60 , nonusers of hormone therapy in the past 12 months and with no personal history of breast cancer. Peripheral blood samples were obtained for DNA extraction and analysis of this polymorphism in intron 1 and promoter [XbaI, PvuII and (GT) n)] of the ERalfa gene region. Results: Among the risk factors for breast cancer there was an association with mammographic density: age (p = 0.005), waist circumference (p = 0.001), number of pregnancies (p = 0.007), age of first birth ( p = 0.035 ), family history (p = 0.035), time after menopause (p = 0.007 ) and body mass index (p = 0.022). Significant difference was observed between the groups of HMD and control only for the distribution of the polymorphism PvuII (p = 0.024 ). XbaI (p = 0.362 ) and for repeat polymorphism (GT) n (p = 0.151) the association was not significant. Conclusion: Only the PvuII polymorphism and clinical factors: age, waist circumference, number of pregnancies, age of first birth, family history of breast cancer, time after menopause and body mass index proved to be associated with high mammographic density after menopause

Diss. (sammanfattning) Stockholm : Karolinska institutet 2006. / Härtill 4 uppsatser.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 160 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Made available in DSpace on 2015-07-22T20:50:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-12-31 / O carcinoma da mama é a neoplasia maligna mais freqüente entre as mulheres com grande impacto na mortalidade. Os estudos de quimioprevenção primária com tamoxifeno têm gerado boas expectativas e consideradas taxas de sucesso. Doses menores do tamoxifeno apresentam eficácia semelhante à dose padrão, com redução de custos e efeitos adversos. Estudou-se a imunorreação do anticorpo monoclonal Ki-67 (MIB-1) e a positividade dos receptores de estrogênio (1D5) e progesterona (PgR 636) no carcinoma de mama de mulheres tratadas com 10 mg de tamoxifeno por um período de 14 dias. Realizou-se estudo prospectivo, randomizado, com 38 mulheres, divididas em dois grupos: Grupo A: N = 20 (Grupo controle - sem medicação) e Grupo B: N = 18 (tamoxifeno 10 mg/dia por 14 dias). Todas as pacientes assinaram termo de consentimento previamente aprovado pelas duas instituições (Universidade Federal de São Paulo – Escola Paulista de Medicina e Hospital Materno Infantil de Goiânia-GO). A seguir foram submetidas à biópsia incisional e, após 14 dias, foi obtida nova amostra do tecido tumoral durante o tratamento cirúrgico definitivo. A positividade foi avaliada quantitativamente, contando-se no mínimo 1.000 células para cada lâmina. Para a análise estatística dos dados, foi utilizado o teste não paramétrico de Wilcoxon, fixandose α em 5%. Os dois grupos (A e B) foram considerados homogêneos em relação às variáveis de controle. No grupo A (controle) não houve redução estatisticamente significativa da positividade do Ki-67 (MIB-1) (p=0,627), e dos receptores de estrogênio (1D5) (p=0,296) e progesterona (PgR 636) (p=0,381). No grupo B (tamoxifeno 10 mg/dia) a porcentagem média de núcleos corados pelo Ki-67 (MIB-1) foi 24,7% antes e 10,4% após. Para o receptor de estrogênio (1D5), 59,5% antes e 25,9% após e para o receptor de progesterona (PgR 636), 59,3% e 29,6%, respectivamente. Houve redução significativa para os três marcadores (p<0,001). O tamoxifeno reduziu significativamente a positividade do anticorpo monoclonal Ki-67 (MIB-1), receptor de estrogênio (1D5) e receptor de progesterona (PgR 636) no epitélio mamário de pacientes com carcinoma, tratadas com tamoxifeno na dose de 10 mg por 14 dias. / Breast carcinoma is the most common malignancy among women, and it has a major impact on mortality. Studies of primary chemoprevention with tamoxifen have generated high expectations and considerable success rates. The efficacy of lower doses of tamoxifen is similar to that seen with the standard dose of the drug, and there is a reduction