Indiana University-Purdue University Indianapolis (IUPUI) / Breast cancer remains the most prevalent malignancy diagnosed in women. More than two thirds of all diagnosed breast cancers are estrogen receptor (ER)-positive and are dependent on estrogen signaling. Drugs for the treatment of ER-positive breast cancer can be divided into three classes: selective estrogen receptor modulators (SERMs), selective estrogen receptor down-regulators (SERDs) and aromatase inhibitors (AIs). However, the efficacy and safety of SERMs, SERDs and AIs are compromised by side effects or tumor resistance. One possible way of improving treatment efficacy and safety profiles is to develop agents with dual aromatase inhibitory and ER modulatory activity. Over the past 30 years, tamoxifen, a SERM, has become the most widely used drug for the adjuvant treatment of breast cancer. The metabolism of tamoxifen has a complex profile involving both active and inactive metabolites, among which endoxifen, 4-hydroxytamoxifen (4-HT) and norendoxifen (Nor) have been shown to have ER modulatory activity. Previous studies have also shown that norendoxifen is a potent AI in vitro. These preliminary studies support the utilization of tamoxifen metabolites as lead compounds for the development of dual AI/SERM(D) agents. Hydroxynorendoxifen (Hdn) was identified as a novel tamoxifen metabolite, with an average plasma concentration of 0.82 nM. Nor and Hdn were potent and relatively selective AIs, with Kis of 70 nM and 20 nM, respectively. Nor and Hdn have high binding affinity for ER-α and ER-β, with EC50 values less than 35 nM. Nor and Hdn can inhibit breast cancer cell proliferation with high potency, with IG50s of 25 nM and 9 nM, respectively. Nor and Hdn can suppress progesterone receptor (PGR) mRNA expression level by reducing it by 68% and 86%. Moreover, a series of Nor analogues were shown to have both potent aromatase inhibitory activity and high ERs binding affinity. Results from this dissertation will contribute to three aspects: 1) the identification of Hdn as a tamoxifen metabolite illustrated a more comprehensive metabolism profile of tamoxifen; 2) the data suggest Nor and Hdn possess dual aromatase inhibitory and ER antagonistic activity; 3) a series of Nor analogues were characterized as lead compounds for the development of dual AI/SERM(D) agents.

No description available.

Age-related declines of androgens and libido in males have been observed for decades. This dissertation sought to elucidate the mechanisms by which hormones may act differentially upon their receptors in the hypothalamus of aging compared to young males. I also examined how sexual experience modulates the ability of hormones to facilitate sexual behavior with aging. Experiment one measured androgen receptors (AR) and estrogen receptor α (ERα) cells in male rats at young, middle-aged and old age. I found that AR cell numbers in hypothalamic regions studied underwent significant age-related increases. Numbers of heavily ERα labeled cells, but not total ERα cells, increased with age. This study demonstrates that the aging brain has the capacity to synthesize hormone receptors which is increased possibly due to decreased testosterone concentrations. Experiment two examined the effect of sexual experience on serum hormones and cells of AR and ERα in hypothalamic regions in young and middle-aged males. The results showed that AR cell numbers increased with aging but did not change with experience. No age- or experience-related alteration in ERα expression occurred. However, serum testosterone increased and estradiol decreased with age. Experience increased total and free testosterone. Interactions of age and experience on total testosterone, estradiol, and luteinizing hormone were found. These results show long-lasting effects of sexual experience on hormones, but not on their receptors in the hypothalamus. Experiment three investigated effects of exogenous testosterone on sexual behavior in young and middle-aged males. The results showed a decline in sexual behavior parameters with age. After castration with testosterone treatment, there were few differences in sexual behavior measures between young and middle-aged males. AR cell numbers were higher and ERα cell numbers lower in testosterone compared to vehicle-treated males of both ages, and few effects of age occurred. These findings indicate that testosterone and aging interact in a complex manner to control numbers of cells expressing hormone receptors in the brain and on the subsequent control of sexual behavior. This insight provides a better understanding of the relationship between molecular changes in the brain and behavior, and suggests new therapeutic targets to human testosterone treatment. / text

