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Le cancer de la prostate est le cancer le plus fréquemment diagnostiqué chez les hommes canadiens et la troisième cause de décès relié au cancer. Lorsque diagnostiqué à un stade précoce de la maladie, le cancer de la prostate est traité de manière curative par chirurgie et radiothérapie. Par contre, les thérapies actuelles ne peuvent éradiquer la maladie lorsqu’elle progresse à des stades avancés. Ces thérapies, comme la chimiothérapie et l’hormonothérapie, demeurent donc palliatives. Il est primordial d’optimiser de nouvelles thérapies visant l’élimination des cellules cancéreuses chez les patients atteints des stades avancés de la maladie. Une de ces nouvelles options thérapeutiques est l’immunothérapie. L’immunothérapie du cancer a fait des progrès considérables durant les dernières années. Cependant, les avancements encourageants obtenus lors d’essais précliniques ne se sont pas encore traduits en des résultats cliniques significatifs. En ce qui concerne le cancer de la prostate, les résultats négligeables suivants des interventions immunothérapeutiques peuvent être causés par le fait que la plupart des études sur le microenvironnement immunologique furent effectuées chez des modèles animaux. De plus la majorité des études sur l’immunologie tumorale humaine furent effectuées chez des patients atteints d’autres cancers, tels que le mélanome, et non chez les patients atteints du cancer de la prostate. Donc, le but central de cette thèse de doctorat est d’étudier le microenvironnement immunologique chez les patients atteints du cancer de la prostate afin de mieux définir les impacts de la tumeur sur le développement de la réponse immunitaire antitumorale. Pour réaliser ce projet, nous avons établi deux principaux objectifs de travail : (i) la caractérisation précise des populations des cellules immunitaires infiltrant la tumeur primaire et les ganglions métastatiques chez les patients atteints du cancer de la prostate; (ii) l’identification et l’étude des mécanismes immunosuppressifs exprimés par les cellules cancéreuses de la prostate. Les résultats présentés dans cette thèse démontrent que la progression du cancer de la prostate est associée au développement d’un microenvironnement immunosuppressif qui, en partie, est régulé par la présence des androgènes. L’étude initiale avait comme but la caractérisation du microenvironnement immunologique des ganglions drainant la tumeur chez des patients du cancer de la prostate. Les résultats présentés dans le chapitre III nous a permis de démontrer que les ganglions métastatiques comportent des signes cellulaires et histopathologiques associés à une faible réactivité immunologique. Cette immunosuppression ganglionnaire semble dépendre de la présence des cellules métastatiques puisque des différences immunologiques notables existent entre les ganglions non-métastatiques et métastatiques chez un même patient. La progression du cancer de la prostate semble donc associée au développement d’une immunosuppression affectant les ganglions drainant la tumeur primaire. Par la suite, nous nous sommes intéressés à l’impact de la thérapie par déplétion des androgènes (TDA) sur le microenvironnement immunologique de la tumeur primaire. La TDA est associée à une augmentation marquée de l’inflammation prostatique. De plus, les protocoles d’immunothérapies pour le cancer de la prostate actuellement évalués en phase clinique sont dirigés aux patients hormonoréfractaires ayant subi et échoué la thérapie. Cependant, peu d’information existe sur la nature de l’infiltrat de cellules immunes chez les patients castrés. Il est donc essentiel de connaître la nature de cet infiltrat afin de savoir si celui-ci peut répondre de manière favorable à une intervention immunothérapeutique. Dans le chapitre IV, je présente les résultats sur l’abondance des cellules immunes infiltrant la tumeur primaire suivant la TDA. Chez les patients castrés, les densités de lymphocytes T CD3+ et CD8+ ainsi que des macrophages CD68+ sont plus importantes que chez les patients contrôles. Nous avons également observé une corrélation entre la densité de cellules NK et une diminution du risque de progression de la maladie (rechute biochimique). Inversement, une forte infiltration de macrophages est associée à un plus haut risque de progression. Conjointement, durant cette étude, nous avons développé une nouvelle approche informatisée permettant la standardisation de la quantification de l’infiltrat de cellules immunes dans les échantillons pathologiques. Cette approche facilitera la comparaison d’études