研究背景和研究目的 / 前列腺癌是許多西方國家男性人群中最常見的惡性腫瘤。最新癌症統計結果表明，前列腺發病例和致死率在亞洲國家尤其是中國和香港地區呈迅猛上升趨勢(2009年，本港前列腺癌發病率列所有腫瘤發病率中第三位，致死率列第五位)。目前前列腺癌治療策略主要集中在拮抗雄激素信號通路。然而，臨床實踐表明，這種治療方式除了引起由於體內激素水平失調產生的一系列副作用之外，往往導致疾病進展到令人棘手的去勢治療無效階段。因此，從分子水平更為深入的理解前列腺癌疾病進展過程對於最終攻克前列腺癌具有重要的研究價值。雌激素相關受體是孤兒核受體的亞組之一，包括 α, β, γ三個亞型。該組受體在結構上與α亞型雌激素受體具有很高的同源性。已有研究表明，α亞型雌激素相關受體直接调控涉及氧化磷酸化，線粒體生物發生和脂肪酸氧化的相關基因表達，從而在細胞能量代謝調節中發揮至關重要作用。最新研究發現， α亞型雌激素相關受體的高表達在包括乳腺癌和前列腺癌在內的一系列腫瘤中與疾病的進展和不良預後高度相關。這提示該受體可能參與這些腫瘤的惡性進展。腫瘤細胞對低氧環境的耐受是實體腫瘤的標誌性表型之一，同時也有研究表明這一機制可能在癌細胞的惡性克隆選擇中發揮了重要作用。在眾多低氧耐受的機制中，細胞能量代謝方式轉換被研究人員看作重要的調節通路之一。考慮到前列腫瘤的低氧微環境以及α亞型雌激素相關受體在能量代谢過程的重要調節作用，有理由推測在該受體可能在前列腺癌細胞低氧耐受中發揮了積極的作用進而促進前列腺癌的惡性進展。 / 材料和方法 / 為了研究α亞型雌激素相關受體在前列腺癌細胞低氧耐受中的功能，本次研究採取了下列實驗方法：1）用免疫組化方法考察α亞型雌激素相關受體在人前列腺癌組織中的表達情況；2）用合適的前列癌細胞系建立α亞型雌激素相關受體穩定過表達細胞系同時研究這些穩轉細胞系的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在低氧环境下的體外生長表型；）研究雌激素相關受體穩定過表達細胞系在免疫缺陷小鼠中的致瘤能力同時用免疫組化方法考察其腫瘤血管生成情況；）用定量 PCR和免疫印跡(Western blot)方法檢測低氧誘導因子-1α亞基(HIF-1α)及其信號通路中相關基因在α亞型雌激素相關受體穩定過表達細胞系中的表達水平，同時用雙螢光素酶報告基因方法考察α亞型雌激素相關受體對低氧誘導因子‐1(HIF-1)靶基因啟動子的轉錄激活效應；5）用 shRNA介導的基因阻斷的方法進一步考察α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；6）通過觀考察用α亞型雌激素相關受體選擇性抑製劑 XCT790處理細胞對其在低氧環境下的體外生長情況的作用，進一步闡明 α亞型雌激素相關受體對前列腺癌細胞低氧耐受的影響；7）用免疫印跡 (Western blot)，免疫共沉澱 (Co-IP)和熒光能量共振轉移(FRET)分析的方法考察α亞型雌激素相關受體對低氧誘導因子‐1α亞基表蛋白表達和穩定性以及對低氧誘導因子 -1信號通路的影響。 / 結果 / 本研究所得得到的結果簡要總結如下：1）α亞型雌激素相關受體在前列癌組織中的免疫反應性呈現隨著惡性程度升高而增加的趨勢；2）α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能提升其在常氧和低氧環境下的體外細胞增殖，細胞集落形成，細胞對胞外基質的粘附以及細胞侵襲能力； 3) α亞型雌激素相關受體在人前列腺癌細胞系 LNCaP中的過表達能促進其體內腫瘤形成及腫瘤血管生成； 4）過表達 α亞型雌激素相關受體能上調低氧誘導因子-1α亞基的蛋白水平並提高其轉錄活性；5）shRNA介導的α亞型雌激素相關受體 mRNA阻斷可以削弱人前列腺癌細胞系 LNCaP細胞在低氧環境下的體外生長能力；6）在体外用α亞型雌激素相關受體選擇性抑製劑 XCT790处理人前列腺癌細胞系 LNCaP細胞可能通過減少低氧誘導因子‐1α亞基蛋白表達水平從而抑制其在低氧環境下的細胞生長能力；7）α亞型雌激素相關受體可以直接與低氧誘導因子-1α亞基相互作用，並且這種相互作用可能有助於抑制低氧誘導因子-1 α亞基的蛋白降解。 / 結論 / 本研究獲得結果提示，α亞型雌激素相關受體可能通過提高低氧誘導因子-1α亞基的蛋白水平及激活低氧誘導因子-1信號通路從而促進前列腺癌細胞在低鹽環境下的細胞生長能力。体外用 shRNA介導的α亞型雌激素相關受體 mRNA阻斷方法和α亞型雌激素相關受體選擇性抑製劑处理都有可能通過阻止低氧誘導因子‐1α亞基以削弱前列腺癌細胞在低鹽環境下的細胞生長能力。同時， α亞型雌激素相關受體能直接與低氧誘導因子-1 α亞基相互作用而這種相互作用有可能有助於抑制其蛋白降解，這些結果提示 α亞型雌激素相關受體可能在前列腺癌進展過程中的低氧耐受中發揮積極作用。 / Background and aims of study / Prostate cancer is the most common cancer in many Western counties among the male populations. Latest cancer statistics also show that its incidence and mortality rates are rapidly increasing in China and Hong Kong (Prostate cancer ranked the 3rd common cancer and 5th cancer causing death in Hong Kong in 2009). Current therapeutic strategies of prostate cancer mainly target to the antagonizing androgen signaling pathway, which usually drives the disease to the impasse of castration resistance albeit the side effects caused by the imbalance of hormone. The substantial clinical significance of prostate cancer is urgent to better understand the progression of this disease. Estrogen-related receptors (α,β,γ) are a subgroup of ligand-independent orphan nuclear receptors, which is constitutively activated without binding any physiological ligands and all share high homology with the estrogen receptor alpha (ER α) structurally. Previous studies indicates that ERR α plays a pivotal role in cellular energy home stasis regulation, target genes of which are involved in the procedures of oxidative phosphorylation, mitochondrial biogenesis and fatty acid oxidation. Recent studies reveals that high expression of ERR α may be useful as a poor prognostic marker in both hormone-dependent and hormone-independent cancers (including breast cancer and prostate cancer), which implicates this nuclear receptor may be involved in the advanced malignant progression of these cancers. Adaptation to hypoxia is one of the hallmark features of solid tumors and it is conceived to play an important role in malignant clonal selection of cancer cells. Among the diverse mechanisms on cellular hypoxia adaptation, energy metabolism reprogramming is characterized and considered as a critical