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.

Philosophiae Doctor - PhD / Esophageal cancer (EC) ranks among the ten most frequent cancers worldwide. Mortality rates associated with EC are very similar to the incidence rates due to the relatively late stage of diagnosis and the poor efficacy of treatment. The aim of this study was to enhance our insights of putative transcriptional circuitry of EC genes, thereby potentially positively impacting our knowledge of therapeutic targets, providing indications as to more appropriate lines of treatment, and additionally allowing for the determination of putative candidate diagnostic markers for the early stage detection of EC. This thesis reports on the development of a novel comprehensive database (Dragon Database of Genes Implicated in Esophageal Cancer, DDEC) as an integrated knowledge database aimed at representing a gateway to esophageal cancer related data. More importantly, it illustrates how the biocurated genes in the database may represent a reliable starting point for divulging transcriptional regulation, diagnostic markers and the biology related to esophageal cancer. DDEC contains known and novel information for 529 differentially expressed EC genes compiled using scientific publications from PubMed and is freely accessible for academic and non-profit users at http://apps.sanbi.ac.za/ddec/. The novel information provided to users of the DDEC is the lists of putative transcription factors that potentially control the 529 manually curated genes. The value of the information accessible through the database was further refined by providing precompiled text-mined and data-mined reports about each of these genes to allow for easy exploration of information about associations of EC-implicated genes with other human genes and proteins, metabolites and enzymes, toxins, chemicals with pharmacological effects, disease concepts and human anatomy. This feature has the capacity to display potential associations that are rarely reported and thus difficult to identify, and it enables the inspection of potentially new ‘association hypotheses’ generated based on the precompiled reports. This study further illustrates how the biocurated esophageal squamous cell carcinoma (ESCC) genes in the database may represent a reliable starting point for exploring beyond current knowledge of the transcriptional circuitry of estrogen related hormone therapy. The genes were used to develop a method that identified 44 combinations of transcription factors (TFs) that characterize the promoter sequence of estrogen responsive genes implicated in ESCC. These significantly over-represented combinations of TFs were then used to increase confidence in the 47 novel putative estrogen response genes that may be related to ESCC too. Coincidently, two of the novel putative estrogen response genes were verified by current (2009), experimental publications. / South Africa

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.

We develop machine learning approaches to predict context specific enhancer-promoter interactions using evidence from changes in genomic protein occupancy over time. Occupancy of estrogen receptor alpha (ER-alpha), RNA polymerase (Pol II) and histone marks H2AZ and H3K4me3 were measured over time using ChIP-Seq experiments in MCF7 cells stimulated with estrogen. Two Bayesian classifiers were developed, unsupervised and supervised. The supervised approach uses the correlation of temporal binding patterns at enhancers and promoters and genomic proximity as features and predicts interactions. The method was trained using experimentally determined interactions from the same system and achieves much higher precision than predictions based on the genomic proximity of nearest ER-alpha binding. We use the method to identify a confident set of ER-alpha target genes and their regulatory enhancers genome-wide. Validation with publicly available GRO-Seq data shows our predicted targets are much more likely to show early nascent transcription than predictions based on genomic ER-alpha binding proximity alone. Accuracy of the predictions from the supervised model was compared against the second more complex unsupervised generative approach which uses proximity-based prior and temporal binding patterns at enhancers and promoters to infer protein-mediated regulatory complexes involving individual genes and their networks of multiple distant regulatory enhancers.

Os estrógenos exercem importantes efeitos fisiológicos através dos dois subtipos dos receptores de estrógeno humanos (hERs), alfa (hER?) e beta (hER?). Enquanto hER? é um importante alvo macromolecular no desenvolvimento de fármacos para o tratamento do câncer de mama, hER? é um alvo promissor no desenvolvimento de agentes terapêuticos para a terapia de reposição hormonal. O progresso no planejamento de moduladores apresentando maior potência, afinidade e seletividade, entretanto, requer a otimização múltipla de interações intermoleculares fármaco-receptor. A Química Medicinal moderna, de forte caráter multidisciplinar, fornece um arsenal de alternativas e estratégias úteis no processo de planejamento de novos fármacos. As ferramentas de modelagem molecular e de estudos das relações quantitativas entre a estrutura e atividade (QSAR) estão integradas a esse processo, sendo de extremo valor na busca por moléculas bioativas com propriedades múltiplas otimizadas. Para a realização deste trabalho, conjuntos padrões de dados foram organizados para diferentes classes químicas de potentes moduladores dos ERs. Esses conjuntos padronizados para os subtipos do hER, contendo a informação qualificada sobre a estrutura química dos ligantes associada a medida da propriedade farmacológica correspondente, estabeleceram as bases para o desenvolvimento de modelos empregando os métodos holograma QSAR, CoMFA e GRID/PCA. Os modelos finais de HQSAR e CoMFA possuem elevada consistência interna e externa, apresentando bom poder de correlação e predição das propriedades alvo. Juntamente com as informações obtidas pelos mapas de contribuição 2D e de contorno 3D, os modelos de QSAR e GRID/PCA construídos são guias químico-medicinais úteis no planejamento de novos moduladores seletivos do ER possuindo maior afinidade e potência. / Estrogens exert important physiological effects through two human estrogen receptor subtypes (hERs), alpha (hER?) and beta (hER?). While hER? is a macromolecular target of great importance for breast cancer therapy, hER? is an attractive drug target for the development of novel therapeutic agents for hormone replacement therapy. Progress towards the design of modulators having improved potency, affinity and selectivity requires the optimization of multiple ligand-receptor interactions. The strong multidisciplinary character of modern Medicinal Chemistry supplies a rich arsenal of useful rational strategies for the design of new drug candidates. Molecular modeling tools and quantitative structure-activity relationships (QSAR) are integrated into the drug design process in the search of bioactive molecules having optimized properties. In this study, standard data sets were organized for different chemical classes of ER modulators, integrating the qualified information about chemical structure associated to the corresponding pharmacological property. The data sets established the scientific basis for the development of models employing the hologram QSAR, CoMFA and GRID/PCA methods. The final HQSAR and CoMFA models possess high internal and external consistency, with good correlative and predictive power. The generated QSAR and GRID/PCA models as well as the information gathered from the 3D contour maps provide useful guidelines for the design of new selective ER modulators having improved affinity and potency.

