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Submitted by Caroline Falcao (caroline.rfalcao@ufpe.br) on 2017-04-04T19:33:58Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JulianaVasconcelos.pdf: 1176748 bytes, checksum: bb96bcabc13f8fef386568e90cfdc033 (MD5) / Made available in DSpace on 2017-04-04T19:33:58Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) 2012-Dissertação-JulianaVasconcelos.pdf: 1176748 bytes, checksum: bb96bcabc13f8fef386568e90cfdc033 (MD5) Previous issue date: 2012 / Câncer é um conjunto de alterações celulares, que leva a uma divisão celular sem controle, podendo invadir tecidos adjacentes através da circulação sanguínea e do sistema linfático. O Câncer de Próstata (CP) é o segundo tumor mais comum entre a população masculina, e é considerado o câncer da terceira idade. A carcinogênese é um mecanismo complexo no qual ocorre mudanças na expressão de proteínas e glicoconjugados. Glicosilação é mediada por glicosiltransferases, enzimas que tem função de inserir resíduos de carboidratos específicos, e é um dos mais importantes processos biológicos pós-tradução de modificações na estrutura final e função de lipídios e proteínas. As fucosiltransferases (FUT) participam da transferência de resíduos de L-fucose, um sacarídeo associado ao câncer e a processos inflamatórios, da GDP-L-fucose. Neste estudo objetivou-se avaliar a expressão dos genes FUT3 e FUT6 através da Imunohistoquímica em Adenocarcinoma Prostático e Hiperplasia Prostática Benigna correlacionando com o padrão de expressão de L-fucose empregando a histoquímica com as lectinas UEA-I (Ulex europaeus) e LTA (Lotus tetragonolobus). As enzimas FUT3 e FUT6 apresentaram-se com uma alta expressão tanto no Adenocarcinoma Prostático como na Hiperplasia Benigna Prostática, principalmente a FUT 6. Os resultados da histoquímica com lectinas mostraram uma baixa distribuição/accessibilidade de L-fucose. Sugere-se que, as enzimas FUT3 e FUT6 possam representar potenciais biomarcadores para avaliar alterações benignas e malignas prostáticas refletindo uma variação no perfil de L-fucose nestes tumores que podem estar associados às suas características biológicas. / Cancer is a set of cellular changes, leading to uncontrolled cell division that may invade surrounding tissues via bloodstream and lymphatic system. Prostate Cancer (PC) is the second most common tumor in men and is considered the cancer of the elderly. Carcinogenesis is a complex mechanism in which changes occur in the expression of proteins and glycoconjugates where glycosylation plays key roles since modulates the carbohydrate moieties in glycoconjugates being one of the most important biological processes of posttranslational modifications in the final structure and function of lipids and proteins. Fucosyltransferases (FUTs) are enzymes that catalyze the transfer of the L-fucose residues, a saccharide which has been linked to cancer and inflammation features, from GDP-Fuc. This study the objective to evaluate the expression of genes FUT 3 and FUT 6 by immunohistochemistry in Prostatic Adenocarcinoma and Benign Prostatic Hyperplasia and to correlates with the expression pattern of L-fucose using lectin histochemistry with UEA-I (Ulex europaeus) and LTA (Lotus tetragonolobus). FUT3 and FUT6 showed a high expression in both prostatic tissues, especially FUT6. The results of lectin histochemistry showed a low distribution/accessibility of L-fucose residues. It is suggested that FUT3 and FUT6 may represent potential biomarkers to evaluate benign and malignant alterations in prostate reflecting a variation in the profile of L-fucose residues in these tumors which can be associated to their biological features.