regulatory pathway. Given the hypoxic microenvironment of prostate cancer and the energy regulatory role of ERR α, it is hypothesized that ERR α might play an active role in the cellular hypoxic adaptation of prostate cancer hence advancing the progre sion of this disease. / Materials and methods / To investigate the functional significance of ERR α in cellular hypoxic adaptation of prostate cancer, the following experimental approaches were employed and performed in my thesis study: 1) to survey the expression pattern of ERR α in human prostate cancer tissues by immunohistochemical staining; 2) to generate ERR α-stable expressing cell lines in selected prostate cancer cell lines and functionally characterize their in vitro phenotypes under normoxia condition; 3) to characterize in vitro hypoxic-response phenotypes of ERR α-infectants; 4) to determine the tumorigenicity of ERR α-infectants in immuno-deficient SCID mice and to investigate their tumor angiogenesis by immunohistochemical staining; 5) to determine the HIF-1α signal cohort in ERR α-infectants by both RT-PCR and immuno blot analysis and to investigate the transactivation effect of ERR α on HIF-1 targeting genes promoters by dual luciferase reporter assay; 6) to further characterize the hypoxic adaptation phenotypes induced by ERR α transduction using shRNA-mediated gene knockdown approach; 7) to further elucidate the effect of ERR α on the hypoxic cell growth regulation of prostate cancer by treating ERR α-infectants with an ERR α-selective antagonist XCT790; 8) to further investigate the mechanisms via which ERR α interferes with the protein expression or stabilization of HIF-1α as well as HIF-1 signal cohort using immuno blot analysis, immunoprecipitation assays and fluorescence resonance energy transfer (FRET) analysis. / Results / My results are briefly summarized as follows: 1) ERR α exhibited an increased immuno expression pattern in high-grade prostate cancer; 2) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vitro cell proliferation, clonal formation, cell-extracellular matrix attachment and cell invasion capacities under both normoxic and hypoxic conditions; 3) Ectopic expression of ERR α in LNCaP prostate cancer cell line could promote its in vivo tumorigenicity and tumor angiogenesis; 4) Overexpression of ERR α could up-regulate protein level of hypoxia regulatory transcriptional factor-1(HIF-1) α subunit (HIF1-α) and enhance its transcriptional activity; 5) mRNA knock-down of ERR α could attenuate in vitro cell growth capacity of LNCaP prostate cancer cell line under hypoxic condition; 6) Treatment with an ERR α specific antagonist XCT790 could inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α; 7) ERR α could physically interact with HIF-1α and such ERR α-HIF1-α interaction might help to inhibit protein degradation of HIF-1α. / Conclusion / The results obtained in this study indicated that ERR α could promote the hypoxic cell growth of prostate cancer via its enhancing the protein level of HIF-1α and activation of HIF-1 signal cohort. Both treatment with ERR α selective antagonist and down-regulating of ERR α by shRNA-mediated gene knockdown approach could attenuate the hypoxia adaptation of prostate cancer cells, which might be mediated by their suppression of the protein level of HIF1α. ERR α could directly interact with HIF-1α and such interaction might help to suppress the protein degradation of HIF1α, suggesting that ERR α may play an active role in hypoxic adaptation in advancing of prostate cancer. / 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. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Zou, Chang. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 138-160). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / ACKNOWLEDGEMENTS --- p.viii / PUBLICATIONS --- p.ix / CONTENTS --- p.x / ABBREVIATIONS --- p.xiii / Chapter CHAPTER 1 --- Introduction --- p.1 / Chapter 1.1 --- Prostate cancer --- p.2 / Chapter 1.1.1 --- Epidemiology --- p.2 / Chapter 1.1.2 --- Risk factors --- p.3 / Chapter 1.1.3 --- Patho-physiology --- p.6 / Chapter 1.1.4 --- Diagnosis and treatment --- p.8 / Chapter 1.2 --- Androgen,androgen receptor and prostate cancer --- p.10 / Chapter 1.2.1 --- Androgen and androgen receptor --- p.10 / Chapter 1.2.2 --- Castration Resistance Prostate Cancer (CRPC) --- p.12 / Chapter 1.2.2.1 --- Overexpression of AR --- p.13 / Chapter 1.2.2.2 --- Increasing sensitivity to and rogen --- p.13 / Chapter 1.2.2.3 --- AR mutation --- p.14 / Chapter 1.2.2.4 --- Deregulation of AR regulator factors --- p.15 / Chapter 1.2.2.5 --- Outlaw pathway --- p.15 / Chapter 1.2.2.6 --- AR-independent pathway --- p.16 / Chapter 1.3 --- Estrogen and prostate cancer --- p.17 / Chapter 1.3.1 --- Overview of estrogen and estrogen receptors --- p.17 / Chapter 1.3.2 --- Estrogen signaling pathway andprostatecancer --- p.18 / Chapter 1.4 --- Nuclear receptors --- p.20 / Chapter 1.4.1 --- Overview of NRs superfamily --- p.20 / Chapter 1.4.2 --- Classification --- p.21 / Chapter 1.4.3 --- NRs as therapeutic targets for cancer treatment --- p.23 / Chapter 1.5 --- Estrogen-related receptors --- p.25 / Chapter 1.5.1 --- NR3B subgroup --- p.25 / Chapter 1.5.2 --- Isoforms --- p.26 / Chapter 1.5.3 --- Structure --- p.27 / Chapter 1.5.4 --- Ligand --- p.28 / Chapter 1.5.5 --- Co-regulators --- p.31 / Chapter 1.5.6 --- Tissue-specific expression pattern and identifiedfunction --- p.32 / Chapter 1.5.6.1 --- Tissue-specific expression pattern --- p.32 / Chapter 1.5.6.2 --- Identified physiological function of ERRs --- p.33 / Chapter 1.5.7 --- ERRs and cancer --- p.35 / Chapter 1.5.7.1 --- ERRβ/γ and cancer --- p.35 / Chapter 1.5.7.2 --- Expression of ERRα in cancer --- p.37 / Chapter 1.5.7.3 --- Identified functional roles of ERRα in cancer --- p.40 / Chapter 1.5.7.4 --- Regulation of ERRα in cancer cells --- p.42 / Chapter 1.6 --- Hypoxiaadaptation andcancer --- p.47 / Chapter 1.6.1 --- HIFs isoforms and structure --- p.47 / Chapter 1.6.2 --- Structure --- p.48 / Chapter 1.6.3 --- Regulation of HIF-1α expression --- p.49 / Chapter 1.6.3.1 --- Regulation of HIF-1α mRNA transcription --- p.49 / Chapter 1.6.2.2 --- Regulation of HIF-1α mRNA transcription --- p.50 / Chapter 1.6.2.3 --- O₂-dependent regulation of stability of HIF-1α protein --- p.51 / Chapter 1.6.2.4 --- O₂-independent regulation of HIF-1α --- p.52 / Chapter 1.6.2.5 --- Genetranscriptional regulation role of HIFs --- p.54 / Chapter 1.6.3 --- HIFs and cancer --- p.55 / Chapter 1.6.3.1 --- Overview --- p.55 / Chapter 1.6.3.2 --- Expression of HIF-1α in cancer progression --- p.55 / Chapter 1.6.3.2 --- Functional roles of HIF-1α in cancer progression --- p.56 / Chapter CHAPTER 2 --- Aims of study --- p.58 / Chapter CHAPTER 3 --- Materials and methods --- p.61 / Chapter 3.1 --- Cell lines and cell culture --- p.62 / Chapter 3.2 --- Human Prostatic Tissues --- p.64 / Chapter 3.3 --- RNA isolation and Reverse transcriptional-PCR --- p.64 / Chapter 3.3.1 --- Total RNA extraction --- p.64 / Chapter 3.3.2 --- Reverse transcription reaction --- p.65 / Chapter 3.3.3 --- Polymerase Chain Reaction for gene expression detection --- p.66 / Chapter 3.4 --- Plasmids construction --- p.69 / Chapter 3.4.1 --- Genomic DNA extraction --- p.69 / Chapter 3.4.2 --- PCR for cloning and sub-cloning --- p.70 / Chapter 3.4.3 --- PCR for mutant generation --- p.70 / Chapter 3.4.4 --- Restriction enzymes cut and ligation --- p.71 / Chapter 3.5 --- Antibody and reagents --- p.73 / Chapter 3.6 --- Immunohistochemistry --- p.74 / Chapter 3.7 --- Western Blot Analysis --- p.75 / Chapter 3.7.1 --- Protein extraction --- p.75 / Chapter 3.7.2 --- Electrophoresis, Protein blotting and Colorimetric detection --- p.76 / Chapter 3.8 --- Retroviral transduction