Orientador: Aparecida Machado de Moraes / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-13T11:25:06Z (GMT). No. of bitstreams: 1 Cordeiro_RaquelCristinaTancsik_D.pdf: 1883576 bytes, checksum: c00bc86b625d6d904784f53f3f6041e6 (MD5) Previous issue date: 2009 / Resumo: A estria atrófica cutânea ou striae distensae (SD) é uma afecção muito comum, sendo causa freqüente de procura por consultas dermatológicas. Ainda que não representem qualquer risco à saúde física, produzem impacto emocional e induzem busca por tratamentos trabalhosos, caros e dolorosos e, com freqüência, inadequados. Além disso, o surgimento de striae distensae pode refletir alteração do tecido conjuntivo e indicar condições patológicas locais e sistêmicas. Alguns autores descrevem as estrias cutâneas como uma condição de estiramento ou distensão da pele, com perda ou ruptura das fibras elásticas na região acometida. Entretanto, vários autores observam que as estrias não surgem com frequência sobre a pele acima de tumores abdominais, ascites, hemorragias extensas ou grandes hérnias. Atualmente admite-se que sua etiopatogenia é multifatorial, englobando aspectos mecânicos, bioquímicos e genéticos. No entanto, considerando-se a multiplicidade de fatores envolvidos, a literatura é divergente e inconclusiva. Portanto, através do estudo de fatores clínicos associados às SD e dos receptores hormonais (estrógeno, andrógeno e glicocorticóide), pretendeu-se entender melhor a participação dos hormônios na fisiopatogênese das estrias. Para o estudo clínico foram selecionados pacientes com queixa de estrias cutâneas e a comparação foi feita com grupo controle de número semelhante, atendido aleatoriamente por outras queixas no Ambulatório Geral de Dermatologia do HC, FCM/UNICAMP. O estudo da expressão dos receptores hormonais foi realizado por Western blot em oito amostras de pele de estrias recentes, com menos de um ano de evolução, comparando com a avaliação de pele sem lesão de região palpebral de oito pacientes que se submenteram a blefaroplastia. Observou-se que fatores como adolescência, gestação e obesidade estão significativamente relacionados ao surgimento das SD. Constatou-se ainda que a idade materna jovem e o ganho ponderal durante a gestação são importantes fatores associados ao desenvolvimento das lesões. Além disso, a localização das lesões correlaciona-se ao fator causador das estrias. Em relação ao estudo dos receptores hormonais, observou-se que na SD recentes há duas vezes mais expressão de receptores de estrógeno e 1,7 vezes mais expressão receptores de andrógeno e glicocorticóide. Alguns autores interpretam SD como cicatrizes. Após influência hormonal, haveria uma reação inflamatória inicial que determinaria a destruição de fibras elásticas e colágenas. O processo seria seguido de regeneração das fibras, um fenômeno de remodelação dinâmica,ou seja, um balanço entre síntese de colágeno e sua quebra, o qual reestrutura o tecido para acomodar as forças que agem sobre ele, resultando na formação das SD. O balanço entre a expressão de receptores de estrógeno, andrógeno e glicocorticóide poderia induzir as modificações específicas da matriz extracelular, o que levaria a esse fenômeno de remodelação. Em concordância com outros estudos, observamos que as estrias surgem em situação de grande modificação sistêmica, como adolescência e gestação. Através das observações morfológicas e moleculares, nota-se que as SD estão correlacionadas com intensas alterações do tecido conectivo. Os resultados mostram-se relevantes e representam mais um passo na compreensão do mecanismo fisiopatológico da estrias cutâneas e abrem espaço para novas linhas de pesquisa, relacionadas às SD e a outras alterações do tecido conectivo. / Abstract: Stretch marks or striae distensae (SD) are a very common condition which often results in persons searching dermatological treatment. While not presenting any risk to one's physical health, the emotional impact often induces those so affected to demand medical treatments, which are often laborious, expensive and painful, and frequently ineffective. Beyond this, the appearance of SD may indicate other alterations in conjunctive tissues, including both local and systemic pathologies. Some authors described cutaneous strias as a condition of stretching or distension of the skin which results in the loss or rupture of elastic fibers in the affected areas. However, many authors have also observed that SD do not occur frequently associated with abdominal tumors, ascites, extensive hemorrhages or large hernias. Current medical opinion is that its etiopathogenesis is multifactored, englobing mechanical, biochemical and genetic aspects. Nevertheless, in view of the multiplicity of the factors involved, the literature is divergent and inconclusive. This study was proposed to look for better understanding of the role played by hormones in the physiopathology of SD studying clinical factors associated with SD and hormone receptors (estrogen, androgen and glycocorticoid). Patients complaining of SD were selected for inclusion in this clinical study, and comparisons were made with a control group of a similar number of persons who had been treated for other medical conditions at the General Ambulatory Dermatology Care Facility at HC, FCM/UNICAMP. The expression of hormone receptors was undertaken of the Western Blot testing of recent skin striations, in comparison with lesion-free skin taken from the eyelids of patients who had undergone blepharoplasty. The study revealed that factors such as adolescence, pregnancy and obesity are significantly related to the appearance of SD. It was further established that age (the younger the gravid, the greater the possibility of SD) and significant weight gain during pregnancy are important factors associated with the development of SD lesions. In addition, there was a positive correlation with the location of the lesions. In relation to the study of hormone receptors, it was observed that recently-formed SD have two times more expression of estrogen receptors, and 1,7 times more expression of androgen and glycocorticoid receptors. Some authors classify SD as scars. Under hormonal influence, there is an initial inflammatory reaction which results in the destruction of elastic fibers and collagens. This is followed by the regeneration of the destroyed fibers, a phenomenon of dynamic remodeling, a balance between the synthesis and destruction of collagens, which restructures the tissue in order to accommodate the forces acting upon it, resulting in the formation of SD. The balance between the expression of estrogen, androgen and glycocorticoid may elicit the specific modifications of the extracellular matrix which leads to the phenomenon of remodalation. In agreement with other studies, we observed that the strias arise under conditions of significant system modification, such as adolescence and pregnancy. The observation of morphological and molecular changes showed that there is a correlation between SD and with intense alterations in connective tissue. The results of this study are relevant and represent an important step in the understanding of physiopathological mechanisms in stretch marks, opening new horizons for new directions in research of SD and other alterations in connective tissues as well. / Doutorado / Clinica Medica / Doutor em Clínica Médica

