Estrogen is the natural agonist of the estrogen receptor (ER). However, certain plant-derived compounds or phytoestrogens have been identified that mimic estrogens and act as agonists and/or antagonists of ERs, depending on subtype and target tissue. Understanding how phytoestrogens interact with ERs, and therefore effect the estrogenic response, may prove beneficial in hormone replacement therapy and in the prevention and treatment of hormone-related diseases. Using Thin Layer Chromatography, gas chromatography/mass spectrometry (GC/MS), and proton nuclear nagnetic resonance (HNMR), I identified 4-ethoxymethylphenol (4EM) found in Maclura pomifera. While most phytoestrogens are heterocyclic compounds, 4EM is a simple phenol that acts as an agonist of ER-alpha and -beta in HeLa and MCF-7 cells. To study the effect of 4EM on ER-alpha and -beta activity, I performed transient transfection assays and showed that 4EM activates ER dependent gene transcription in a dose dependent manner in both ER subtypes. Further, 4EM- mediated transcription in ER-alpha, like estrogen, was enhance in the presense of co-activators, SRC-1 (steroid receptor coactivator-1), CBP (CREB binding proteins), and E6-AP (E6-associated protein) and inhibited by trans-4- hydroxytamoxifen (4HT). I found that 4EM was specific for ER and did not activate transcription of the progesterone receptor in HeLa cells.

No presente estudo, observamos aumentada expressão de iNOS e de proteínas s-nitrosiladas (S-NT) em aorta (AO) e concentração de NO no plasma de ratas diabéticas (DB), que foram corrigidas pelo tratamento com insulina (INS), que foi incapaz de normalizar a glicemia. O tratamento com o inibidor da iNOS L-NIL reduziu a expressão de S-NT e a concentração de NO. A contração induzida por agonistas adrenérgicos (ADRs), mas não por cloreto de potássio, que estava reduzida em anéis de AO sem endotélio de ratas DB, foi corrigida pelos tratamentos com INS e L-NIL. A expressão dos receptores de estrógeno ESR2 e GPER estava aumentada em AO de ratas DB e foi corrigida pelo tratamento com INS. Nossos resultados mostram que o aumento da expressão da iNOS/geração de NO é responsável pela redução da contração mediada por receptor ADR, mas não daquela induzida por despolarização direta da membrana. A INS modula negativamente a expressão da iNOS e dos receptores ESR2 e GPER em aorta de ratas DB, efeito que pode contribuir com a restauração da contração induzida por agonistas ADRs. / In the present study, we observed that in aortas (AO) of diabetic (DB) female rats have an increase in iNOS and S-nitrosilated (S-NT) proteins expression along with higher levels of plasmatic NO. Although insulin (INS) treatment did not normalize blood glucose levels, it corrected protein expression and NO concentrations. The iNOS inhibitor treatment reduced the altered expression of S-NT proteins and NO levels. The reduced adrenergic agonists (ADR)-induced contractions in DB without endothelium were corrected by INS and L-NIL treatments, such treatments did not affect the reduced vasoconstriction response to KCl in DB AO. The increase expression of estrogen receptors GPER and ESR2 found in DB AO was recovered by INS treatment. Our results showed that the increased expression of iNOS/NO generation is responsible for reducing ADR-induced contraction, but not for membrane depolarization-induced contraction. INS negatively modulates protein expression of iNOS, ESR2, and GPER receptors in DB AO; such effect may contribute to restore ADR-induced vascular contraction.

Made available in DSpace on 2016-08-10T10:39:27Z (GMT). No. of bitstreams: 1 RITA DE CASSIA PEREIRA DA COSTA E SILVA.pdf: 1242407 bytes, checksum: 6e72b473663dff3c1314681f84f66edc (MD5) Previous issue date: 2010-06-30 / Endometriosis is defined by the appearance of foci of endometrial tissue with glandular features and / or stromal identical to the uterine cavity at locations other than the endometrium. And these foci of tissue are usually functioning and sensitive to the action of hormones showing a strong genetic component to disease correlated with several polymorphisms. It focuses primarily on women of reproductive age. Is present in 10% of general population and infertility in 30% to 40% of cases. The degree of involvement of endometriosis is based on a points system proposed by the American Society for Reproductive Medicine (1978), based on the findings of laparoscopy. The action of estrogen is mediated by intracellular receptors that, with the binding of ligands are translocated to the nucleus where it activates gene transcription and REβ is one of the isoforms of these receptors. The REβ gene was mapped and located on the long arm of chromosome 14 in the loco 2 among subloco 23:24 (14q22-24) and the polymorphism consists of a silent G1082A transition in the binding domain of exon 5. The intent of this study was to determine the frequency of the RsaI polymorphism of the gene REβ in two groups of patients with endometriosis and without symptoms. The study included 54 samples of peripheral blood of women with endometriosis from a referral center in laparoscopy and infertility in the city of Goiania (FÉRTILE) and 46 peripheral blood samples from women without endometriosis clinic called the control group. Later the group with endometriosis were divided into two groups fertile (n = 25) and infertile (n = 27). The RsaI polymorphism of the gene REβ (AA, AG, GG) and the gene p53 in codon 72 was assessed using PCR. The frequency of heterozigous genotype AG was nine times higher in patients with endometriosis (59.3%) than in the control group (6.5%). The fertile patients (68%) had a frequency of AG 10.5 times higher than in the control group (6.5%) and infertile (55.6%) 8.5 times higher than in the control group. Social habits like smoking, alcohol, contraceptives and physical activity was not found a significant association. Of the patients with genotypes ArgPro/ProPro (51.5%) of the polimorphism of p53 gene the AG frequency was 8.6 times higher than in the control group (6%). We conclude that the AG frequency of the RsaI polymorphism of the gene REβ is associated with the presence of endometriosis. / A endometriose é definida pelo aparecimento de focos de tecido endometrial com características glandulares e/ou estromais idênticos aos da cavidade uterina em outras localizações, que não o endométrio. E esses focos de tecido geralmente são funcionantes e sensíveis à ação de hormônios apresentando um forte componente genético correlacionando a doença com diversos polimorfismos. Incide principalmente em mulheres em idade reprodutiva. Está presente em 10% na população geral e com infertilidade em 30% a 40% dos casos. O grau do comprometimento da endometriose baseia-se num sistema de pontos proposta pela American Society for Reproductive Medicine (1978), com base nos achados de laparoscopia. A ação estrogênica é mediada por receptores intracelulares, que com a ligação dos ligantes são translocados para o núcleo onde ativam a transcrição gênica e o REβ é uma das isoformas destes receptores. O gene do REβ foi mapeado e localizado no braço longo do cromossomo 14 no loco 2 entre os sublocos 22 e 24 (14q22-24) e o polimorfismo consiste em uma transição silenciosa G1082A no domínio de ligação do éxon 5. A intenção deste estudo foi determinar a freqüência do polimorfismo RsaI do gene REβ, em dois grupos de pacientes com endometriose e sem sintomas da doença. O estudo incluiu 54 amostras de sangue periférico de mulheres com endometriose proveniente de um centro de referência em videolaparoscopia e infertilidade de Goiânia (FÉRTILE) e 46 amostras de sangue periférico de mulheres sem clínica de endometriose chamado de grupo controle. Posteriormente o grupo com endometriose foi subdividido em dois grupos férteis (n= 25) e inférteis (n= 27). O polimorfismo RsaI do gene REβ (AA, AG, GG) e do gene p53 no códon 72 foi avaliado por PCR. A freqüência do genótipo heterozigoto AG foi 9 vezes maior nas pacientes com endometriose (59,3%) do que no grupo controle (6,5%). As pacientes férteis (68%) apresentaram uma freqüência AG de 10,5 vezes maior que no grupo controle (6,5%) e as inférteis (55,6%) 8,5 vezes maior que no grupo controle. Dos hábitos sociais como o fumo, álcool, anticoncepcionais e atividade física não foi encontrado uma associação significante. Das pacientes com genótipos ArgPro/ProPro (51,5%) do polimorfismo do gene p53 a frequência AG foi 8,6 vezes maior do que no grupo controle (6%). Conclui-se que, a freqüência AG do polimorfismo RsaI do gene REβ está associada a presença da endometriose.

Fan, Dahua. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 117-155). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Afin de mettre en évidence les principaux éléments de la voie androgénique et œstrogénique dans l’épididyme du rat des sables adulte, capturé dans la région de Beni Abbès, en Algérie, l’aromatase, l’œstradiol, les récepteurs des androgènes (RA) et des œstrogènes (REα, REβ, GPR30) ont été recherchés chez des animaux en saison d’activité, en saison de repos sexuel, chez des animaux castrés, castrés puis traités par la testostérone et chez des animaux ayant subi la ligature des canaux efférents. En saison d’activité, les RA sont ubiquitaires, l’aromatase est cytoplasmique par contre l’œstradiol est nucléaire et cytoplasmique. Les REα et le GPR30 sont principalement dans le cytoplasme apical par contre les REβ sont nucléaires. En saison de repos sexuel, les RA, l’aromatase, l’œstradiol, les REα et le GPR30 persistent, cependant, les REβ subissent une translocation cytoplasmique. Chez les animaux castrés, les RA, l’aromatase et l’œstradiol sont réduits par contre les REα persistent avec une faible intensité. Le GPR30 est cytoplasmique et nucléaire. Chez les animaux castrés puis traités, les RA, l’aromatase, l’œstradiol, les REα, les REβ et le GPR30 sont restaurés. Chez les animaux ligaturés, le RA est faiblement conservé uniquement dans l’épididyme proximal. L’aromatase et l’œstradiol sont conservés. Le signal des REα, des REβ et du GPR30 est fortement exprimé dans le noyau et le cytoplasme dans l’épididyme proximal par contre il est fortement exprimé uniquement pour les REα dans l’épididyme distal. Par western blot, les RA, REα, REβ et GPR30 sont de 122, 64, 55 et 55 kDa respectivement. / In order to highlight the main elements of androgen and estrogen pathway in the epididymis of sand rat, captured in Beni Abbès area, in Algeria, androgen receptor (AR), aromatase, estradiol, estrogen receptors (ERα, ERβ and GPR30) were explored in breeding season, in resting season and in animals underwent castration, castration then testosterone treatment and ligation of efferent ducts. In breeding season, AR has a ubiquitous distribution, aromatase is exclusively cytoplasmic and estradiol is nuclear and cytoplasmic. The ERα and GPR30 were distributed with a high intensity in the apical cytoplasm contrarily to ERβ which were nuclear. In resting season, AR, aromatase, estradiol, ERα persist with lower staining. However, ERβ undergo cytoplasmic translocation and GPR30 persist in cytoplasm. In castrated animals, AR, aromatase and estradiol are reduced. ERα persist with low intensity in the apical cytoplasm. GPR30 is distributed in the cytoplasm and the nucleus. In castrated then treated animals, AR is restored; aromatase and estradiol reappear with a cytoplasmic localization for aromatase, nuclear and apical for ERα. ERβ and GPR30 are restored and have a cytoplasmic localization. In ligatured, RA is preserved in the caput, aromatase and estradiol persist caput and cauda. The signal of ERα, ERβ and GPR30 is highly expressed in the nucleus and cytoplasm of caput epididymis and highly expressed of ERα exclusively in cauda. By Western blot, RA, ERα, ERβ and GPR30 are found with molecular weights of 122, 64, 55 and 55 kDa respectively.