and generation of ERRα poolandstable clones --- p.77 / Chapter 3.9 --- In vitro Cell Growth Assays --- p.77 / Chapter 3.9.1 --- Cell counting --- p.77 / Chapter 3.9.2 --- 5-Bromodeoxyuridine (BrdU) incorporation assay --- p.78 / Chapter 3.9.3 --- MTT assay --- p.79 / Chapter 3.9.4 --- In vitro clonal formation assay --- p.79 / Chapter 3.10 --- Cell attachment assay --- p.80 / Chapter 3.11 --- Transwell cell invasion assay --- p.81 / Chapter 3.12 --- In vivo tumorigenicity assay --- p.81 / Chapter 3.13 --- RNA interference --- p.82 / Chapter 3.14 --- Transient Transfection and Luciferase Reporter Assay --- p.83 / Chapter 3.15 --- Immuno-precipitation (IP) assay --- p.84 / Chapter 3.16 --- Fluorescence Resonance Energy Transfer (FRET) detection --- p.85 / Chapter 3.17 --- In vitro treatment with XCT790, cycloheximide and MG-132 --- p.86 / Chapter CHAPTER 4 --- Reuslts --- p.88 / Chapter 4.1 --- ERRα exhibits an increased expression pattern in high grade prostate cancer --- p.89 / Chapter 4.2 --- Ectopic expression of ERRα in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacity under normoxic condition --- p.91 / Chapter 4.3 --- Ectopic expression of ERR α in LNCaP prostate cancer cell line can promote its in vitro cell proliferation, clonal formation, cell attachment and cell invasion capacities under hypoxic condition --- p.94 / Chapter 4.4 --- Ectopic expression of ERR α in LNCaP prostate cancer cells can promote their in vivo tumorigenicity and tumor angiogenesis. --- p.97 / Chapter 4.5 --- Overexpression of ERRα can up‐regulate protein level of HIF-1α and enhance its transcriptional activity --- p.99 / Chapter 4.6 --- mRNA Knock-down of ERRα can attenuate in vitro cell growth of LNCaP prostate cancer celll line under hypoxic condition --- p.107 / Chapter 4.7 --- Treatment with an ERRα specific antagonist XCT790 can inhibit in vitro hypoxic cell growth of LNCaP cells via its effect on decreasing the protein level of HIF-1α --- p.110 / Chapter 4.8 --- ERRα can physically interact with HIF-1α and such ERRα-HIF-1α interaction helps to inhibit protein degradation of HIF-1α --- p.114 / Chapter CHAPTER 5 --- Discussion --- p.119 / Chapter CHAPTER 6 --- Summary --- p.134 / References --- p.138

Because of the structural resemblance to the female hormone, phytoestrogen is another important class of endocrine disruptor. In the present project, we evaluated the effects of phytoestrogens isoliquiritigenin (ILN), hesperetin (HES), genistein, (GEN) and naringenin (NAR) on estrogen metabolism and also their effects on MCF-7 tumor growth in ovariectomized nude mice. We found that these phytoestrogens had differential effect on MCF-7 xenografts. NAR and GEN had totally different responses in the tumor growth. In contrast, ILN and HES only deterred MCF-7 xenograft growth when CYP19 was overexpressed in the graft. / Breast cancer is one of the most prevalent female cancers in Hong Kong and western countries. Prolonged exposure to estrogen has been associated with increased risk of breast cancer. Many enzymes are responsible for estrogen metabolism, for instance, aromatase (CYP19) is responsible for biosynthesis; CYP1 family enzymes hydroxylate estrogen; COMT (catechol-O-methyltransferase) inactivates the hydroxyestrogen; and UDP-glucuronosyltransferase 1A1 (UGT1A1) eliminates the estrogen metabolites. In this project, we employed cell and animal models to address estrogen metabolism-related questions under the influence of endocrine disruptors. / TCDD is a prototype compound of a whole class of halogenated aromatic hydrocarbons termed dioxin-like contaminants, which are also known to be endocrine disruptors. Because of their persistence in the environment dioxins are one of the most concerned classes of carcinogens. Humans can be exposed to this pollutant through contaminated food, air, drinking water, etc. We found that pre-ovariectomy administration of TCDD could significantly reduce aromatase expression in the brain but increase the expression in the adipose tissue. Our results suggested that the timing of exposure to the toxicant could determine the estrogenicity of TCDD. / The present project indicated that endocrine disruptors can alter the metabolism of estrogen; however, the significance of this alteration may be specific to tissues' phenotype and the timing of exposure. / Ye Lan. / Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 169-192). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

