Uterine leiomyomas are benign tumors within the uterus, where patients present with symptoms such as abnormal bleeding, urinary retention, and pelvic pressure. The exact etiology of uterine leiomyomas is unknown, but numerous theories have been proposed, indicating a multifactorial mechanism, including lifestyle and steroid hormones. Uterine leiomyomas have become a public health concern due to the high cost of treatment as well as the high prevalence within African American communities. Currently, many treatment options exist, ranging from conservative treatments that address symptoms, to surgical intervention to remove the uterus. Research efforts thus far have determined the relationship between the role of estrogen in the growth of uterine leiomyomas (which has led to development of medications that target different approaches to estrogen synthesis) and its effects in the pathogenesis. Studies have shown that estrogen acts on estrogen receptor subtypes, ER and ER. This study examines the role of these two receptors in estrogenic effects, and how these effects relate to the development of uterine leiomyomas. Available research has shown that each receptor has its unique functions and impacts the growth of tumors differently. There is conflicting evidence in how the number of receptors and surrounding environment modulate leiomyomas, with some studies reporting that it is the corepressors and/or coactivators that ultimately determine the influence of estrogenic effects. However, the general consensus of such studies suggests that estrogen receptor-specific therapeutic intervention is a novel area with great potential. The primary benefit of estrogen receptor-specific treatment, such as selective estrogen receptor modulators, is the ability to regulate physiological processes that contribute to the growth of uterine leiomyomas. Future directions of research include confirming the exact roles of ER and ER and harnessing the effects of their differing functions to manage uterine leiomyomas.

Philosophiae Doctor - PhD / Estrogens play an extraordinary role in the endocrine system regulation through the stimulation and regulation of endocrine pathways. 17β-estradiol is one of the final metabolites in estrogen regulation by hydroxylase enzymes that are well recognized for their metabolic role in hormone fragmentation and dissociation, through hydroxylation reactions that reversibly convert a series of androgens to estrogens (i.e. or one estrogen to the other). However, the 17β-estradiol hormone has been classified as one of the estrogenic endocrine disrupting compounds {i.e. EDC (s)} that show significant adverse effects in the estrogen pathways of male and female animal species. Estrogen receptor alpha (ER-α) is significantly activated by 17β-estradiol, which is a steroid hormone. A biosensor system for the determination of 17β-estradiol was developed based on the highly selective and specific physiological substrate level activation of the ER-α biomolecule by the (17β-estradiol) compound. The chemically-tuned tin selenide quantum dots capped with 3-mercaptopropionic acid were produced at room temperature and employed to capture the ER-α micro-molecule onto the electrode surfaces. These quantum dots possessed average particle size (APS) diameters between 4.6 ± 0.6 nm and an indirect band gap energy (Eg) of 3.14 eV. Surface modification on the quantum dots permitted the formation of efficient amide bonds between the capping molecules of the quantum dots and the estrogen receptor-alpha. The tin selenide quantum dots platform enhanced the surface bio-reactivity of the receptorsensor film. The receptorsensor’s sensitivity towards 17β-estradiol was 5.9 μA/μM associated with a response time (tResponse) of less than 1.2 s. The formal potential, Ep˚ˈ, of the receptorsensor-substrate complex was 149 mV. A detection limit (DL) of 1.9 nM was obtained for the electrochemical biosensing methodology. 17β-estradiol–receptorsensor response kinetics were also evaluated, where a dissociation rate (kd) of 7.6 μM/s, a 50 % inhibition concentration (IC50) value of 3.4 nM and a binding efficiency (Bmax) of 7 nM were obtained. Effective measure of 17β-estradiol concentrations as low as 3.8 nM present in surface waters have been reported to induce feminisation in male aquatic species. The receptorsensor’s dynamic linear range (DLR) nevertheless showed capability of screening a minimum of 0.2 nM to a maximum of 8 nM of the 17β-estradiol concentrations. Furthermore, during the estrogen replacement therapy (ERT), 17β-estradiol concentration levels are monitored at frequent phases, wherein 17β-estradiol concentrations from as low as 0.37 nM are recovered in the serum (i.e. this value was also evaluated to be within the receptorsensor’s-DLR), determining its future capability to be developed for; clinical-diagnosis screening of the 17β-estradiol.