Plusieurs éléments de la pathogenèse de la scoliose idiopathique de l’adolescent indiquent que les estrogènes pourraient intervenir dans le développement et la progression de cette maladie. Ce projet avait donc pour but d’explorer l’expression et la fonctionnalité des récepteurs aux estrogènes ERα et ERβ ainsi que leurs isoformes dans les ostéoblastes de patients scoliotiques et sains. L’induction des gènes de facteurs influençant la minéralisation et la différenciation des ostéoblastes par les estrogènes a également été étudié. Par immunofluorescence, nous avons remarqué une augmentation de la présence protéique de ERβ dans les ostéoblastes de patients SIA comparé aux sujets contrôles. Les récepteurs aux estrogènes provenant des ostéoblastes des patients sont fonctionnels tout comme ceux des contrôles et aucune différence dans l'interaction ADN-protéine n’a été observée. Il y a également une augmentation de l’expression génique de l’ostéopontine, l’ostéocalcine, le collagène de type I, la phosphatase alkaline et BMP2 dans les ostéoblastes des patients SIA. Un début de minéralisation in vitro a été observé dans les ostéoblastes de patients SIA et contrôles. ERα et ERβ sont présents et fonctionnels dans les ostéoblastes des patients SIA et sains. Leur expression est variable, mais ces variations existent chez les patients SIA et les contrôles. L’implication des estrogènes dans la SIA ne serait donc pas au niveau des récepteurs aux estrogènes mais au niveau de l’interactions des estrogènes avec d’autres facteurs étiologiques tels que la mélatonine, la formation/résorption osseuse ou autres facteurs neuro-endocriniens. / Recent developments in the research on pathogenesis of Adolescent Idiopathic Scoliosis indicate that estrogens could intervene in the development and progression of this disease. Therefore, this project focussed on the expression and functionality of estrogen receptors ERα and ERβ and their isoforms in osteoblasts of scoliotic patients and controls. The induction by estrogens of the expression of factors influencing mineralization and osteoblast differentiation was also studied. We observed by immunofluorescence, an increase of the presence of ERβ in osteoblasts of AIS patients compared to controls. A higher level of expression of osteopontin, osteocalcin, type I collagen, alkaline phosphatase and BMP2 was found in osteoblasts of AIS patients compared to controls. Estrogen receptors in osteoblasts from AIS patients are functional, as they were in controls and no difference in DNA-binding activity was observed. ERα et ERβ are present and functional in osteoblasts from AIS patients and controls. Their expression is variable, but these variations exist in both populations. The implication of estrogens in AIS would therefore be in the interaction between these hormones and other factors influencing the aetiology of AIS like melatonin, bone formation and resorption and neuro-endocrin factors, rather than in a default in the estrogens receptors themselves.

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.