The estrogen receptor alpha (ERa) and estrogen receptor beta (ER~) have been demonstrated to be important for immune system regulation and studies have suggested an antiproliferative effect of ER~ in lymphoid malignancies. We have studied the expression of ERa and ER~ in peripheral blood mononuclear cells from patients with chronic lymphocytic leukemia (CLL). Expression of ERa was low, while ER~ was highly expressed in CLL cells. In order to investigate the possible inhibitory effect of ligand-activated ER~ , we treated CLL cells and Mecl cell lines with the selective ER~ agonist diarypropionitrile (DPN) in culture. Treating Mecl cell lines with DPN showed an antiproliferative effect of ER~ agonist by significantly inhibit the growth of Mec 1 cell lines. This suggests that ER~ agonist may be useful in the treatment of CLL.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Reproductive Biology))--University of Pretoria, 2005. / Urology / unrestricted

No description available.

Endocrine resistance is a significant clinical problem in the treatment of estrogen (E2) receptor positive breast cancers. There are two ER subtypes, ERα and ERβ, which promote and inhibit breast cancer cell proliferation respectively. While ER positive breast cancers typically express a high ratio of ERα to ERβ, the acquisition of antiestrogen resistance in vitro and in vivo is associated with increased relative expression of the ERβ. On some gene enhancers ERβ has been shown to function in opposition to the ERα in the presence of E2. Here we demonstrate that exposure to two different ERβ agonists results in decreased cell viability, and produced a marked reduction in G2/M phase in antiestrogen resistant breast cancer cell line in conjunction with altered cyclin D1, and cyclin E expression relative to E2. ERβ agonists also strongly downregulated Bcl-2 expression and recruited both ERs to the Bcl-2 and pS2 E2-response elements resulting in a reduction in mRNA transcripts from both of these genes. Bcl-2 reduction correlated with increased lipidation of LC3-I to LC3-II, indicative of increased autophagic flux. Although ERβ agonist treatment alone did not induce apoptosis, remarkably, the coaddition of ERβ agonist and the autophagy inhibitor, chloroquine, resulted in robust cell death. Lastly, in vivo studies demonstrate that preferential-ERβ agonists are not estrogenic in the uterus or mammary gland. Together, these observations suggest that combined therapies including an ERβ agonist and an autophagy inhibitor may provide the basis for a safe, novel approach to the treatment of antiestrogen-resistant breast cancers.