Background: Estrogen receptor (ER)- and progesterone receptor (PR)-negative (ER-PR-) breast cancer is associated with higher grade and poorer prognosis compared with other breast cancer subtypes. High parity, coupled with lack of breastfeeding, has been associated with an increased risk of ER-PR- cancer. The mechanism of this etiology is unclear, and may be obfuscated by ER and PR correlation with each other as well as other prognostic tumor characteristics. Methods: Using population-based and clinic-based ascertained cases and controls from the Breast Cancer Family Registry, I examined reproductive risk factors, including parity, breastfeeding, and oral contraceptive (OC) use, in relation to ER and PR status, using polytomous logistic regression (for the population-based data) and the method of generalized estimating equations (GEE) (for the clinic-based data) as well as the pseudo-conditional likelihood approach, which accounts for correlated outcome variables. Results: High parity (≥ 3 live births) combined with lack of breastfeeding, was positively associated with ER-PR- tumors (odds ratio [OR]=1.57, 95% confidence interval [CI] 1.10-2.24, population-based cases vs. controls) relative to nulliparity. There was no association with ER-PR- tumors and parity in women who breastfed (OR=0.93, 95%CI 0.71-1.22) relative to nulliparous women. Associations with ER-PR- cancer were higher across all races/ethnicities among women who did not breastfeed compared with women who did. Population-based and clinic-based data were generally in agreement (OR=2.07, 95% CI 1.09-3.91, clinic-based cases vs. controls, relative to nulliparity). When adjusted for the correlation of PR-status and grade, to ER-status, the association between high parity +lack of breastfeeding and ER- status, was maintained. OC use before year 1975 was associated with an increased risk of ER-PR- tumors (OR=1.32, 95% CI 1.04-1.67, population-based data, cases vs. controls) relative to never use of OCs. For women who began OC use in 1975 or later there was no increased risk. Analysis of OC use in clinic-based data agreed with the findings of the population-based data. Conclusions: My findings support that there are modifiable factors for ER-PR- breast cancer, and that breastfeeding in particular may mitigate the increased risk of ER-PR-cancers seen from multiparity. The mechanism of both risk and risk mitigation may operate primarily through the estrogen, rather than progesterone, pathway.