Orientadores: Hernandes Faustino de Carvalho, Paulo Roberto Eleutério de Souza / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T14:18:25Z (GMT). No. of bitstreams: 1 AlmeidaNeto_Adauto_D.pdf: 4167026 bytes, checksum: ca3afd135b583810d6996e33016f9e69 (MD5) Previous issue date: 2014 / Resumo: A próstata é alvo de afecções severas que comprometem a função urinária, a qualidade de vida e que consistem em risco de vida aos indivíduos do sexo masculino, particularmente com o avançar da idade. Interações dinâmicas entre o epitélio prostático e o estroma, regulam vários aspectos do desenvolvimento, da função e das patologias prostáticas. O gene WFDC1 é expresso pelas células musculares lisas do estroma prostático normal e tem função na regulação do comportamento do epitélio, na organização da matriz extracelular e na regulação da angiogênese. Dois transcritos principais são oriundos de splicing alternativo do transcrito primário: um com todos os éxons (WFDC1) e outro sem o éxon 3 (Delta 3). Neste trabalho, investigamos as relações quantitativas entre estas duas variantes, com emprego de qRT-PCR (Taqman) e sondas para as junções dos éxons 2-3 e 2-4, em amostras de hiperplasia prostática benigna (BPH) e de câncer de próstata (PCA) provenientes de bancos de tecidos. A expressão do gene marcador MYH11 foi utilizada como estimativa do conteúdo de células musculares lisas nas amostras. Os resultados demonstraram que as amostras puderam ser dividas em dois grupos com expressão diferencial da MYH11(um com baixa expressão e outro com alta miosina, sendo o primeiro correspondente ao quartil inferior da distribuição dos valores de expressão). Foi demonstrada correlação entre a expressão de WFDC1 e MYH11 em BPH, mas não em PCA, enquanto não houve correlação entre Delta 3 e WFDC1 e nem com MYH11. O conteúdo de Delta 3 variou em cinco ordens de magnitude em comparação ao de WFDC1. A razão entre as duas variantes apresentou variação exponencial, distribuições discretas e intercaladas das amostras de BPH e de PCA, que se distribuíram em populações que preservaram as relações 10:1; 1:1 e 1:3. Poucas amostras estiveram livres de cada uma destas variantes. Em conclusão, a expressão do gene WFDC1 e de sua variante WFDC1 correlaciona-se com a diferenciação das células musculares lisas, mas não está condicionada a ela, enquanto a expressão de Delta 3 é completamente independente deste parâmetro e tem correlação positiva com o progressão do PCA, quando o sistema de classificação de Gleason (Gleason 1 + Gleason 2) foi considerado. Adicionalmente, fatores independentes da idade, incidência de BPH ou PCA, são mais influentes na determinação da quantidade total e da proporção entre as duas variantes / Abstract: The prostate gland is intimately related with reproductive and urinary functions, commonly disturbed by a series of diseases. Besides reducing the quality of life, they consist in serious life risk particularly to the aging men. Dynamic interactions between the epithelium and stroma in the gland regulate various aspects of development, function and pathologies. The WFDC1 gene is expressed by smooth muscle cells in the prostate stroma and its product ps20 was shown to control epithelial cell behavior, extracellular matrix organization and angiogenesis. It is supposed to function as a serine protease inhibitor, as other members of its family do. Two transcripts are produced as a result of alternative splicing. The first (WFDC1) retains all exons and the second (Delta 3) lacks the exon 3. In this work, we investigated the quantitative relationship among these two splicing variants, using qRT-PCR (Taqman) probing the junctions between exons 2-3 and 2-4, in benign prostatic hyperplasia (BPH) and prostate cancer (PCA) samples from tissue banks. The expression of the MYH11 gene was used to estimate the content of smooth muscle cells in the samples. The results demonstrate that the samples could be divided in two groups with low or high expression of MYH11, the first corresponding to the lower quartile of expression values). WFDC1 and MYH11 expression were correlated in the BPH samples. Delta 3 expression was independent of both WFDC1 and from WFDC1. The ratio between the variants WFDC1 and Delta 3 varied exponentially in five orders of magnitude. The the ratio between the two variants also varied exponentially, with BPH and PCA samples arranged in discrete and intercalated subgroups. The distribution of populations with different expression levels preserved the ratios 10:1, 1:1 and 1:3. Either variant was absent in only a few samples. In conclusion, the expression of WFDC1 and it WFDC1 variant correlates with but is not conditioned to the differentiation of smooth muscle cells, while Delta 3 is completely independent and is positively correlated with PCA grade (as assessed by the summed Gleason score). Unknown factors independent of age, BPH or PCA incidence are likely influencing Delta 3 expression / Doutorado / Biologia Celular / Doutor em Biologia Celular e Estrutural