O Bisfenol A (BPA) é usado na fabricação de plásticos de policarbonato e resinas epóxi. A exposição pré-natal a esse agente pode causar diversos efeitos, tais como: antecipação da puberdade, hiperplasia de próstata, diminuição do número de espermatozoides, diminuição dos níveis de testosterona, alteração do desenvolvimento e organização tecidual da glândula mamária, diminuição da resposta celular induzida por hormônios, câncer de mama, diabetes, doenças cardiovasculares, alterações das funções de enzimas hepáticas, além de efeitos sobre o desenvolvimento cognitivo. Poucos estudos avaliam os efeitos do BPA sobre as células neuronais, porém existem evidências de que este agente induza a apoptose. O presente trabalho tem como objetivo estudar a neurotoxicidade do BPA, avaliando vias de sinalização que levam a indução da apoptose em cultura primária de hipocampo. As células foram expostas ao BPA nas concentrações de 50, 100, 150, 200, e 250 µM (0,1% DMSO v/v) pelos períodos de 6, 12, 24, e 48 horas para a realização dos ensaios da atividade mitocondrial (MTT) e citotoxicidade pela liberação da enzima Lactato Desidrogenase (LDH). A partir dos resultados de MTT e LDH, foram adotados novos horários de exposição (3, 6 e 9 horas) utilizando somente as concentrações de 200 e 250 µM. Neste novo desenho experimental, foi realizada a quantificação da concentração de BPA na cultura primária por HPLC-PDA, determinação da concentração de Ca2+ intracelular pela quantificação da fluorescência do Fluo-4 AM, caracterização dos mecanismos envolvidos na morte celular por citometria de fluxo e Western Blotting, e avaliação dos receptores de estrógeno ER-α e ER-β por Western Blotting. Nossos resultados apontam que aproximadamente 20% de BPA na concentração de 250 µM após 6 horas de exposição e 18% para a concentração de 200 µM com 9 horas de exposição foram absorvidos pela cultura celular. O ensaio do MTT mostrou que as células expostas a 200 e 250 µM de BPA, por 12, 24 e 48 horas, apresentaram diminuição significativa da função mitocondrial em relação ao controle. Porém, não houve liberação de LDH para o meio de cultura para nenhuma das concentrações de BPA em nenhum dos períodos de incubação, o que sugere que não houve rompimento da membrana plasmática. Foi observada atividade apoptótica somente com a concentração de 250 µM no período de exposição de 6 horas por citometria de fluxo. Não foram encontradas células em necrose, nem alteração na concentração de cálcio intracelular em nenhuma das condições estudadas. Na avaliação dos marcadores de morte celular, observamos aumento da razão de Bax/Bcl-2 para a concentração de 250 µM em todos os períodos de exposição e aumento das caspases 8, 9 e 3 para a concentração de 250 µM no período de exposição de 6 horas, indicando que o BPA deve ativar tanto a via intrínseca como a extrínseca no processo de apoptose. Verificamos ainda, por Western Blotting, que a cultura primária de hipocampo apresenta os receptores de estrógeno ER-α e ER-β. A exposição ao BPA aumentou os ER-α e ER-β avaliados por Western Blotting para as duas concentrações estudadas no período de 6 horas de exposição e, para o período de exposição de 9 horas, houve um aumento do ER-α para a concentração de 250 µM e do ER-β para a concentração de 200 µM. É possível concluir que o BPA pode levar a morte das células neuronais hipocampais por apoptose por ambas as vias intrínseca e extrínseca, sendo o processo de morte celular mais evidente para a concentração de 250 µM no período de 6 horas de exposição. Sugerimos ainda que o aumento observado em ambos os receptores de estrógeno possa representar uma tentativa de interrupção ou reversão do processo de morte celular. / Bisphenol A (BPA) is used in the manufacture of polycarbonate plastics and epoxy resins. The prenatal exposure to this agent may cause several effects, such as anticipation of puberty, prostate hyperplasia, reduced number of sperm, reduced testosterone levels, alteration in the development and tissue organization of the mammary gland, decreased cellular response induced by hormones, breast cancer, diabetes, cardiovascular disease, changes in the functions of liver enzymes, and effects on cognitive development. Few studies have evaluated the effects of BPA in neuronal cells, however there are evidences that this agent may induce apoptosis. This work aims to study the neurotoxicity of BPA, by analyzing the signaling pathways of apoptosis in hippocampus primary culture. Cells were exposed to BPA at 50, 100, 150, 200, and 250 µM (0.1% DMSO v/v) for 6, 12, 24, and 48 hours for the assay of mitochondrial activity (MTT) and the release of the enzyme lactate dehydrogenase (LDH). From the results of MTT and LDH, new exposure times (3, 6 and 9 hours) and only 200 and 250 µM were used. In this new experimental design we performed the quantification of the BPA concentration in the primary culture by HPLC-PDA, intracellular Ca2+ quantification by Fluo-4 AM assay and the characterization of the mechanisms involved in cell death by flow cytometry and Western Blotting assays. Furthermore, evaluation of the estrogen receptor ER-α and ER-β was done by Western Blotting. Our results demonstrate that about 20% of the BPA concentration of 250 µM after 6 hours of exposure and 18% for the concentration of 200 µM with 9 hours of exposure were absorbed by the cell culture. Cells exposed to 200 and 250 µM of BPA for 12, 24 and 48 hours, showed a significant decrease in mitochondrial function, by the MTT assay, compared to control. However, there was no release of LDH into the culture medium for any of the BPA concentrations in any of incubation times studied, which suggests no rupture of the plasma membrane by BPA. Apoptotic activity was observed after 6 hours of exposure to 250µM BPA by flow cytometry. It was not observed cell necrosis and changes in intracellular calcium concentration in any of the studied conditions. Regarding the cell death markers, exposure to 250 µM BPA in all periods of exposure resulted in an increased Bax/Bcl-2 ratio; moreover, an increase in caspase 8, 9 and 3 was detected after exposure to 250 µM BPA for 6 hours. Taken together, these findings indicate that BPA activates both the intrinsic and the extrinsic pathway during the apoptotic process. We also verified by Western Blotting the presence of the estrogen receptors ER-α and ER-β at the primary culture of hippocampus, and that they can be modulated by BPA. The exposure to 200 and 250 µM BPA for 6 hours caused an increase of ER-α and ER-β, however, 9 hours of exposure to 200 µM and 250 µM BPA increased the expression of ER-α and ER-β, respectively. In conclusion, BPA can lead hippocampal neuronal cells to death by both, intrinsic and extrinsic, apoptotic pathways and this process is more evident at 250 µM BPA after 6 hours of exposure. Furthermore, we suggest that the increase of both estrogen receptors might represent an attempt to interrupt or reverse the cell death process.