骨科醫生面臨著老年婦女的骨修復受損或者癒合延遲的挑戰，這使得康復過程變長，甚至引發高死亡率。至今為止，臨床上仍然沒有促進老年骨癒合的滿意治療方法，因此亟需其他治療策略。骨癒合重現了胚胎後的骨骼發育過程。直接由骨外膜成骨（膜內骨化）以及通過軟骨介質成骨（軟骨內骨化）是骨癒合中的兩個重要過程。 雌激素受體β（ERβ基因敲除雌性小鼠的研究表明ERβ信號通路在骨骼發育過程中同時參與了抑制膜內骨化和軟骨內骨化這兩個過程。臨床活檢的資料顯示，在絶經後婦女的骨痂中，ERβ陽性的增生軟骨細胞數量增加。然而，ERβ在正常和老年骨癒合的作用還沒有研究。 / 本研究通過下述部分檢查了ERβ在正常和老年骨癒合的作用，以及其將來的藥物應用：1) 建立一個以膜內骨化為主的骨癒合模型。2) 通過連個骨癒合模型，檢查ERβ在正常骨癒合中的作用。3) 檢查ERβ在老年骨癒合中的作用並檢查ERβ拮抗劑PHTPP 對老年骨癒合的潛在藥物療效。 / 實驗1是建立一個以膜內骨化為主的骨癒合模型。以前建立的小鼠股骨中段骨折模型是軟骨內骨化為主的骨癒合模型。由於技術難度，該模型可重複性不高，而且其金屬內固定器會造成金屬偽影，進而不能應用高解析度微焦點CT跟蹤觀察的技術。為了檢查ERβ在膜內骨化中的作用，並且應用微焦點CT跟蹤觀察技術，我們首先建立了一個小鼠鑽孔缺損模型。該實驗同時也確認了去勢誘導的骨質疏鬆小鼠相比正常小鼠，在鑽孔缺損模型中骨癒合受阻。 / 實驗2檢驗了阻斷ERβ能促進正常骨癒合的假設。本實驗應用ERβ基因敲除小鼠，在兩個模型中檢驗了實驗假設。第一個是傳統的小鼠股骨中段骨折模型，第二個是由實驗1建立的鑽孔缺損模型。兩個模型都證實ERβ基因敲除小鼠骨癒合和野生型小鼠相比，早期的血管新生和中期的礦化有所增強，末期的骨癒合沒有明顯差異。 / 實驗3 進一步研究ERβ在老年骨癒合中的作用。實驗應用老年小鼠股骨中段骨折模型，比較ERβ基因敲除小鼠和野生型小鼠之間的癒合過程。結果顯示ERβ基因敲除小鼠骨癒合和野生型小鼠相比，早期的血管新生，中期的礦化以及末期的力學性能都有所增強。該結果預示阻斷ERβ能作為另一種治療老年骨折癒合的治療策略。同時，我們也檢測了ERβ的拮抗劑PHTPP（4 - [2 - 苯基- 5,7 -二（三氟甲基）吡唑並[1,5 - A]嘧啶3 - 基]苯酚, 在老年骨癒合中的治療效果。 通過比較用藥組小鼠與安慰劑組小鼠的骨癒合品質，顯示PHTPP治療小鼠血管新生，骨痂礦化和最終的力學性質均優於對照安慰劑組小鼠。 / 綜上所述，本研究描述了ERβ在正常和老年骨癒合中的作用。骨癒合的關鍵過程包括血管新生，膜內骨化以及軟骨內骨化在阻斷ERβ後都得到增強，從而加快正常骨和老年骨的骨痂形成，礦化並增強力學性質。ERβ的拮抗劑PHTPP在老年小鼠骨折模型中能促進骨癒合。本研究提出了一個新的骨癒合治療策略，並為將來的臨床實驗提供了堅實的基礎。 / Orthopaedic surgeons are challenged by impaired or delayed bone healing in elderly women, which requires prolongation of rehabilitation process or even induces high mortality. Up to date, there are no satisfactory therapeutic modalities for promoting aged bone healing clinically, and alternative therapeutic stratagem is therefore desirable. Bone healing recapitulates postnatal bone development. Direct periosteam-dependent bone formation (intramembranous ossification) and the formation of bone through a cartilage intermediate (endochondral ossification) are the two important processes during bone healing. Evidences from Estrogen Receptor β (ERβ), gene knockout female mouse studies have demonstrated that ERβ signaling participates in inhibiting both intramembranous and endochondral ossification during bone development. Clinical biopsy data demonstrated that the number of ERβ positive proliferative chondrocytes within fracture callus was increased in postmenopausal women. However, the role of ERβ in normal and aged bone healing is not examined yet. / This study examined role of ERβ in normal and aged bone healing and the future pharmaceutical application though the following part: 1) Establish an intramembranous ossification-dominant bone healing model. 2) Examine the role of ERβ in normal bone healing though two models. 3) Examine the role of ERβ in aged bone healing and investigate the potential therapeutical efficacy of an ERβ antagonist PHTPP in aged bone healing. / Study I was to establish an intramembranous ossification dominant bone healing mouse model. Previous available mouse femoral shaft fracture model was a endochondral ossification dominant bone healing model. This model was technically difficult to generate high reproducibility and the inside metal stabilization devices prevented the application of high-resolution in vivo micro-CT monitoring due to the metal artifact. In order to examine the role of ERβ in intramembranous ossification and apply the micro-CT monitoring technique, a drill-hole defect mouse model was developed. The study also confirmed bone healing was impaired in mice with ovariectomy -induced osteoporosis in drill-hole defect model. / Study II was to test the hypothesis that blockade of ERβ could promote normal bone healing. ERβ knockout mice were employed in this study and the hypothesis was examined in two models, the first is the traditional mouse femoral shaft fracture model, and the second is the drill-hole defect model that was developed in study I. Both models demonstrated that the bone healing in ERβ knockout mice was enhanced in the early stage of neovascularization and the middle stage of ossification but not by the end of healing compare to the wild type mice. / Study III was designed to further investigate the role of ERβ in aged bone healing. Femoral shaft fracture model was created in aged mice. The healing process was compared between the ERβ knockout mice and wild type mice. The results demonstrated that ERβ knockout mice was enhanced in the early stage of neovascularization, the middle stage of ossification and end stage of mechanical strength. The findings implied blockade of ERβ can be considered as another therapeutic