indépendantes sur la densité de l’infiltrat immun. Ces résultats nous ont donc permis de confirmer que les effets pro-inflammatoires de la TDA chez les patients du cancer de la prostate ciblaient spécifiquement les lymphocytes T et les macrophages. L’hypothèse intéressante découlant de cette étude est que les androgènes pourraient réguler l’expression de mécanismes immunosuppressifs dans la tumeur primaire. Dans le chapitre V, nous avons donc étudié l’expression de mécanismes immunosuppressifs par les cellules cancéreuses du cancer de la prostate ainsi que leur régulation par les androgènes. Notre analyse démontre que les androgènes augmentent l’expression de molécules à propriétés immunosuppressives telles que l’arginase I et l’arginase II. Cette surexpression dépend de l’activité du récepteur aux androgènes. Chez les patients castrés, l’expression de l’arginase II était diminuée suggérant une régulation androgénique in vivo. Nous avons observé que l’arginase I et l’arginase II participent à la prolifération des cellules du cancer de la prostate ainsi qu’à leur potentiel immunosuppressif. Finalement, nous avons découvert que l’expression de l’interleukin-8 était aussi régulée par les androgènes. De plus, l’interleukin-8, indépendamment des androgènes, augmente l’expression de l’arginase II. Ces résultats confirment que les androgènes participent au développement d’une microenvironnement immunosuppressif dans le cancer de la prostate en régulant l’expression de l’arginase I, l’arginase II et l’interleukin-8. En conclusion, les résultats présentés dans cette thèse témoignent du caractère unique du microenvironnement immunologique chez les patients atteints du cancer de la prostate. Nos travaux ont également permis d’établir de nouvelles techniques basées sur des logiciels d’analyse d’image afin de mieux comprendre le dialogue entre la tumeur et le système immunitaire chez les patients. Approfondir les connaissances sur les mécanismes de régulation du microenvironnement immunologique chez les patients atteint du cancer de la prostate permettra d’optimiser des immunothérapies mieux adaptées à éradiquer cette maladie. / Prostate cancer is the most frequently diagnosed cancer in Canadian men and the third cause of cancer related death. When diagnosed at an early stage, prostate cancer can be effectively cured by surgery and radiotherapy. However, current therapies do not eradicate the advanced stages of the disease. Treatment of prostate cancer via chemotherapy or hormonotherapy remains palliative. It is thus essential to optimize novel therapies whose goal is to eliminate tumor cells in patients with advanced prostate cancer. One such approach is immunotherapy. Cancer immunotherapy has made important strides in recent years. The encouraging progress observed in pre-clinical trials has nonetheless not translated to significant results in the clinical setting. Concerning prostate cancer, the limited clinical efficacy of current immunotherapeutic protocols could be explained by the lack of studies directly evaluating the immunological microenvironment in prostate cancer patients and not in animal models or in patients afflicted by other malignancies, such as melanoma. Thus, the fundamental goal of this Ph.D. thesis is to study the immunological microenvironment in prostate cancer patients in order to better understand the impact of the tumor on the development of the anti-tumoral immune response. To realize this project, we established two main working objectives: (i) to precisely characterize the immune cell populations in tumor draining lymph nodes (LNs) and in the primary tumor of prostate cancer patients; (ii) to identify and to study the immunosuppressive pathways expressed by prostate cancer cells. The results detailed in this thesis demonstrate that prostate cancer progression is associated with the development of an immunosuppressive microenvironment, which is regulated, in part, by the presence of androgens. The initial study was based on the characterization of the immunological microenvironment of tumor draining LNs of prostate cancer patients. The results presented in chapter III allowed us to demonstrate that metastatic lymph nodes displayed cellular and histopathological evidence associated with a reduced immunological reactivity. This LN immunosuppression seemed to be dependant on the presence of metastatic cells as we noted significant immunological differences between non-metastatic and metastatic lymph nodes of the same patient. Prostate cancer progression was thus associated with the development of an immunosuppressive state, which