Submitted by Morgana Andrade (morgana.andrade@ufes.br) on 2016-04-11T20:38:10Z No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Capa - Doutorado Leticia Soncini de Souza.pdf: 5208177 bytes, checksum: 19d82e927ab0bb3e8f7dde3d954c6d35 (MD5) / Approved for entry into archive by Patricia Barros (patricia.barros@ufes.br) on 2016-05-16T14:40:15Z (GMT) No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Capa - Doutorado Leticia Soncini de Souza.pdf: 5208177 bytes, checksum: 19d82e927ab0bb3e8f7dde3d954c6d35 (MD5) / Made available in DSpace on 2016-05-16T14:40:15Z (GMT). No. of bitstreams: 2 license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Capa - Doutorado Leticia Soncini de Souza.pdf: 5208177 bytes, checksum: 19d82e927ab0bb3e8f7dde3d954c6d35 (MD5) / FACITEC, FAPES, CNPq / Uma vez admitindo-se que o conhecimento da regulação dos mecanismos de formação e reabsorção óssea é crucial na busca de alternativas terapêuticas em doenças como a osteoporose, a biologia molecular surge como ferramenta interessante e, indispensável para alcançar tal objetivo. Dentre esses marcadores genéticos, os polimorfismos associados ao gene do receptor de estrogênio alfa (REα) e ao gene da Apolipoproteína “E” (ApoE) têm recebido maior atenção nos últimos anos. O objetivo deste estudo foi analisar a expressão dos polimorfismos PvuII (CT nt -397) e XbaI (GA nt -351) presentes no gene do REα e dos polimorfismos presentes no gene da ApoE (HhaI - ε2, ε3 e ε4) em populações de mulheres pós-menopausadas, associando estas alterações gênicas, seus perfis clínicos e bioquímicos com a osteoporose. Após análise dos resultados obtidos, o SNP PvuII, do gene do RE, está relacionado com a baixa Densidade Mineral Óssea (DMO), sendo este efeito mais observado em mulheres com idade mais avançada. O alelo P, contudo, correlaciona-se fortemente com alta DMO (p<0,05) em toda a população estudada, e se reproduz quando analisada a população com idade acima de 65 anos, sugerindo um papel protetor à perda de massa mineral óssea. No SNP XbaII do mesmo gene, observou-se uma associação significativa do alelo x em concentrações de triglicerídeos e de lipídios totais, além da dependência da idade dos pacientes e do Índice de Massa Corporal (IMC). Já o SNP HhaI, no gene da APOE, o alelo E2 pode estar relacionado como um fator de risco para a baixa DMO, e o alelo E3 pode estar relacionado como um fator protetor em relação à DMO. Esses resultados contribuem para uma melhor compreensão sobre a expressão de genes relacionados à osteoporose e podem fornecer subsídios para uma melhor determinação prognóstica da enfermidade, além de racionalização da conduta terapêutica a ser escolhida, proporcionando uma melhor qualidade de vida de pacientes já na pós-menopausa. / Since that assuming the knowledge of the regulatory mechanisms of bone formation and resorption is crucial in the search for alternative therapies in diseases such as osteoporosis, molecular biology emerges as interesting tool and essential to achieving this goal. Among these genetic markers, gene polymorphisms associated with estrogen receptor alpha (ERα) and the gene Apolipoprotein " E" (ApoE) have received increased attention in recent years. The aim of this study was to analyze the expression of polymorphisms PvuII (CT nt -397) and XbaI (GA nt -351) gene present in REα and polymorphisms in the gene ApoE (HhaI - ε2 , ε3 and ε4) in populations of postmenopausal women, associating these gene alterations, and their clinical and biochemical profiles with osteoporosis. After analyzing the results obtained, the PvuII SNP in the gene of ERα is related to low BMD, and this effect is most noticeable in women with advanced age. The P allele, however, correlates strongly with high BMD (p < 0.05) in the whole population studied and reproduced when analyzing the population aged over 65 years, suggesting a protective role in the loss of bone mineral. In XbaII SNP, of same gene, a significant association of allele x at concentrations of triglycerides and total lipids and dependence on patient age and Body Mass Index (BMI) was observed. On the other hand, for SNP HhaI, ApoE gene, the E2 allele may be associated as a risk factor for low BMD, and E3 allele may be associated as a protective factor in relation to BMD. These results contribute to a better understanding of the expression of genes related to osteoporosis and can provide information for better prognostic determination of the disease as well as rationalization of therapeutic conduct to be chosen, providing a better quality of life for postmenopausal patients.