strategy for aged fracture healing. PHTPP (4-[2-Phenyl-5,7-bis(trifluoromethyl)pyrazolo[1,5-a]pyrimidin-3-yl] phenol), an ERβ antagonist, was employed in aged mice femoral shaft fracture model. The bone healing quality of treated mice was compared with that of the vehicle control mice. It showed PHTPP treated mice had enhanced neovascularization, callus ossification and finally better mechanical properties than vehicle mice. / The present study depicted the role of ERβ in normal and aged bone healing. Key processes including neovascularization, intramembranous and endochondral ossification were all enhanced by blockade of ERβ, which led to fast callus formation, mineralization in normal bone and better mechanical properties in aged bone. ERβ antagonist PHTPP could promote aged bone healing in mouse osteotomy model. This study raised an alternative therapeutic stratagem for bone healing and provided solid basis for future clinical trials. / 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. / He, Yixin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 147-167). / 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 / 中文摘要 --- p.iv / PUBLICATIONS AND AWARDS --- p.vi / ACKNOWLEDGEMENTS --- p.xi / TABLE OF CONTENTS --- p.xii / LIST OF ABBREVIATIONS --- p.xvi / LIST OF FIGURES --- p.xviii / LIST OF TABLES --- p.xx / Chapter CHAPTER 1 --- INTRODUCTION AND LITERATURE REVIEW --- p.1 / Chapter 1.1 --- Fracture and Bone Healing --- p.2 / Chapter 1.1.1 --- Epidemiology and Impacts of Fractures --- p.2 / Chapter 1.1.2 --- Current Management and Limitations --- p.3 / Chapter 1.1.3 --- Bone Structures --- p.5 / Chapter 1.1.4 --- Bone Healing --- p.7 / Chapter 1.1.5 --- Aged Bone Healing --- p.12 / Chapter 1.1.6 --- Enhancements of Bone Healing --- p.17 / Chapter 1.2 --- Estrogen and Estrogen Receptors --- p.19 / Chapter 1.2.1 --- Estrogen Receptors α and β --- p.19 / Chapter 1.2.2 --- Molecular Actions of Estrogens --- p.20 / Chapter 1.2.3 --- Estrogen receptors in bone homeostasis --- p.24 / Chapter 1.3 --- Hypothesis --- p.28 / Chapter 1.4 --- Study Plan and Objectives --- p.32 / Chapter 1.4.1 --- Bone Healing Models --- p.32 / Chapter 1.4.2 --- Study Outline --- p.32 / Chapter 1.5 --- Figures and Tables --- p.34 / Chapter CHAPTER 2 --- ESTABLISHMENT OF DRILL-HOLE DEFECT HEALING MODEL IN MICE --- p.39 / Chapter 2.1 --- Introduction --- p.40 / Chapter 2.1.1 --- Limitations in currently available mouse models of osteoporotic bone healing --- p.40 / Chapter 2.1.2 --- Creation of a drill-hole defect at the mid-diaphysis of the femur for in vivo monitoring of bone healing in mice --- p.40 / Chapter 2.2 --- Materials and Methods --- p.43 / Chapter 2.2.1 --- Experimental animals --- p.43 / Chapter 2.2.2 --- Surgical protocol and experimental design --- p.43 / Chapter 2.2.3 --- Micro-CT analysis of intact femur --- p.44 / Chapter 2.2.4 --- In vivo micro-CT analysis of new bone formation in the drill-hole site --- p.45 / Chapter 2.2.5 --- Micro-CT-based angiography --- p.45 / Chapter 2.2.6 --- Histological examination --- p.46 / Chapter 2.2.7 --- Immunohistochemistry --- p.46 / Chapter 2.2.8 --- Quantitative real-time PCR --- p.47 / Chapter 2.2.9 --- Analysis of bone formation and resorption markers --- p.47 / Chapter 2.2.10 --- Mechanical testing --- p.48 / Chapter 2.2.11 --- Statistical analysis --- p.48 / Chapter 2.3 --- Results --- p.51 / Chapter 2.3.1 --- Confirmation of osteoporotic bone prior to generation of a drill-hole defect --- p.51 / Chapter 2.3.2 --- General observation of mice following drill-hole surgery --- p.51 / Chapter 2.3.3 --- In vivo micro-CT analysis of new bone in the drill-hole site of mouse femurs --- p.51 / Chapter 2.3.4 --- In vivo micro-CT analysis of new bone in drill-hole sites is highly reproducible --- p.52 / Chapter 2.3.5 --- Micro-CT angiography --- p.52 / Chapter 2.3.6 --- Histological observation of bone healing --- p.53 / Chapter 2.3.7 --- Immunohistochemical analysis of ER expressions during bone healing --- p.54 / Chapter 2.3.8 --- Quantitative real-time PCR analysis of gene expression during bone healing --- p.54 / Chapter 2.3.9 --- Analysis of bone formation and resorption markers during bone healing --- p.54 / Chapter 2.3.10 --- Mechanical testing of femurs from Sham and OVX mice --- p.55 / Chapter 2.4 --- Discussion --- p.56 / Chapter 2.4.1 --- Bone healing with dominant intramembranous ossification --- p.56 / Chapter 2.4.2 --- Impaired