affected tumor-draining lymph nodes. Next, we studied the impact of androgen deprivation therapy (ADT) on the immunological microenvironment of the primary tumor. Following ADT, there is a marked augmentation in intra-prostatic inflammation. Immunotherapeutic protocols currently evaluated in clinical trials are targeted at hormone refractory patients, which have received and failed ADT. However, very little information is available regarding the nature of the post-ADT immune infiltrate. Thus, it becomes essential to understand whether this post-ADT infiltrate could positively react to immunotherapy. In chapter IV, I present the results of the quantification of the immune cell abundance within the primary tumor. In patients who have received ADT prior to surgery, there was an elevated density of CD3+ and CD8+ T lymphocytes as well as CD68+ macrophages compared to control patients. We also observed an inverse correlation between the NK cell density and the risk of disease progression (biochemical recurrence). Conversely, an elevated macrophage infiltration was associated with a higher risk of progression. Furthermore, for this study, we developed a novel computerized approach allowing for the standardization of the quantification of immune cell infiltrate. This approach could facilitate the interpretation of results from independent studies on the density of immune cells within pathological specimens. This study confirmed that the pro-inflammatory impact of androgen deprivation therapy in prostate cancer patients target specifically the T lymphocyte and macrophage populations. The interesting hypothesis arising from this study was that androgens could positively regulate the expression of immunosuppressive pathways within the primary tumor. In chapter V, we evaluated the immunosuppressive mechanisms expressed by prostate cancer cells and regulated by androgens. Our analysis demonstrate that androgens increase the expression of molecules with immunosuppressive properties, such as arginase I and arginase II in an androgen receptor dependent manner. This androgen regulated expression of arginase II was also observed in prostate cancer patients treated by ATD. We observed that arginase I and arginase II participate in prostate cancer cell proliferation as well as in their immunosuppressive potential. Finally, we discovered that interleukin-8 expression was also regulated by androgens. Moreover, interleukin-8, independently of androgens, increased the expression of arginase II. Altogether, these results confirmed that androgens participate in the development of an immunosuppressive microenvironment in prostate cancer by regulating the expression of arginase I, arginase II and interlukin-8. In conclusion, the results presented in this thesis attest to the unique character of the immunological microenvironment in prostate cancer patients. Our work has also allowed to establish novel software-based analysis methods in order to better understand the dialogue between the tumor and the immune system. Further understanding of the regulatory pathways involved in the immunological microenvironment will allow for the optimization of immunotherapies better suited to eradicate prostate cancer.

A deficiência androgênica acomete um percentual de homens idosos ainda não bem definido na literatura. São usados parâmetros séricos hormonais de homens jovens, e apesar disto, a terapia de reposição da testosterona vem sendo defendida e utilizada largamente por muitos autores. Várias vias de reposição são usadas tentando simular o ciclo fisiológico de produção das testosteronas. Parece que a transdérmica é a que mais se aproxima do ideal. A relação câncer de próstata e testosterona é pouco compreendida. O receio do estímulo de um câncer oculto pela terapia de reposição não foi devidamente afastado. O tratamento deve ser instituído quando o quadro clínico e laboratorial for evidente. / The androgenic deficiency attacks a percentage of elderly men not well defined in literature yet. Hormonal serum parameters of young men are used and, in spite of that, the testosterone replacement therapy has been widely supported and used by many authors. Various ways of replacement are used trying to simulate the physiological cycle of the testosterone production. It seems that the transdermic is the one closest to the ideal. The relation of prostate cancer and testosterone is little understood. The fear of the stimulation of a concealed cancer by the replacement therapy has not been properly eliminated. The treatment should be indicated when the clinical and laboratorial was defined.