Christopher John Haines. / Thesis (M.D.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 239-279). / LIST OF TABLES --- p.xviii / LIST OF FIGURES --- p.xxi / LIST OF ABBREVIATIONS --- p.xxii / Chapter CHAPTER1 --- INTRODUCTION --- p.1 / Problems related to the menopause / Research plan / Chapter CHAPTER2 --- OVERVIEW --- p.15 / Introduction / Atherosclerosis and the lipid profile / Coronary artery disease and lipid abnormalities in women / "Exogenous oestrogens, progestogens and coronary artery disease " / Lipoprotein (a) / Chapter CHAPTER3 --- GENERAL METHODOLOGY --- p.134 / Recruitment of cases / Pharmacokinetics / Data collection and analysis of samples / Ethical considerations / Chapter CHAPTER4 --- STUDY I -THE SHORT TERM EFFECTS OF ORAL OESTROGEN --- p.157 / Crossover analysis of effects of oral oestrogen on lipoprotein (a) and other lipoproteins / Relationship between lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER5 --- STUDY II -THE SUSTAINED EFFECTS OF ORAL OESTROGEN --- p.186 / Analysis of prolonged effects of oral oestrogen on lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER6 --- STUDY III -THE EFFECTS OF COMBINED CYCLICAL HORMONE REPLACEMENT THERAPY --- p.196 / Analysis of effect of combined cyclical hormone replacement therapy on lipoprotein (a) and other lipids and lipoproteins / Comparison between sampling during oestrogen alone and combined phase of treatment / Chapter CHAPTER7 --- STUDY IV -THE EFFECTS OF PERCUTANEOUS OESTROGEN --- p.214 / Analysis of effect of percutaneous on lipoprotein (a) and other lipids and lipoproteins / Chapter CHAPTER8 --- SUMMARY AND CONCLUSIONS --- p.228 / BIBLIOGRAPHY --- p.239

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: O câncer de mama é a neoplasia maligna mais comum no sexo feminino e a segunda causa de óbito por câncer na mulher. Apesar dos avanços no conhecimento da patogênese e dos aspectos moleculares da doença, a exposição prolongada tanto ao estrogênio endógeno quanto exógeno constitui importante fator na carcinogênese mamária. Produtos da degradação e inativação do estrogênio podem ter ação proliferativa e causar danos ao DNA. Genes de baixa penetrância relacionados ao metabolismo hormonal, atuando em conjunto com fatores comportamentais e endógenos, podem ser responsáveis por parte dos casos de câncer de mama, atuando em conjunto a genes de alta penetrância. OBJETIVOS: Analisar polimorfismos no receptor de estrogênio e em genes relacionados ao seu metabolismo em mulheres com e sem câncer de mama, observando suas frequências em cada um dos grupos. CASUÍSTICA E MÉTODOS: Foram incluídas mulheres portadoras de carcinoma mamário recém-diagnosticado, com idade superior a 40 anos e sem histórico de outros tipos de neoplasia maligna (grupo estudo) e mulheres sem câncer de mama, apresentando exame mamográfico normal realizado há no máximo 12 meses, com mais de 40 anos de idade e sem histórico de câncer (grupo controle). Foi realizada coleta de sangue periférico para extração e análise de DNA genômico. Avaliaram-se os polimorfismos em CYP1A1 MspI, CYP3A4*1B, COMT L/L, ESR1 PvuII e XbaI e UGT1A1*28. RESULTADOS: Os polimorfismos em UGT1A1*28 e ESR1 PvuII foram mais frequentes no grupo de mulheres com câncer de mama, porém sem atingir significância estatística. As mutações em ESR1 