The circadian clock is a molecular mechanism that synchronizes physiological changes with environmental variations. Disruption of the circadian clock has been linked to increased risk in diseases and a number of disorders (e.g. jet lag, insomnia, and cancer). Period 2 (Per2), a circadian protein, is at the center of the clock's function. The loss or deregulation of per2 has been shown to be common in several types of cancer including breast and ovarian [1, 2]. Epidemiological studies established a correlation between circadian disruption and the development of estrogen dependent tumors. The expression of estrogen receptor alpha (ERα) mRNA oscillates in a 24-hour period and, unlike Per2, ERα peaks during the light phase of the day. Because up regulation of ERα relates to tumor development, defining the mechanisms of ERα expression will contribute to our comprehension of cellular proliferation and regulation of normal developmental processes. The overall goal of this project is to investigate the molecular basis for circadian control of ERα transcription. Transcriptional activation of ERα was measured using a reporter system in Chinese hamster ovary (CHO) cell lines. Data show that Per2 influences ERα transcription through a non-canonical mechanism independent of its circadian counterparts. Breast cancer susceptibility protein 1 (BRCA1) was confirmed to be an interactor of Per2 via bacterial two-hybrid assays, in accordance with previous studies [2]. BRCA1 is a transcriptional activator of ERα promoter in the presence of octamer transcription factor-1 (OCT-1) [3]. Our results indicate that the DNA binding domain of OCT-1, POU, to directly interact with Per2 and BRCA1, in vitro. Pull-down assays were used to map direct interaction of various Per2 and BRCA1 recombinant proteins and POU. Chromatin immunoprecipitation assays confirmed the recruitment of PER2 and BRCA1 to the estrogen promoter by OCT-1 and the recruitment of Per2 to the ERα promoter decreases ERα mRNA expression levels in MCF-7 cells. Our work supports a circadian regulation of ERα through the repression of esr1 by Per2 in MCF-7 cells. / Ph. D.

<p>Sweden has one of the highest incidences of osteoporotic fractures in the world. A more sedentary lifestyle is one of several proposed reasons for the increase in osteoporosis seen in the developed countries. The aim of this thesis was primarily to study the influence of muscle strength, and body composition, on bone mineral density, BMD, in young adults. The second aim was to evaluate the possible influence of estrogen on muscle strength in women.</p><p>A population-based study of 113 subjects (53 men and 60 women) aged 22-85 showed associations for premenopausal, but not postmenopausal women, between isometric quadriceps muscle strength and BMD in the total body, lumbar spine, and femoral neck. In men there was only an association between muscle strength and BMD in the total body. Another population-based study of 125 randomly selected young adults (64 women and 61 men) showed that total body BMD, TBMD, is influenced by isokinetic knee flexion and extension strength in women but not in men where body composition influenced TBMD. In 159 randomly selected young adult women (20-39 years) knee flexion and extension strength influenced not only TBMD but also total hip BMD, and heel BMD. However, lean body mass and body weight were better predictors for BMD at these skeletal sites. An extension of this study involving 335 women again demonstrated that lean body mass is the best predictor of BMD. This study also showed that Uppsala women aged 20-39 years have a BMD that is approximately 0.1-1.2 SD (2-12 %) above international/national references. In addition marked variations in BMD T-scores between various skeletal sites were noted. </p><p><i>In Conclusion: </i>The association between muscle strength and BMD is evident in women in their early twenties but with age lean body mass and body weight becomes better predictors for BMD. In men lean body mass and body composition but not muscle strength predicted BMD. Hormone replacement therapy does not influence muscle strength and there is no association between allelic variations in the estrogen receptor alpha and muscle strength in women.</p>

Sweden has one of the highest incidences of osteoporotic fractures in the world. A more sedentary lifestyle is one of several proposed reasons for the increase in osteoporosis seen in the developed countries. The aim of this thesis was primarily to study the influence of muscle strength, and body composition, on bone mineral density, BMD, in young adults. The second aim was to evaluate the possible influence of estrogen on muscle strength in women. A population-based study of 113 subjects (53 men and 60 women) aged 22-85 showed associations for premenopausal, but not postmenopausal women, between isometric quadriceps muscle strength and BMD in the total body, lumbar spine, and femoral neck. In men there was only an association between muscle strength and BMD in the total body. Another population-based study of 125 randomly selected young adults (64 women and 61 men) showed that total body BMD, TBMD, is influenced by isokinetic knee flexion and extension strength in women but not in men where body composition influenced TBMD. In 159 randomly selected young adult women (20-39 years) knee flexion and extension strength influenced not only TBMD but also total hip BMD, and heel BMD. However, lean body mass and body weight were better predictors for BMD at these skeletal sites. An extension of this study involving 335 women again demonstrated that lean body mass is the best predictor of BMD. This study also showed that Uppsala women aged 20-39 years have a BMD that is approximately 0.1-1.2 SD (2-12 %) above international/national references. In addition marked variations in BMD T-scores between various skeletal sites were noted. In Conclusion: The association between muscle strength and BMD is evident in women in their early twenties but with age lean body mass and body weight becomes better predictors for BMD. In men lean body mass and body composition but not muscle strength predicted BMD. Hormone replacement therapy does not influence muscle strength and there is no association between allelic variations in the estrogen receptor alpha and muscle strength in women.