Síndrome dos Ovários Policísticos (PCOS), caracterizada por disfunção ovulatória e hiperandrogenismo, é a endocrinopatia mais frequente entre mulheres em idade reprodutiva, afetando aproximadamente de 6 a 19% desta população, conforme o critério diagnóstico empregado. Além dos distúrbios reprodutivos, as mulheres com PCOS frequentemente apresentam maior prevalência de fatores de risco cardiometabólico como obesidade abdominal, dislipidemia, hipertensão, tolerância diminuída à glicose e diabetes mellitus tipo 2. A resistência insulínica e a hiperinsulinemia compensatória são consideradas como o ponto central destas alterações metabólicas. Os critérios atuais para diagnóstico de PCOS, a partir do Consenso de Rotterdam, indroduziram diferentes fenótipos. Os mais frequentes são o fenótipo “clássico” (hiperandrogenismo e anovulução, com ou sem a aparência policística do ovário), e o fenótipo “ovulatório” (hiperandrogenismo e aparência policística do ovário). Evidências sugerem que as mulheres com PCOS fenótipo “clássico” tenham alterações metabólicas mais severas comparadas às mulheres com o fenótipo ovulatório. Em razão disto, o objetivo do primeiro artigo original foi avaliar o desempenho da circunferência abdominal, da razão cintura-estatura, do índice de conicidade, do produto da acumulação lipídica (LAP) e do índice de adiposidade visceral (VAI) com base no modelo de homeostasia de à insulina (HOMA-IR)≥ 3,8 como padrão de referência para rastreamento de alterações Este estudo mostrou que, dentre os índices de adiposidade avaliados, os que apresentaram maior acurácia foram o LAP entre as mulheres com PCOS fenótipo “clássico” e o VAI entre as com fenótipo “ovulatório”. Aplicando o ponto de corte do LAP< 34, identificamos um subgrupo de pacientes sem alterações cardiometabólicas no grupo de mulheres com PCOS com fenótipo “clássico”, população com maior risco de hipertensão, de dislipidemia e de tolerância diminuída à glicose. Dentre as mulheres com PCOS classificadas com o fenótipo “ovulatório”, VAI≥ 1,32 foi capaz de detectar mulheres com pressão arterial significativamente mais alta e variáveis glicêmicas e lipídicas menos favoráveis em relação às mulheres com PCOS com fenótipo “ovulatório” com VAI abaixo deste ponto de corte. Atualmente, mudanças de estilo de vida (dieta e/ou exercício físico) são consideradas como primeira opção de tratamento não farmacológico nas mulheres com PCOS. Embora a prática de, pelo menos, 30 minutos por dia de exercício físico estruturado seja recomendada e tenha mostrado um potencial efeito na melhora da resistência insulínica e das variáveis antropométricas e hormonais, a manutenção deste hábito a longo prazo permanece como um ponto crítico. Neste sentido, o objetivo do segundo artigo foi avaliar o efeito da atividade física habitual (AFH) nos perfis metabólico e hormonal de mulheres com PCOS e controles pareadas por idade e índice de massa corporal (IMC). A AFH das participantes foi avaliada por meio de pedômetro digital e, conforme o número de passos diário, a participante foi classificada como ativa (≥ 7500 passos/dia) ou sedentária (< 7500 passos/dia). Mulheres ativas, em ambos os diagnósticos, apresentaram menor IMC, circunferência abdominal e LAP. No grupo PCOS, as mulheres ativas apresentaram menores valores de testosterona total, androstenediona e índice de androgênios livres (IAL) em comparação às sedentárias. Além disto, o aumento de 2000 passos foi um preditor independente de redução do IAL nas mulheres com a síndrome. Este estudo mostrou que ser mais ativa foi associado a um perfil antropométrico e metabólico mais saudável, portanto encorajar um estilo de vida mais ativo pode ser benéfico para mulheres com PCOS, especialmente para as obesas e sedentárias. / Polycystic Ovary Syndrome (PCOS) is characterized by ovulatory dysfunction a hyperandrogenism. The prevalence of PCOS in women of reproductive age range from 6- 19%. Women with PCOS present higher prevalence of cardiometabolic risk factors as abdominal obesity, dyslipidemia, hypertension, impaired glucose tolerance and diabetes mellitus type 2. These metabolic abnormalities have been primarily linked to insulin resistance. Currently, the diagnosis of PCOS is confirmed according the Rotterdam Consensus. The more frequent phenotypes are the classic phenotype (hyperandrogenism and anovulation with or without polycystic ovary appearance), and the ovulatory phenotype (hyperandrogenism and polycystic ovary appearance). Evidence suggests that women with classic PCOS phenotype present more severe metabolic alterations compared to women with ovulatory PCOS phenotype. The aim of the first study was to evaluate the performance of the waist circumference (WC), waist-to-height ratio, conicity index, lipid accumulation product (LAP), and visceral adiposity index (VAI) based on homeostasis model assessment of insulin resistance (HOMA- IR)≥ 3.8 as standard reference for screening preclinical metabolic alterations and cardiovascular risk factors in classic PCOS and ovulatory PCOS phenotypes. Among these indexes, LAP had the best accuracy for screening metabolic alterations in classic PCOS phenotype, while VAI had the best accuracy for ovulatory PCOS phenotype. cardiometabolic alterations in the group with classic PCOS, a phenotype which is characterized by higher risk for hypertension, dyslipidemia, and impaired glucose tolerance. In ovulatory PCOS, VAI≥ 1.32 was useful to detect women with significantly higher blood pressure and less favorable glycemic and lipid variables as compared to ovulatory PCOS with lower VAI. In addition, lifestyle intervention (diet and/or exercise) is the first-line treatment for PCOS. Although structured exercise (at least 30 minutes daily) has been recommended and has shown a potential effect on improving insulin resistance, anthropometric, and hormonal variables, the long-term maintenance of exercise remains a critical point. Therefore, the aim of the second study was to objectively examine the effect of habitual PA on metabolic, hormonal, BMI, and anthropometric variables of women with PCOS and a control group, matched by age and BMI. The PA was assessed by digital pedometer, and according to the number of steps/day, participants were classified as active (≥ 7500 steps) or sedentary (< 7500 steps). This study showed that BMI, WC, and LAP were lower in active women in both groups. In the PCOS group, total testosterone, free androgen index (FAI), and androstenedione levels were lower in active when compared to sedentary women. Besides that, a 2,000 daily step increment was an independent predictor of the FAI reduction in PCOS group. The present study showed that a more active lifestyle is associated with healthier anthropometric and metabolic profile, and may be beneficial to women with PCOS, especially for those which are obese and sedentary.

Orientador: José Eduardo Tanus dos Santos / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-11-07T16:45:40Z (GMT). No. of bitstreams: 1 Gomes_ValeriaAguiar_D.pdf: 9967733 bytes, checksum: 0f5a9488cc56b0e1272b80bd9cb6ea0b (MD5) Previous issue date: 2012 / Resumo: A síndrome do ovário policístico (SOP) é a endocrinopatia mais comum em mulheres na idade reprodutiva e está frequentemente associada a alguns fatores de risco cardiovascular. A grande maioria das doenças cardiovasculares (DCV) ocorre inicialmente com o remodelamento vascular, em que as metaloproteinases de matriz (MMPs) são os principais mediadores. Sendo assim, o objetivo do presente estudo foi comparar os níveis plasmáticos