XbaI, CYP3A4*1B e CYP1A1 MspI foram mais frequentes no grupo controle, também sem significância estatística. Já o genótipo L/L do gene COMT foi mais significativamente mais frequente no grupo controle. CONCLUSÕES: A frequência de polimorfismos em UGT1A1*28 e ESR1 PvuII foi de 16,8% e 39,4% nas mulheres com câncer de mama e 10,8% e 18,3% nas sem câncer. A frequência de mutações em ESR1 XbaI, CYP3A4*1B e CYP1A1 MspI foi de 15,1, 44,2 e 3,6% nas mulheres sem câncer de mama e de 25, 22,2% e 0 nas com câncer de mama. O genótipo L/L do gene COMT foi significantemente mais frequente no grupo controle (28,9 vs. 8,6%), sugerindo que este polimorfismo poderia ser um fator protetor ao câncer de mama na população estudada / INTRODUCTION: Breast cancer is the most common malignancy in women and second leading cause of cancer death in women. Despite advances in studies on the pathogenesis and molecular aspects of the disease, prolonged exposure to endogenous and exogenous estrogen is an important factor for mammary carcinogenesis. Products of degradation and inactivation of estrogen may have proliferative action and cause DNA damage. Low penetrance genes related to hormone metabolism, acting in conjunction with behavioral and endogenous factors may be responsible for most cases of breast cancer, and together with high penetrance genes. OBJECTIVES: To analyze polymorphisms in genes related to the estrogen receptor and metabolism in patients with and without breast cancer, observing the frequencies in each group. MATERIALS AND METHODS: We included women with newly diagnosed breast cancer, aged 40 years or more and without history of other types of malignancy (study group) and women without breast cancer, with a normal mammography performed for no more than 12 months, with more than 40 years old and no history of cancer (control group). A sample of peripheral was collected for genomic DNA extraction and analysis. The polymorphisms in CYP1A1 MspI, CYP3A4*1B, COMT L/L, ESR1 PvuII and XbaI and UGT1A1*28 were evaluated. RESULTS: The polymorphisms in UGT1A1*28 and ESR1 PvuII were more frequent in the group of women with breast cancer, without reaching statistical significance. The mutations in ESR1 XbaI, CYP3A4*1B and CYP1A1 MspI were more frequent in the control group, also not statistically significant. The COMT genotype L/L was significantly more frequent in control group. CONCLUSIONS: The frequency of polymorphisms in UGT1A1*28 and ESR1 PvuII was 16.8% and 39.4% in women with breast cancer and 10.8% and 18.3% in those without cancer. The frequency of mutations in ESR1 XbaI, CYP3A4*1B and CYP1A1 MspI was 15.1, 44.2 and 3.6% in women without breast cancer and 25, 22.2 and 0% in breast cancer. The L/L genotype of the COMT gene was significantly more frequent in the control group (28.9 vs. 8.6%), suggesting that this polymorphism could be a protective factor against breast cancer in this population

Chan, Ming Yan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 92-106). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / ABSTRACT --- p.ii / 摘要 --- p.iv / LIST OF ABBREVIATIONS --- p.vi / TABLE OF CONTENTS --- p.viii / Chapter CHAPTER 1 --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- Aromatase --- p.1 / Chapter 1.2 --- Tissue Specific Promoter for Aromatase Expression --- p.4 / Chapter 1.3 --- Signaling Pathway --- p.7 / Chapter CHAPTER 2 --- MATERIALS AND METHODS --- p.9 / Chapter 2.1 --- Chemicals And Materials --- p.9 / Chapter 2.2 --- Mammalian Cell Culture --- p.9 / Chapter 2.2.1 --- Maintenance of Cells --- p.10 / Chapter 2.2.2 --- Preparation of Cells Stock --- p.10 / Chapter 2.2.3 --- Cell Recovery from Liquid Nitrogen Stock --- p.11 / Chapter 2.3 --- Tritiated Water Release Assay --- p.11 / Chapter 2.3.1 --- Aromatase Activity in Intact Cell --- p.11 / Chapter 2.3.2 --- Aromatase Assay on Recombinant Supersomes --- p.12 / Chapter 