Abstract The stem cell is a primitive cell that is capable of dividing to reproduce itself and can give rise to a selection of differentiated progeny. Stem cells are thought to be involved in or even main factors in many diseases. In postnatal humans, mesenchymal tissues have the capacity to regenerate from stem cells called mesenchymal stem cells (MSC). It is currently thought that these cells will become the basis of therapy for many diseases. In the present study, a novel in vitro method was developed to examine human bone marrow derived MSC differentiation into osteoblast lineage, and to study the role of MSC in a variety of mesenchymal diseases. The ability of MSCs to differentiate into osteoblasts was investigated during aging. In addition, the interindividual variability in the osteogenesis of MSCs and in the osteoblastic response of MSC to estrogen and testosterone was studied. Furthermore, an ex vivo model using a human aortic valve microenvironment was developed to explore whether the extracellular matrix influences the osteoblastic differentiation of the MSC. Finally, the role of MSC in neurofibromatosis type 1 (NF1) related congenital pseudarthrosis of the tibia (CPT) was studied. It was found that after menopause the osteogenic potential of MSCs does not decrease. It was also found that estrogen receptor (ER) alpha genotype confers interindividual variability of response to estrogen and testosterone in MSC derived osteoblasts. In addition, it was found that the non-calcified valves with living valve cells inhibited osteogenesis of co-cultured MSCs, whereas the calcified and devitalised valves promoted differentiation towards an osteoblastic lineage. Finally, MSCs from NF1-related pseudarthrosis showed altered NF1 gene expression, poor osteoblastic differentiation and bone formation. In conclusion, MSC can be easily isolated from the bone marrow and MSC has the capacity to regenerate tissue even at later stages of life. These results could help explain the contradictory effects of 17β-estradiol (E2) on osteoblasts in vitro and might also provide new insights into understanding the differences in responses to hormone replacement therapy. It seems that adult stem cells from bone marrow undergo milieu-dependent differentiation to express phenotypes that are similar to cells in the local microenvironment. Finally, the NF1 gene was shown to have a role in bone development and remodelling.

Des études épidémiologiques ont démontré que l’exposition aux pesticides tels que le chlordécone augmente le risque du cancer, en particulier de la prostate. Il avait été précédemment montré au laboratoire que le chlordécone possède des propriétés pro-angiogéniques impliquant la libération de NO et la production de VEGF suite à l’activation du récepteur aux oestrogènes de type alpha (ERα). Les processus angiogéniques pouvant impliquer les espèces réactives de l’oxygène (EROs), produites notamment par la mitochondrie, l’objectif de ce travail a été d’évaluer la contribution de la biogenèse mitochondriale et du stress oxydatif dans l’angiogenèse induite par le chlordécone.Les résultats obtenus montrent que la biogenèse mitochondriale n’est pas essentielle pour la réponse angiogénique du chlordécone puisqu’elle n’est retrouvée que pour de forte concentration de chlordécone. Les mécanismes mis en jeu ont été identifiés au niveau des cellules endothéliales humaines. Ils impliquent une régulation spatio temporellede la production des EROs impliquant dans un premier temps la NADPH oxydase, elle même capable de stimuler la production mitochondriale d’EROs via la voie impliquant la NO synthase. Le chlordécone serait par ailleurs capable de favoriser la localisation périnucléaire des EROs afin de favoriser la production de VEGF. L’ensemble de ces effets implique le récepteur aux oestrogènes. Ce travail a donc permis d’identifier les mécanismes cellulaires impliqués dans la modulation de l’angiogenèse par le chlordécone. Ces mécanismes moléculaires pourraient contribuer à identifier de nouvelles cibles permettant de réguler les processus angiogéniques et la tumorigenèse induites par ce toxique. / Epidemiological studies report that exposure to pesticides like chlordecone increases risk of prostate cancer and tumorigenesis. We have reported recently that the pro-angiogenic effect of chlordecone involving NO release and VEGF production is mediated through activation of α isoform of the estrogen receptor (ERα). Since mitochondria and ROS have been implicated inthe process of angiogenesis, this study aims to determine the contribution of mitochondrial biogenesisand oxidative stress in chlordecone-induce dangiogenesis. Firstly, our results indicate that mitochondrial biogenesis is not essential for chlordecone angiogenic response. We also identified the molecular mechanism involved; chlordecone induces endothelial cells angiogenesis by a spatiotemporal regulation of ROS production involving NADPH oxidase then mitochondrial O2 -via a NO sensitive pathways through activation of ERα.These findings propose that a molecular mechanism may partly explain the epidemiological evidence implicating chlordecone as risk factor of prostatic cancer.