osteoporotic bone healing --- p.57 / Chapter 2.4.3 --- Reproducibility of the in vivo micro-CT method for analysis of bone healing --- p.58 / Chapter 2.4.4 --- Dysregulated expression of estrogen receptors and bone healing in OVX mice --- p.59 / Chapter 2.4.5 --- Study limitations --- p.60 / Chapter 2.4.6 --- Conclusions --- p.60 / Chapter 2.5 --- Figures and Tables --- p.61 / Chapter CHAPTER 3 --- ROLE OF ERβ IN NORMAL BONE HEALING --- p.72 / Chapter 3.1 --- Introduction --- p.73 / Chapter 3.2 --- Materials and Methods --- p.75 / Chapter 3.2.1 --- Part I Study --- p.75 / Chapter 3.2.1.1 --- Experimental animals --- p.75 / Chapter 3.2.1.2 --- Fracture model and experimental design --- p.75 / Chapter 3.2.1.3 --- Radiographic Analysis --- p.76 / Chapter 3.2.1.4 --- Micro-CT-based angiography --- p.76 / Chapter 3.2.1.5 --- Micro-CT analysis of callus --- p.77 / Chapter 3.2.1.6 --- Histological examination --- p.78 / Chapter 3.2.1.7 --- Dynamic Bone histomorphometric analysis --- p.78 / Chapter 3.2.1.8 --- Mechanical testing --- p.79 / Chapter 3.2.1.9 --- Quantitative real-time PCR --- p.80 / Chapter 3.2.1.10 --- Analysis of bone formation and resorption markers --- p.80 / Chapter 3.2.1.11 --- Statistical analysis --- p.81 / Chapter 3.2.2 --- Part II Study --- p.81 / Chapter 3.2.2.1 --- Experimental animals and design --- p.81 / Chapter 3.2.2.2 --- Evaluation protocols --- p.82 / Chapter 3.2.2.3 --- Statistical analysis --- p.82 / Chapter 3.3 --- Results --- p.83 / Chapter 3.3.1 --- Part I Study --- p.83 / Chapter 3.3.1.1 --- Radiographic Analysis --- p.83 / Chapter 3.3.1.2 --- Micro-CT angiography --- p.83 / Chapter 3.3.1.3 --- Micro-CT analysis of callus --- p.83 / Chapter 3.3.1.4 --- Histological and dynamic histomorphometric analysis --- p.84 / Chapter 3.3.1.5 --- Mechanical testing of the callus --- p.85 / Chapter 3.3.1.6 --- Quantitative real-time PCR analysis of gene expression --- p.85 / Chapter 3.3.1.7 --- Analysis of bone formation and resorption markers during bone healing --- p.85 / Chapter 3.3.2 --- Part II Study --- p.86 / Chapter 3.3.2.1 --- In vivo micro-CT analysis of new bone in the drill-hole site of mouse femurs --- p.86 / Chapter 3.3.2.2 --- Micro-CT angiography --- p.87 / Chapter 3.3.2.3 --- Histological observation of bone healing --- p.87 / Chapter 3.3.2.4 --- Quantitative real-time PCR analysis of gene expression --- p.88 / Chapter 3.3.2.5 --- Analysis of bone formation and resorption markers during bone healing --- p.88 / Chapter 3.3.2.6 --- Mechanical testing of femurs from WT and KO mice --- p.88 / Chapter 3.4 --- Discussion --- p.90 / Chapter 3.4.1 --- Angiogenesis --- p.90 / Chapter 3.4.2 --- Fracture Healing --- p.91 / Chapter 3.4.3 --- Estrogen receptor β and endochondral and intramembranous ossification --- p.93 / Chapter 3.4.4 --- Estrogen receptor β in aged bone --- p.94 / Chapter 3.4.5 --- Conclusions --- p.94 / Chapter 3.5 --- Figures and Tables --- p.95 / Chapter CHAPTER 4 --- ROLE OF ERβ AND ITS ANTAGONIST PHTPP IN AGED BONE HEALING --- p.113 / Chapter 4.1 --- Introduction --- p.114 / Chapter 4.2 --- Materials and Methods --- p.116 / Chapter 4.2.1 --- Experimental animals --- p.116 / Chapter 4.2.2 --- Fracture model and experimental design --- p.116 / Chapter 4.2.3 --- Radiographic Analysis --- p.117 / Chapter 4.2.4 --- Micro-CT-based angiography --- p.118 / Chapter 4.2.5 --- Micro-CT analysis of callus --- p.118 / Chapter 4.2.6 --- Histological examination --- p.119 / Chapter 4.2.7 --- Dynamic Bone histomorphometric analysis --- p.120 / Chapter 4.2.8 --- Mechanical testing --- p.120 / Chapter 4.2.9 --- Quantitative real-time PCR --- p.121 / Chapter 4.2.10 --- Analysis of bone formation and resorption markers --- p.122 / Chapter 4.2.11 --- Statistical analysis --- p.122 / Chapter 4.3 --- Results --- p.123 / Chapter 4.3.1 --- Radiographic Analysis --- p.123 / Chapter 4.3.2 --- Micro-CT angiography --- p.123 / Chapter 4.3.3 --- Micro-CT analysis of callus --- p.123 / Chapter 4.3.4 --- Histological and dynamic histomorphometric analysis --- p.124 / Chapter 4.3.5 --- Mechanical testing of the callus --- p.125 / Chapter 4.3.6 --- Quantitative real-time PCR analysis of gene expression during fracture healing --- p.125 / Chapter 4.3.7 --- Analysis of bone formation and resorption markers during bone healing --- p.126 / Chapter 4.4 --- Discussion --- p.127 / Chapter 4.4.1 --- Angiogenesis --- p.127 / Chapter 4.4.2 --- Fracture Healing --- p.128 / Chapter 4.4.3 --- Estrogen receptor β and endochondral ossification --- p.129 / Chapter 4.4.4 --- ERβ antagonist PHTPP --- p.130 / Chapter 4.4.5 --- Conclusions --- p.130 / Chapter 4.5 --- Figures and Tables --- p.131 / Chapter CHAPTER 5 --- STUDY LINITATIONS, FURTHER RESEARCH AND CONCLUDSIONS --- p.142 / Chapter 5.1 --- Limitations --- p.143 / Chapter 5.1.1 --- Bone healing model --- p.143 / Chapter 5.1.2 --- Estrogen receptors and transgenic mouse --- p.143 / Chapter 5.1.3 --- ERβ antagonist PHTPP --- p.144 / Chapter 5.2 --- Further Research --- p.144 / Chapter 5.2.1 --- ERβ signaling --- p.144 / Chapter 5.2.2 --- Preclinical Trial --- p.145 / Chapter 5.3 --- Conclusions --- p.146 / BIBLIOGRAPHY --- p.147

In order to further investigate the functions of ERRbeta/gamma in prostate cancer, the following aspects were explored in my study. My results show that: (1) Expressions of ERRbeta/gamma was down-regulated in prostate cancer cell lines and prostate cancer tissues. And only the short-form but not other ERRbeta isoforms was expressed in prostatic cells. (2) Overexpression of ERRbeta/gamma significantly inhibited the cell proliferation in vivo and in vitro. (3) Cell cycle analysis showed that S-phase fraction of ERRbeta/gamma stable clones was significantly decreased, while there was no significantly induced apoptosis by ERRbeta/gamma overexpression. This was confirmed by BrdU incorporation assay. (4) Expressions of two cyclin-CDK inhibitors p21Cip1/Waf1 and p27Kip1 were increased significantly in ERRgamma clones, but only p21 in ERRbeta clones. (5) P21 and p27 gene promoters could be transactived by ERRgamma, but only p21 by ERRbeta. The transactivity of p21 by ERRbeta can be potently enhanced by PGC-1alpha (6) Deletion mutants of ERRgamma showed the transaction of p21 required an intact DNA-binding domain. (7) DY131, the ERRbeta/gamma agonist, further potentiated the growth inhibition in ERRbeta/gamma-stable clones in a dose-dependent manner. (8) There were increase in number of giant potential-active mitochondria and accumulation of lipid droplets in ERRbeta-clones. / Prostate cancer is the most common diagnosed cancers in men in western countries. Despite the substantial clinical significance, the mechanisms underlying the development and progression of prostate cancer are poorly understood. ERRs(alpha, beta, gamma) belong to orphan nuclear. All ERR subtypes share significant homology with estrogen receptors (ERs) in their protein structures. Functionally ERRalpha shares, regulates same target genes with ERalpha and is involved in carcinogenesis, while the ERRbeta and ERRgamma are still unknown. / The results obtained indicate that ERRbeta/gamma inhibit proliferation of prostate cancer cells by arresting of cell cycle progression, suggesting a tumor suppressor function for ERRbeta/gamma in prostate cancer. p21 may be the key mediator of this suppressor function, and the p21 is the target gene of ERRbeta/gamma. The selective ERRbeta/gamma agonist, DY131, potently inhibited the proliferation of ERRbeta/gamma-positive prostate cancer cells, suggesting ERRbeta and gamma could be a potential therapeutic target for prostate cancer therapy. / Yu, Shan. / "July 2007." / Adviser: Franky L. Chan. / Source: Dissertation Abstracts International, Volume: 69-01, Section: B, page: 0238. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (p. 140-165). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Maternal behaviours are those that increase offspring survival. Estrogens affect maternal behaviour by activating Estrogen Receptors (ER) in the brain. Maternal brain plasticity was explored by characterizing the effects of exposure to a mixture of environmental pollutants on number of ERs. Following exposure to the toxicants during pregnancy and lactation, brains of female rats were collected, sectioned at 30 μm and immunohistochemistry for ERα performed. Immuno-positive cells in the mPOA, VTA and NAc were counted. A two way ANOVA revealed no main effect of Treatment on the number of immunopositive cells for all three brain regions. However, a significant difference between the High and Low Doses with the high dose reducing the number of ERα+ cells in the mPOA and VTA. Our work showcases the importance of studying the effects of multiple chemical co-exposures on the mother's brain, as maternal brain changes impact maternal behaviour consequently affecting offspring neurodevelopment.