Orientadores: Wagner Eduardo Matheus, Ubirajara Ferreira / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:15:24Z (GMT). No. of bitstreams: 1 Alonso_JoaoCarlosCardoso_M.pdf: 3539592 bytes, checksum: 0be37d55627566f90e50845617162ed4 (MD5) Previous issue date: 2013 / Resumo: Introdução: Os andrógenos, além de desempenhar um papel importante no desenvolvimento e no crescimento da próstata, também são responsáveis pelo surgimento e progressão de lesões prostáticas. Portanto, as ações desses hormônios podem ser antagonizadas impedindo a conversão irreversível de testosterona em dihidrotestosterona, por meio da inibição da 5?-redutase. Neste contexto, os objetivos deste estudo foram caracterizar os efeitos clínicos e morfofuncionais da dutasterida, um inibidor de 5?-redutase, em receptores de hormônios esteroides no tecido da próstata humana normal, bem como para verificar a viabilidade desses receptores como potenciais marcadores para o manejo clínico dos pacientes em uso de dutasterida. Métodos: estudo prospectivo, duplo-cego e randomizado que avaliou 49 homens com idades entre 45 e 70 anos, sem alterações no exame de toque retal e com dosagens de PSA entre 2,5 e 4,0 ng/ml. Estes pacientes foram submetidos a biópsia de próstata guiada pelo ultrassom transretal, e após ter sido descartada neoplasia de próstata, foram divididos em dois grupos recebendo dutasterida (n=25) ou placebo (n=24). Os pacientes foram avaliados clinicamente a cada trimestre e, ao final de 12 meses, submetidos a novos testes laboratoriais e análise histopatológica por nova biópsia da próstata. Resultados: Os resultados não mostraram variações significativas nos níveis de estrógeno, testosterona séricos e índice de massa corporal em ambos os grupos, bem como de ER? (receptor estrogênico ?). No entanto, a imunorreatividade para AR (receptor androgênico) e ER? (receptor estrogênico ?) foram significativamente maiores no grupo dutasterida em relação ao grupo placebo, acompanhado por uma redução significativa do volume da próstata e dos níveis de PSA séricos no grupo dutasterida. Além disso, ambos os índices de proliferação e apoptose também foram significativamente maiores no grupo dutasterida, porém a razão proliferação/apoptose foi significativamente menor neste grupo, indicando assim predominância de apoptose. Conclusão: O tratamento com dutasterida mostrou distintas reatividades no tecido prostático normal, apontando a importância da ativação de ER? no mecanismo apoptótico, propiciando efeito protetor no tecido prostático normal, indicando ser este receptor um importante mediador para o seguimento clínico de pacientes em uso de dutasterida / Abstract: Introduction: The androgens, besides playing an important role in prostate development and growth, are also responsible for the development and progression of prostatic lesions. Therefore, preventing the irreversible conversion of testosterone into dihydrotestosterone by inhibiting 5?-redutase can antagonize the actions of these hormones. In this context, the aims of this study were to characterize the clinical and morphofunctional effects of 5?-redutase inhibitor, dutasteride, on steroid hormone receptors in the human normal prostate tissue, as well as to verify the viability of these receptors as potential markers to clinical management of patients on dutasteride use. Methods: Prospective, randomized and double-blind study, evaluating 49 men with ages between 45 and 70 years, no alterations in digital rectal examination and PSA levels between 2.5 and 4.0 ng/ml. These patients underwent prostate biopsy guided by transretal ultrasound (TRUS) with prostate neoplasia ruled out and it has divided into two groups receiving dutasteride (n=25) or placebo (n=24). Patients were clinically assessed every three months and at the end of 12 months undergoing new laboratory tests, prostate rebiopsy, histopathological and clinical analysis. Results: The results did not show significanty variations in serum estrogen and testosterone levels and body mass index in both dutasteride and placebo groups, as well as their ER? (? estrogen receptor). However, AR (androgen receptor) and ER? (? estrogen receptor) immunoreactivity were significantly higher in the dutasteride group in relation to placebo group, followed by a significant reduction in prostate volume performed by TRUS and total serum PSA levels in the dutasteride group when compared to placebo group. Furthermore, both proliferative and apoptotic indices were significantly higher in the dutasteride group in relation to placebo group. However, the proliferation/apoptotic ratio was significantly lower in the dutasteride group, indicating predominance of apoptotic process. Conclusion: The dutasteride treatment led to distinct reactivities in the normal prostate tissue, indicating different signals to the dynamics of the prostate and pointed out the importance of ER? pathways in the activation of apoptosis. Taken together, these data demonstrated that dutasteride treatment exerted protective effect in the normal prostate via ER?, indicating this receptor as important mediator to clinical management of patients on dutasteride use / Mestrado / Fisiopatologia Cirúrgica / Mestre em Ciências