da MMP-2 e da MMP-9 e dos inibidores teciduais de MMPs (TIMPs) das pacientes com SOP com as controles saudáveis e examinar se os níveis desses biomarcadores estão associados com às características clínicas e bioquímicas da SOP. Além disso, avaliar o efeito do anticoncepcional oral sobre os níveis plasmáticos de MMPs e respectivos inibidores endógenos nas mulheres com SOP. Para isso, na primeira parte do estudo, avaliamos 65 controles ovulatórias e 80 pacientes com SOP. As concentrações plasmáticas de MMP-8, MMP-9, TIMP-1, TIMP-2 foram medidas por Elisa e, as de MMP-2, por zimografia. Os níveis de MMP-2, MMP-8, MMP-9 e TIMP-1 não foram significativamente diferentes entre os grupos (p? 0,05). Pacientes com SOP apresentaram menores níveis plasmáticos de TIMP-2 do que as controles saudáveis (182,30 ± 5,60 vs. 204,20 ± 7,28 ng/ml; p ?0,05). Além disso, a testosterona foi preditor independente dos níveis de TIMP-2 (estimativa = -0,35, p = 0,04) e da razão MMP-9/TIMP-1 (estimativa = 0,01, p = 0,04). Para avaliar se a redução do hiperandrogenismo iria promover alguma alteração no perfil das MMPs, foram analisadas 20 mulheres com SOP que queriam contracepção hormonal (grupo SOP- ACO), 20 mulheres ovulatórias que desejavam contracepção hormonal (grupo controle- ACO) e 15 mulheres ovulatórias que desejavam contracepção não-hormonal (grupo controle). O tratamento com ACO contendo 30 mcg de etinilestradiol/2mg de acetato de clormadinona durante 6 meses reduziu significativamente as concentrações plasmáticas de MMP-2 no grupo controle ( de 1,44 ± 0,11 unidades arbitrárias no tempo basal para 1,22 ± 0,07 unidades arbitrárias após 6 meses; p = 0,01), e no grupo SOP ( de 1,43 ± 0,08 unidades arbitrárias no tempo basal para 1,25 ± 0,09 unidades arbitrárias após 6 meses; p = 0,007). O ACO reduziu as concentrações de TIMP-2 e TIMP-1 no grupo controle (todos p ?0,05), mas não teve efeitos na MMP-9 plasmática e nas razões MMP-2/TIMP-2 e MMP-9/TIMP-1 (todos p? 0,05) nos grupos avaliados. Os achados do presente estudo indicam que as mulheres com SOP possuem um desequilíbrio nas razões MMP-2/TIMP-2 e MMP-9/TIMP-1, bem como níveis reduzidos de TIMP-2. Parte desses achados estão relacionados ao hiperandrogenismo presente nessas mulheres. Na segunda parte do estudo, observamos que a redução do hiperandrogenismo, promovido pelo tratamento em longo prazo com o ACO, reduziu as concentrações plasmáticas de MMP-2. Considerando o desequilíbrio no perfil das MMPs apresentado pelas mulheres com SOP e, as possíveis consequências decorrentes desse cenário, o tratamento com ACO se mostra benéfico nessas pacientes, podendo reduzir os riscos de futuras complicações cardiovasculares / Abstract: The polycystic ovary syndrome (PCOS) is the most common endocrinopathy in women of reproductive age and it is often associated with some cardiovascular risk factors. The majority of cardiovascular disease (CVD) occurs initially with vascular remodeling in which matrix metalloproteinases (MMPs) are key mediators. Therefore, the aim of this study was to compare plasma levels of MMP-2 and MMP-9 and tissue inhibitors of MMPs (TIMPs) of PCOS patients with healthy controls and to examine whether the levels of these biomarkers are associated with clinical and biochemical characteristics of PCOS. In addition to it, our goal was to evaluate the effect of oral contraceptives on plasma levels of MMPs and their endogenous inhibitors in women with PCOS. In order to prove it, in the first part of the study we evaluated 65 controls and 80 patients with ovulatory PCOS. The plasma concentration of MMP-8, MMP-9, TIMP-1 and TIMP-2 were measured by Elisa, and MMP-2 by zymography. The levels of MMP-2, MMP-8, MMP-9 and TIMP-1 were not significantly different between groups (p? 0.05). PCOS patients