in medical care costs and adverse effects. The immune reaction to monoclonal antibody Ki-67 (MIB-1) and the expression of estrogen receptors (1D5) and progesterone receptors (PgR 636) in breast carcinoma were studied in patients treated with 10 mg of tamoxifen for a period of 14 days. A prospective randomized clinical trial was conducted with 38 patients divided into two groups: Group A: N = 20 (control group–without medication) and Group B: N = 18 (tamoxifen/10 mg/day for 14 days). All patients signed an informed consent term previously approved by both institutions (UNIFESP-EPM and Hospital Materno Infantil, Goiânia-GO). Patients underwent incisional biopsy before treatment and 14 days later a sample of tumor tissue was obtained during surgical treatment. Positivity was quantitatively assessed, counting at least 1.000 cells per slide. For statistical data analysis, a Wilcoxon non-parametric test was used, and α was set at 5%. Both groups (A and B) were considered homogeneous regarding control variables. In Group A (control), there was no statistically significant reduction in Ki-67 (MIB-1) (p=0.627), estrogen receptor (1D5) (p=0.296) and progesterone receptor positivity (PgR 636) (p=0.381). In Group B (tamoxifen 10 mg/day), the mean percentage of nuclei stained by Ki- 67 (MIB-1) was 24.7% before and 10.4% after tamoxifen treatment. Mean percentage of nuclei stained by estrogen receptor (1D5) was 59.5% before and 25.9% after tamoxifen treatment. Mean percentage of nuclei stained by progesterone receptor (PgR 636), was 59.3 before and 29.6% after tamoxifen treatment. A statistically significant reduction was found with the three markers (p<0.001). Tamoxifen significantly reduced monoclonal antibody Ki-67 (MIB-1), estrogen receptor (1D5) and progesterone receptor positivity (PgR 636) in the breast epithelium of patients with carcinoma, treated with a 10 mg dose of tamoxifen for 14 days. / TEDE / BV UNIFESP: Teses e dissertações

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

O diabetes mellitus tipo 2 (DM2) é caracterizado pela presença de resistência à insulina, a qual pode ser modulada pelo estrógeno, tanto em fêmeas como em machos. Nesse processo, o transportador de glicose GLUT4 (gene Slc2a4, solute carrier family 2 member 4) desempenha papel importante, pois aumento da expressão do GLUT4 melhora o controle glicêmico. Estradiol (E2) regula a expressão do Slc2a4 por meio do balanço dos efeitos contrários de seus receptores (ERs): ER-alfa estimula e ER-beta inibe a expressão. Efeitos transcricionais dos ERs envolvem a participação de co-reguladores, destacadamente o SP1 (transcription factor Sp1), potente estimulador do Slc2a4. Entretanto, o papel do SP1 na regulação do Slc2a4 mediada pelos ERs é desconhecido; e este foi o objetivo do presente estudo. Investigou-se adipócitos maduros 3T3-L1, tratados por 24 horas com E2, agonista de ER-alfa (PPT) ou agonista de ER-beta (DPN). Avaliou-se: a expressão gênica (RT-qPCR) de Slc2a4 e Sp1; o conteúdo (Western blotting) total de GLUT4 e o nuclear de ER-alfa/beta e SP1; a atividade de ligação do SP1 no Slc2a4 (ensaio de mobilidade eletroforética); e a formação de complexos SP1/ER-alfa (imunoprecipitação). Os resultados confirmaram que E2 aumenta a expressão de Slc2a4/GLUT4 pela ação preponderante do ER-alfa. O agonista PPT aumentou: o conteúdo nuclear de SP1, a interação SP1/ER-alfa e a atividade de ligação do SP1 no Slc2a4. O agonista DPN indicou que a ação repressora do ER-beta não envolve o SP1. Conclui-se que o efeito estimulador do ER-alfa na expressão do Slc2a4 envolve mecanismo de transativação gênica via SP1. Essas observações colocam a cooperação ER-alfa/SP1 como um novo alvo para o desenvolvimento de medidas terapêuticas para resistência à insulina e diabetes mellitus tipo 2 / Type 2 diabetes mellitus (T2DM) is characterized by insulin resistance, which can be modulated by estrogen in both females and males. In this process, the