BACKGROUND: Prostate cancer (PCa) is the most commonly diagnosed solid tumor and the second leading cancer death in the United States, and also one of the major cancer-related deaths in Chinese. Androgen deprivation therapy (ADT) is the first line treatment for metastatic PCa. PCa ultimately relapses with subsequent ADT treatment failure and becomes castrate-resistant (CR). It is important to develop effective therapies with a surrogate marker towards CR PCa. METHOD: Histone deacetylase (HDAC) inhibitors were examined to determine their effects in androgen receptor (AR)/ cellular prostatic acid phosphatase (cPAcP)-positive PCa cells, including LNCaP C-33, C-81, C4-2 and C4-2B and MDA PCa2b androgen-sensitive and androgen-independent cells, and AR/cPAcP-negative PCa cells, including PC-3 and DU 145 cells. Cell growth was determined by cell number counting. Western blot analyses were carried out to determine AR, cPAcP and PSA protein levels. RESULTS: cPAcP protein level was increased by HDAC inhibitor treatment. Valproic acid, a HDAC inhibitor, suppressed the growth of AR/cPAcP-positive PCa cells by over 50% in steroid-reduced conditions, higher than on AR/cPAcP-negative PCa cells. Further, HDAC inhibitor pretreatments increased androgen responsiveness as demonstrated by PSA protein level quantitation. CONCLUSION: Our results clearly demonstrate that HDAC inhibitors can induce cPAcP protein level, increase androgen responsiveness, and exhibit higher inhibitory activities on AR/cPAcP-positive PCa cells than on AR/cPAcP-negative PCa cells. Upon HDAC inhibitor pretreatment, PSA level was greatly elevated by androgens. This data indicates the potential clinical importance of cPAcP serving as a useful biomarker in the identification of PCa patient sub-population suitable for HDAC inhibitor treatment.

5α-reductases (5αRs) catalyse reduction of 4-pregnene steroids, most notably the androgen testosterone to its more potent metabolite dihydrotestosterone (DHT). Well-characterised isozymes of 5αR are designated 5αR1 and 5αR2. Inhibitors of 5αR, finasteride (a 5αR2 inhibitor) and dutasteride (a dual 5αR1 and 5αR2 inhibitor), are utilised in conditions where a reduction in androgen action is desired, including benign prostatic hyperplasia. Although 5αR2 is predominantly expressed in reproductive tissues, both isozymes, but particularly 5αR1, are expressed in metabolic tissues including liver and adipose and both metabolise glucocorticoids as well as androgens; therefore inhibition of 5αR may have consequences for metabolic health. This thesis addresses the hypotheses that 5αR1 inhibition with dutasteride decreases insulin sensitivity and causes dysregulation of the HPA axis in humans. Metabolism and the HPA axis were studied in men prior to and following 3 months of dutasteride (0.5 mg daily; n=16), finasteride (5 mg daily; n=16) or control (tamsulosin MR; 0.4 mg daily; n=14). Glucose disposal during hyperinsulinaemia was the primary endpoint, measured during a hyperinsulinaemic euglycaemic clamp, with d2-glucose and d5-glycerol tracers. Peripheral insulin sensitivity for both glucose uptake and NEFA suppression decreased with dutasteride versus both finasteride and control, while hepatic insulin sensitivity was preserved. Body fat increased with dutasteride, though was not accompanied by changes in metabolic or inflammatory gene transcript abundance in subcutaneous adipose biopsies, nor any differences in abdominal adipose depots on post-treatment MRI. Subtle dysregulation of the HPA axis was evident with both 5αR inhibitors, though to a greater degree with dutasteride and changes were largely compensated for. In support of this study, this thesis also describes the development, validation and application of two novel liquid chromatography tandem mass spectrometry assays; establishing compliance by measuring serum drug levels, and demonstrating effects of 5αR inhibitors on androgen metabolism and adrenal steroidogenesis by measurement of testosterone, DHT and androstenedione. In conclusion, 5αR1 inhibition with dutasteride, but not finasteride, induces peripheral insulin resistance and increases body fat. Findings presented may have important implications for patients prescribed dutasteride for benign prostatic hyperplasia.