2.4 --- RNA Isolation and cDNA Synthesis --- p.13 / Chapter 2.5 --- Semi-Quantitative PCR Reaction --- p.13 / Chapter 2.6 --- Quantitative Real Time PCR Using Taqman Probe --- p.15 / Chapter 2.7 --- Western Blotting --- p.17 / Chapter 2.8 --- Measurement of Promoter Activity --- p.18 / Chapter 2.8.1 --- Plasmid Preparation --- p.18 / Chapter 2.8.2 --- Transient Transfection and Dual Luciferase Assay --- p.18 / Chapter 2.9 --- Statistical Methods --- p.19 / Chapter CHAPTER 3 --- Genistein up-regulate aromatase in Estrogen receptor alpha-transfected HepG2 cells --- p.21 / Chapter 3.1 --- Introduction --- p.21 / Chapter 3.1.1 --- Cardiovascular Disease (CVD) --- p.21 / Chapter 3.1.2 --- Phytoestrogen --- p.21 / Chapter 3.1.3 --- Estrogen Receptor --- p.24 / Chapter 3.1.4 --- Protective Mechanism Against CVD Protection --- p.25 / Chapter 3.1.5 --- Effects of genistein on LDL Receptor and Apolipoprotein A-I --- p.26 / Chapter 3.1.6 --- Effects of estradiol on LDL Receptor and Apolipoprotein A-I　 --- p.26 / Chapter 3.1.7 --- Aim of study and hypothesis --- p.27 / Chapter 3.2 --- Result --- p.29 / Chapter 3.2.1 --- ERa increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.2 --- Genistein increased Aromatase Activity in HepG2 cells --- p.29 / Chapter 3.2.3 --- Differential Effect of MAP kinase Inhibitors --- p.35 / Chapter 3.2.4 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in ERa-transfected HepG2 cells" --- p.35 / Chapter 3.2.5 --- Genistein Increased Aromatase Protein Expression in ERa-transfected HepG2 cells --- p.38 / Chapter 3.2.6 --- Genistein Induced Aromatase mRNA Expression Attributed to Induction of Exon ̐ơ.1 Expression --- p.40 / Chapter 3.2.7 --- Genistein Induced Promoter 1.1 Transcriptional Activity in ERa- transfected HepG2 cells --- p.44 / Chapter 3.2.8 --- Genistein Increased ERE and AP-1 Reporter Activity Through Interaction with ERa --- p.47 / Chapter 3.3 --- Discussion --- p.51 / Chapter CHAPTER 4 --- "Effect of 2,3,7,8-tetrachlorodibenzo- para-TCDD (TCDD) on aromatase in MCF-7 cells" --- p.54 / Chapter 4.1 --- Introduction --- p.54 / Chapter 4.1.1 --- Breast Cancer --- p.54 / Chapter 4.1.2 --- TCDD --- p.54 / Chapter 4.1.3 --- CYP Enzymes --- p.55 / Chapter 4.1.4 --- TCDD and Breast Cancer --- p.56 / Chapter 4.1.5 --- Aim of Study --- p.56 / Chapter 4.2 --- Result --- p.57 / Chapter 4.2.1 --- Effect of TCDD on Aromatase Activity in Different Cell Lines --- p.57 / Chapter 4.2.2 --- TCDD Increased Aromatase Activity in MCF-7 Cells --- p.62 / Chapter 4.2.3 --- Effect of TCDD on Human CYP 19 Recombinant Supersomes® and MCF-7aro Cells --- p.66 / Chapter 4.2.4 --- TCDD Increased Aromatase Protein Expression in MCF-7 Cells --- p.66 / Chapter 4.2.5 --- Effect of TCDD in Aromatase mRNA Expression in MCF-7 Cells --- p.70 / Chapter 4.2.6 --- Effect of TCDD in CYP 19 Promoter and AP-1 Promoter Activity in MCF-7 Cells --- p.70 / Chapter 4.2.7 --- Effect of TCDD in CYP 19 mRNA Half-life --- p.75 / Chapter 4.2.8 --- "Role of MAP Kinase, PKA and PKC in Genistein Induced Aromatase Activity in MCF-7 Cells" --- p.78 / Chapter 4.2.9 --- TCDD induced ERK1/2 Activation --- p.78 / Chapter 4.2.10 --- Induction of aromatase activity in MCF-7erk cells --- p.78 / Chapter 4.3 --- Discussion --- p.87 / Chapter CHAPTER 5 --- Summary --- p.90 / BIBLIOGRAPHY --- p.92