RESUMO A similaridade entre o tecido da mama e o da glândula salivar está bem estabelecida. A porção das estruturas acinares e ductais destes órgãos são basicamente semelhantes. Estes aspectos, associados ao fato de que uma coexistência de carcinomas da mama e de glândula salivar, têm sido relatados em uma incidência maior do que a esperada. Guiaram estudos tentando determinar a importância dos receptores de estrógeno e progesterona em adenomas pleomórficos (AP). A neoplasia é mais freqüente nas glândulas salivares e exibe uma predileção para o sexo feminino. Recentemente a presença do receptor de estrógeno (RE) e do receptor de progesterona (RP) tem sido investigada no AP, entre outras neoplasias de glândula salivar, questionando-se a possibilidade da existência da dependência hormonal. A expressão dos receptores hormonais nos carcinomas de mama é importante para determinar o prognóstico e a probabilidade de responder à manipulação hormonal. Neoplasias que apresentam positividade para ambos os receptores, estrógeno e progesterona, exibem maior probabilidade de resposta à terapia anti-estrogênica do que as neoplasias que são negativas para estes receptores. Baseando-se na literatura científica pertinente, o presente trabalho se propõe a investigar a presença da proteína RE e da proteína RP em APs humanos, relacionando-os com a proliferação de linhagens celulares sob a influência destes hormônios. A técnica utilizada foi a imuno-histoquímica para a pesquisa dos RE e RP em 10 APs emblocados em parafina pertencentes ao arquivo do Serviço de Patologia Cirúrgica da FOUSP, e de duas linhagens de APs estabelecidas no mesmo serviço: uma derivada de um paciente do sexo masculino e a outra de um paciente do sexo feminino. No meio de cultivo onde subculturas destas células proliferavam foram diluídos 17-b-estradiol e progesterona. Através de contagens destas células em períodos pré-determinados (24 horas, 48 horas e 72 horas), pretendeu-se verificar a influência dos respectivos hormônios na multiplicação celular. Como controle positivo utilizou-se uma linhagem denominada T-47D, que foi largamente estudada na literatura. A T-47D é derivada de um carcinoma metastático de mama, reconhecidamente hormônio dependente. E como controle negativo, utilizou-se de uma linhagem de carcinoma epidermóide, denominada HN30. Encontrou-se positividade para o RE em 7 de 10 APs estudados (4 em homens e 3 em mulheres) e positividade para o RP em 8 Aps estudados (4 em mulheres e 4 em homens). Pela análise estatística, constatou-se que existe uma diferença significativa no índice proliferativo entre o controle e as células submetidas à ação do 17-b-estradiol e da progesterona. Para a linhagem derivada do paciente do sexo masculino houve diferença entre o controle e as células expostas ao 17-b-estradiol e a progesterona somente nas últimas 72 horas. Para a linhagem derivada do sexo feminino constatou-se uma diferença significativa entre o controle e as células sob a influência da progesterona, a partir de 48 horas de proliferação celular. A diferença significativa entre o controle e as células sob a ação do 17-b-estradiol ocorreu somente a partir das 72 horas, sugerindo que o AP poderia ser uma neoplasia influenciada pela ação hormonal. / SUMARY It is well established the similarity between mammary and salivary glands especially between the acinic and ductile structures. These aspects, associated to the fact of coexistence of breast carcinomas and of salivary gland tumors been described, leaded studies in attempt to determine the importance of the ERs and Pr in pleomorphic adenomas (PA), the most frequent salivary gland tumor and with predilection for the females. Lately, the presence of ERs and of the PRs has been investigated in PA and other salivary gland tumors pointing out their hormonal dependency. The expression of hormone receptors in breast carcinomas is crucial to determine a presence for both receptors. These tumors exhibit better response to anti-estrogenic therapy than the negative ones. Basing on the pertinent scientific literature, the present study proposes to investigate the presence of the RE and of the RP in humans PA and connecting them with cellular proliferation in vitro, under the influence of these hormones. Immunohistochemistry technique was used for the detection of RE and RP in paraffin embedded 10 PAs from the files of the Department of Oral Pathology, School of Dentistry, University of São Paulo, and two PA cell lines one from a male patient and other female. The culture midia was supplied with, 17-b-estradiol and progesterone. A growth curve was performed (24 hours, 48 hours and 72 hours) to verify the influence of the respective hormones in the cellular proliferation. As a positive control T-47-D cells derived from a hormone dependent metastatic breast carcinoma were used, and as negative control HN30 cells, derived from a tongue squamous cell carcinoma. 7 of 10 PAs were positive (4 in men and 3 in women) for RP and 8 of 8 PAs (4 in women and 4 in men) for RE. The statistical analysis verified a significant difference in the proliferative index between the control cells and the ones submitted to the action of the 17-b-estradiol and of the progesterone: for male derived lineage a difference was only observed in the last 72 hours. In the other hand, for the female derived lineage a significant difference was verified starting from 48 hours, suggesting that PA can be influenced by hormonal action.