glucose transporter GLUT4 (solute carrier family 2 member 4 gene - Slc2a4) plays an important role, since increasing GLUT4 expression improves glycemic control. Estradiol (E2) regulates the expression of Slc2a4, by a mechanism in which estrogen receptors (ERs) play opposite effects: ER-alpha stimulates, whereas ER-beta inhibits the expression. Transcriptional effects of ERs involve co-regulators, notably the transcription factor SP1, a powerful enhancer of Slc2a4. However, the role of SP1 in the ERs-mediated regulation of Slc2a4 is unknown; and that was the aim of the present study. Differentiated adipocytes 3T3-L1 were treated (24 hours) with E2, ER-alpha agonist (PPT) or ER-beta agonist (DPN). It was analyzed: gene expression (RT-qPCR) of Slc2a4 and Sp1; total content o GLUT4 and nuclear content of ER-alpha/beta and SP1 (Western blotting); binding activity of SP1 into Slc2a4 promoter (electrophoretic mobility shift assay); and content of nuclear SP1/ER-alpha complexes (immunoprecipitation). Results confirmed that E2 increases the expression of Slc2a4/GLUT4, by the dominant effect of ER-alpha. The ER-alpha agonist PPT increased the nuclear content of SP1, the interaction of SP1/ER-alpha, and the binding activity of SP1 into the Slc2a4. The agonist DPN evinced that ER-beta activity does not involve the SP1. In conclusion, the enhancer effect of ER-alpha upon Slc2a4 gene expression involves a transactivation mechanism via SP1. This observation point outs the cooperation of ER-alpha/SP1 as a new target for the development of approaches to treat insulin resistance and T2DM

No setor de Ginecologia do Hospital das Clínicas da FMUSP, 67 mulheres com leiomiomas do útero e idade de 24 a 39 anos, nuligestas foram estudadas. 31 receberam goserelin a cada 28 dias por 6 meses (grupo I) e 36 não (grupo II). Do grupo I, 16 apresentaram redução volumétrica menor ou igual a 36% (subgrupo Ia) e 15, maior ou igual a 36% (subgrupo Ib). Após a miomectoma, os nódulos foram encaminhados para anatomopatológico. Um único leiomioma de cada mulher foi submetido ao estudo eimuno-histoquímico para avaliação das concentrações de receptores de estrógeno, progesterona, vasos sanguíneos, colágeno, AgNOR e da celularidade. Concluiu-se que o análogo do GnRH está relacionado à diminuição da concentração de receptores de estrógeno. Não apresentou influência uniforme para progesterona, vasos sanguíneos, colágeno e celularidade / From 1994 to 1998, a total of 67 women with leiomyomas in the uterus, aging from 24 to 39, nuliparous and avid for pregnancy were studied in the Department of Gynaecology and Obstetrics of Hospital das Clínicas of Medical School of the University of São Paulo. From these, 31 received Goserelin 3,6mg at each 28 days for six months (group I) and 36 did not received medication (group II or control group). From the pacients who received medication, 16 presented volumetric reduction equal to or less than 36% (subgroup Ia) and the other 15 reduction larger than 36% (subgroup Ib). All women were submitted to myomectomy and the nodes were sent to anatomicopathological study. Only one leiomyoma of each woman was submitted to histochemical and immunohistochemical study to measure the concentrations of receptors of estrogen and progesterone, blood vessels, collagen, AgNOR and cellularity. It was observed that the group that presented larger volumetric reduction after using this medication showed variations of the concentration of receptors of estrogen (p&#61500;0,001), progesterone (p=0.019), blood vessels (p=0.060), collagen (p=0.048), AgNOR (p=0.321) and number of cells (p=0.221), in comparison to the subgroup Ia and the group II (control group). As a result , it was observed that the GnRH analogue is related to the decrease of the concentration of receptors of estrogen, however it did not present uniform influence in the receptors of progesterone, blood vessels, collagen, and cellularity of this tumor