Multiple sclerosis (MS) is an idiopathic neurodegenerative, demyelinating disease of the central nervous system (CNS). MS affects females more than males (3:1) and pregnancy reduces the number of relapses especially during the third trimester when 17-beta-estradiol (E2) and estriol (E3) are at their highest levels. In order to study the role of estrogens as potential therapeutic agents for MS we investigated their role in Theiler's murine encephalomyelitis virus (TMEV)-induced demyelination (TVID). SJL female mice were infected intracranially with Theiler's virus or PBS. The mice in the treatment groups were clinically scored and at week 20 they were ovarectomized (OVx) and given a subdermal pellet containing either 1) 0.1mg of E2, 2) 5mg of E3, or 3) placebo. Four weeks after treatment initiation, the mice were sacrificed and tissue samples were collected and vertebral columns and brains were fixed and placed in paraffin for histological analysis using either hematoxylin and eosin (H&E) stain for general anatomic features or Weil's stain for myelin. No signs of clinical disease developed in any of the sham-infected mice. Prior to ovariectomy, infected mice had developed significant clinical scores indicative of demyelination. Mice in the placebo and E3-treatment groups deteriorated rapidly whereas the E2-treated mice improved significantly during the course of the treatment. Uteri were used to assess hormonal effects post-ovariectomy. Hormone treated groups were significantly different from placebo, indicating hormones were present. Hormone treatment showed significant differences among treatment groups for both inflammation and demyelination. E2-treatment significantly decreased inflammation compared to placebo and E3. E2 was also effective in reducing demyelination compared to placebo groups but not E3. E3 treatment was effective in reducing inflammation compared to placebo, but no significance was found for demyelination. Both E3 and E2 treated mice developed lower antibody levels against TMEV. The improvement in clinical signs, inflammation, demyelination, and the reduction of antibody levels in 17-beta-estradiol-treated mice indicate a therapeutic potential for the treatment of MS.

Estrogenic contamination of waterways is of world-wide concern due to the adverse effects observed in aquatic biota. Recently, wastes from agricultural activities have been identified as likely sources of steroid estrogens released into the environment. Wastes from dairying activities are of particular concern in New Zealand. This project included development of analytical methods to measure free and conjugated estrogens, measurement of estrogens from the source to receiving environments and an investigation of effluent treatment technologies. The analytical method developed in this study was based on GC-MS measurement of free estrogens (17α-estradiol (17α-E2), 17β-estradiol (17β-E2) and estrone (E1)) and LC-IT-MS measurement of their sulfate-conjugates (17α-E2-3S, 17β-3S, E1-3S) in raw and treated farm dairy shed effluents (DSE). Effluents from farms in the Canterbury and Waikato Regions, two regions where dairy farming is the dominant land-use, were collected and analysed. All effluents demonstrated high concentrations of steroid estrogens, particularly 17α-E2 (median 760 ng/L). Estrogenic activity was also elevated, at up to 500 ng/L 17β-E2 equivalents using the E-Screen, an in vitro cell proliferation bioassay. Comparison to the chemical data indicated that for most samples, the highest proportion of estrogenic activity was derived from steroid estrogens naturally excreted by dairy cows. Conjugated estrogens were measured in several raw effluent samples, at similar concentrations to those of free estrogens, particularly E1. Dairy effluent treatment systems reduced free estrogen concentrations by 63-99% and reduced estrogenic activity by up to 89%. In spite of high removal efficiencies, estrogens remained elevated in the treated effluents that are discharged into waterways. Steroid estrogens and estrogenic activity were detected in streams and groundwater in areas impacted by dairy farming. Although concentrations were generally low, in two streams the concentrations were above levels regarded as safe for aquatic biota (<1 ng/L). The results demonstrate that dairy effluents are indeed a major source of estrogens to the environment and to waterways.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.