O câncer de mama corresponde a aproximadamente um quarto das neoplasias malignas em mulheres. A incidência do câncer de mama no Brasil é de cerca de 50.000 novos casos por ano, sendo considerado importante problema de saúde pública. HER2 e receptores hormonais (receptores de estrógeno e progesterona) são considerados os mais importantes marcadores prognósticos e preditivos em carcinoma de mama. A amplificação do gene HER2 ou a superexpressão da proteína HER2, que ocorre em cerca de 20 porcento dos carcinomas de mama, está associada a curso clínico mais agressivo e determina elegibilidade para terapia específica anti-HER2 com trastuzumabe. A terapia hormonal reduz em mais de 50 porcento o risco relativo de recorrência da doença em pacientes com tumores sensíveis a esse tratamento. Os testes de HER2 e receptores hormonais são partes essenciais da avaliação clínica das pacientes com carcinoma de mama; resultados precisos são fundamentais na identificação de pacientes que podem ser beneficiadas por terapias específicas. O presente estudo investigou a concordância nos resultados dos testes de HER2 e receptores hormonais determinados por imuno-histoquímica em 500 carcinomas invasivos de mama entre um laboratório referência e laboratórios locais de todas as regiões geográficas do Brasil. Os resultados demonstram baixa concordância geral (171/500 casos, 34,2 porcento) em relação aos resultados do teste de HER2 entre laboratórios locais e referência, o que pode estar relacionado ao baixo volume de testes de HER2 realizados, inexperiência com o sistema de escores de HER2 e/ou questões técnicas relacionadas à imuno-histoquímica nos laboratórios locais. A concordância nos resultados do teste de receptores de estrógeno e progesterona foi de 89,4 porcento (447/500 casos) e de 85,0 porcento (425/500 casos), respectivamente, entre laboratórios locais e referência. Padronização dos testes de HER2 e receptores hormonais com medidas de controle de qualidade rigorosas por laboratórios locais é fortemente recomendada para se evitar o tratamento inadequado de pacientes com câncer de mama / Breast cancer accounts for approximately one quarter of all cancers in females. The incidence of breast cancer in Brazil is about 50,000 new cases per year, and it is considered an important public health problem. HER2 and hormone receptors (estrogen and progesterone receptors) are considered the main prognostic and predictive markers for breast carcinoma. HER2 gene amplification or HER2 protein overexpression, detected in about 20 percent of breast carcinomas, predicts a more aggressive clinical course and determines eligibility for targeted therapy with trastuzumab. Hormonal therapy reduces the relative risk of recurrence by more than 50% in breast cancer patients with hormone-sensitive tumors. HER2 and hormone receptors testing has become an essential part of the clinical evaluation of all breast carcinoma patients, and accurate results are critical in identifying patients who may benefit from targeted therapy. The present study investigated the concordance in the results of HER2 and hormone receptors immunohistochemistry assays performed in 500 invasive breast carcinomas between a reference laboratory and local laboratories from all geographic regions of Brazil. Our results showed an overall poor concordance (171/500 cases, 34.2 percent) regarding HER2 results between local and reference laboratories, which may be related to the low-volume load of HER2 assays, inexperience with HER2 scoring system, and/or technical issues related to immunohistochemistry in local laboratories. The concordance of estrogen and progesterone receptors results was 89.4 percent (447/500 cases) and 85.0 percent (425/500 cases), respectively, between local and reference laboratories. Standardization of HER2 and hormone receptors testing with rigorous quality control measures by local laboratories is highly recommended in order to avoid erroneous treatment of breast cancer patients