Introdução: Estimativas mostram que mais de dois terços das mulheres com câncer de mama possuem receptores hormonais positivos, e recebem terapia endócrina como tratamento, sendo tamoxifeno (TAM) o tratamento padrão (EBCTCG 2005; Davies et al., 2012). Porém, muitas pacientes se tornam resistentes com o passar do tempo. Estudos prévios mostraram que a expressão do receptor de estrogênio β (REβ) aumenta a resposta ao tratamento com TAM em células de câncer de mama, assim como a coexpressão de REα e REβ esta associada com maior ação proliferativa de TAM (Treeck et al., 2010; Sun et al., 2014). Também foi observada a existência de “cross-talk” entre os RE e a família do receptor do fator de crescimento epidérmico (HER) na resposta ao tratamento com TAM (Lindberg et al., 2011; Blows et al., 2010). Objetivo: Verificar a expressão do REβ, e suas interações com REα e receptores HER, durante o tratamento com TAM e em células resistentes ao TAM. Métodos: A expressão do REβ foi analisada em dois bancos de dados contendo informações de pacientes com câncer de mama. A expressão de RNAm dos RE, receptores HER e vias de sinalização PTEN, Akt e MAPK foram avaliadas após tratamento com TAM, em células resistentes ao TAM e em células silenciadas para os genes dos RE. Também foi avaliada a viabilidade celular após tratamento com TAM e nas células silenciadas para os genes dos RE. Resultados: Pacientes com câncer de mama apresentaram expressão reduzida do REβ, e os subtipos de câncer de mama REα positivos apresentaram baixa expressão do REβ quando comparados aos subtipos REα negativos. Células expressando níveis moderados de REβ apresentaram melhor resposta ao tratamento com TAM. Diminuição nos níveis dos RE é acompanhada por aumento nos níveis dos receptores ErbB2 e ErbB3, aumento de PTEN e diminuição de Akt e MAPK3 após tratamento com TAM. ERβ modula a ação antiproliferativa do TAM através da via de MAPK3. Células resistentes ao TAM apresentaram baixos níveis dos RE e altos níveis dos receptores EGFR, ErbB3 e ErbB4. Conclusão: Estes resultados demonstram que o REβ, e suas interações com REα e receptores HER, possuem papel importante na resposta ao tratamento com TAM. / Introduction: Approximately two-thirds of all breast cancer patients overexpress hormonal receptors, and are treated with endocrine therapy, being tamoxifen (TAM) the standard treatment. However many of initial responders to TAM as first-line experience relapse. Several mechanisms have been proposed to explain the occurrence of acquired TAM resistance. Previous studies showed that estrogen receptor β (ERβ) expression is associated with better response to tamoxifen treatment, as the co-expression of ERα and ERβ is associated with TAM antiproliferative effects. Moreover, there is growing interest about the cross-talk between ERs and ErbB family in response to endocrine therapy. Suggesting that TAM can acts through ERβ and/or ErbB family as compensatory pathways. Objective: To evaluate the expression of ERβ and the relation of ERβ with ERα and ErbB family in response to TAM treatment and in TAM resistant cells. Methods: ERβ expression was analyzed in two different databases of breast cancer patients. The mRNA levels of ER, HER receptors and PTEN, Akt and MAPK signal pathways were measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. The cellular viability was also measured after TAM treatment, in TAM resistance cells and in cells silenced for ER genes. Results: Breast cancer patients presented reduced ERβ expression and the ERα-positive breast cancer subtypes presented lower ERβ levels when compared to ERα-negative breast cancer subtypes. Cells expressing moderates levels of ERβ presented better response to TAM treatment. Down-regulation of ERs induced by TAM treatment are accompanied with an increase in ErbB2 and ErbB3, reduced AKT and MAPK3 mRNA levels and increased PTEN levels. ERβ modulates TAM anti-proliferative effects through MAPK3 pathway. TAM– resistant cells expressed decreased ER mRNA levels and increased EGFR, ErbB3 and ErbB4 levels. Demonstrating that the cross-talk between ERs and HER family influence the response to TAM treatment. Conclusion: These results provide additional data indicating the importance of ERβ, and the relation with ERα and HER receptors, to predict TAM responsiveness.

No description available.