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

Over 200,000 cases of breast cancer are diagnosed every year. Nearly 20% of these patients supplement their diets with some form of vitamin D. This high frequency of vitamin D supplement use may be due in part to research suggesting that cancer patients with higher serum vitamin D3 levels have better prognoses than patients with low serum vitamin D3. However, double-blind clinical trials on the efficacy of vitamin D3 supplementation in breast cancer have been inconclusive. A recent meta-analysis showed evidence of reduced cancer recurrence in patients taking vitamin D3 supplements who had ‘estrogen receptor positive’ (ERα66+) breast cancer, but not those who had estrogen receptor negative’ (ERα66-) breast cancer. Once ingested, vitamin D3 is metabolized in the liver into the circulating pre-hormone 25(OH)D3, which is then further metabolized into 1a,25(OH)2D3 and 24R,25(OH)2D3. 24R,25(OH)2D3 has been shown to activate a number of membrane signaling pathways, some of which overlap with 17b-estradiol (E2) signaling through ERα36, a membrane isoform of ERα66. The central hypothesis of this thesis was that 24R,25(OH)2D3 is tumorigenic in certain cancers and that this tumorigenicity is mediated in part by ERa isoforms. E2 signaling through ERa36 has been described in the ERa66-, ERa36+ breast cancer cell line HCC381. Specific aim 1 determined whether E2 signaling through ERa36 was tumorigenic other cancers with different ERa profiles. Specific aim 2 determined how 24R,25(OH)2D3 affected tumorigenicity in breast cancer using the common breast cancer cell line MCF7 (ERa66+, ERa36+) as a model. Specific aim 3 investigated the role of ERa isoforms in 24R,25(OH)2D3 signaling in breast cancer cell lines by comparing the tumorigenic effects of 24R,25(OH)2D3 in MCF7 cells (ERa66+, ERa36+) and HCC38 cells (ERa66-, ERa36+). To determine whether ERa66 regulates the effects of 24R,25(OH)2D3, ERa66 was expressed in two ERα66- cell lines. The effect of 24R,25(OH)2D3 on apoptosis was assessed in wild-type and ERa-expressing cell lines.

Billions of pounds of bisphenol A (BPA) are produced annually around the globe for the manufacture of numerous consumer products, including polycarbonate food and water containers, the protective resin linings of food cans, thermal printing paper, and dental fillings. BPA exposure during nervous system development has been associated with learning and behavioral impairments in animal models. The mode of action for these effects is not clearly defined. While BPA is a weak estrogen receptor (ER) agonist, it is also an estrogen-related receptor gamma (ERR��) agonist. ERR�� binds BPA with 100 times greater affinity than ERs. This study was designed to test the hypothesis that exposure to human-relevant BPA concentrations impacts nervous system development and behavior through ERR�� activation. To examine whether BPA behaves more like an ER or ERR�� ligand, two positive control compounds were used throughout the study: 17��-estradiol (E2) and GSK4716, ER and ERR�� agonists, respectively. Initial behavior testing results included the observation that neurodevelopmental exposure to 0.01 or 0.1 ��M BPA led to hyperactivity in larvae, while exposure to 0.1 or 1 ��M BPA led to learning deficits in adult zebrafish. Exposure to 0.1 ��M E2 or GSK4716 also led to larval hyperactivity. To identify early molecular signaling events that lead to the observed neurobehavioral phenotypes, a global gene expression analysis using a 135K zebrafish microarray was conducted. The concentrations of compounds tested were anchored to the common larval hyperactivity phenotype they elicited. Gross abnormalities, in the case of higher concentrations of BPA and E2, were also anchored phenotypes included in the analysis. Functional pathway analysis of the BPA versus E2 results predicted an impact on prothrombin signaling from the two lower concentrations of BPA and E2. Both BPA and GSK4716 were also predicted to impact nervous system development, potentially involving inhibition of the upstream regulator, SIM1. Additionally, GSK4716 exposure was predicted to inhibit neuron migration. There were fewer similarities in transcriptional responses between BPA and E2 when the lower versus higher concentrations were compared, suggesting different mechanisms operated at the higher concentrations. Subsequent experiments were focused on the role of ERR�� in the larval hyperactivity phenotype. Transient ERR�� knockdown by antisense oligonucleotide morpholino during the first 24 hours of development abrogated the hyperactive phenotype induced by 0.1 ��M BPA exposure. Transient ERR�� knockdown during the first 48 hours of development resulted in developmental delays, craniofacial defects, pericardial edema, and severe body axis curvature. This work is the first to identify behavioral effects in a fish from developmental BPA exposure. It is also the first study to confirm a role for ERR�� in mediating BPA's neurobehavioral effects in any animal model. The global gene expression analysis identified similarities between BPA, E2, and GSK4716, suggesting that BPA's mode of action may involve crosstalk between ERR�� and other ERs. These results from human-relevant BPA exposures help explain the widely documented in vivo effects of BPA, despite low binding affinity exhibited by nuclear ERs. ERR�� is an evolutionarily conserved vertebrate receptor and the developmental impacts of BPA in the zebrafish are an indication of hazard potential to vertebrates. They are also an important translational step toward knowing the hazard potential from human developmental exposure to BPA and yet unknown environmental ligands of ERR��. / Graduation date: 2013 / Access restricted to the OSU Community, at author's request, from Dec. 21, 2012 - June 21, 2013