had lower their plasma levels of TIMP-2 than healthy controls ones (182,30 ± 5,60 vs. 204,20 ± 7,28 ng/ml; p = 0,02). Furthermore, testosterone was an independent predictor of the levels of TIMP-2 (estimate = -0.35, p = 0.04) and the MMP-9/TIMP- 1 ratio (estimate = 0.01, p = 0.04). To assess whether the reduction of hyperandrogenism would promote a change in the profile of MMPs, we analyzed 20 women with PCOS who wanted to hormonal contraception (OC-PCOS group), 20 ovulatory women who required hormonal contraception (OC-control group) and 15 ovulatory women who wanted non-hormonal contraception wanted a nonhormonal contraception (non-OC control group). Treatment with OC containing 2 mg chlormadinone acetate/30 ?g ethinylestradiol for 6 months significantly reduced plasma MMP-2 concentrations in the OC-control (from 1.44 ± 0.11 arbitrary units at baseline to 1.22 ± 0.07 arbitrary units after 6 months; p = 0.01) and the PCOS groups (from 1.43 ± 0.08 arbitrary units at baseline to 1.25 ± 0.09 arbitrary units after 6 months; p = 0.007) and TIMP-2 and TIMP-1 levels (448.0 ± 66.3 ng/mL versus 349.0 ± 40.9 ng/mL; p = 0.009) in the OC-control group (all p ?0.05) but had no effects on MMP-9 concentrations or on MMP-2/TIMP-2 and MMP-9/TIMP- 1 ratios (all p? 0.05) in any group. The results of this study indicate that women with PCOS have an imbalance in the MMP-2/TIMP-2 and MMP-9/TIMP-1 ratios and reduced levels of TIMP-2. Parts of these findings are also related to hyperandrogenism presence in these women. In the second part of the study, we observed that the reduction of hyperandrogenism promoted by long-term treatment with the OC reduced plasma concentrations of MMP-2. Given the imbalance in the profile of MMPs presented by women with PCOS and the possible consequences of this scenario, treatment with OC shows beneficial in these patients may reduce the risk of future cardiovascular complications / Doutorado / Farmacologia / Doutora em Farmacologia

Conselho Nacional de Desenvolvimento Científico e Tecnológico / A síndrome dos ovários policísticos é uma desordem frequente e complexa, com grande variabilidade fenotípica, predominando os sinais de disfunção ovariana. Alterações metabólicas, inflamatórias e vasculares vinculadas à resistência à insulina são muito prevalentes nessa desordem podendo manifestar-se precocemente. O objetivo principal deste estudo foi investigar a presença de alterações microvasculares em mulheres jovens e não obesas portadoras da síndrome dos ovários policísticos, através de videocapilaroscopia periungueal e dosagem dos níveis séricos de endotelina-1. O objetivo secundário foi verificar a existência de associações entre os achados vasculares, níveis séricos de androgênios, parâmetros clínicos, bioquímicos, metabólicos e inflamatórios relacionados ao risco cardiovascular. Em estudo observacional, transverso e controlado avaliamos 12 mulheres com diagnóstico de síndrome dos ovários policísticos, segundo os critérios estabelecidos pelo consenso de Rotterdam e nove voluntárias saudáveis. A idade (22,82,3 X 24,62,7), o índice de massa corporal (22,53,4 X 23,73,1) e a circunferência da cintura (7510,1 X 77,38,1) foram semelhantes nos dois grupos. As portadoras da síndrome apresentavam hiperandrogenismo clínico. Não foram observadas diferenças significativas entre os grupos quando analisados os níveis séricos de estradiol, testosterona total, androstenediona ou o índice de testosterona livre, entretanto a SHBG mostrou-se significativamente mais baixa no grupo de estudo (p=0,011). A glicemia de jejum, insulina, HOMA-IR e o perfil lipídico foram normais e sem diferença entre os grupos. A amostra com síndrome dos ovários policísticos não apresentava intolerância à glicose ou Diabetes Mellitus pelo teste oral de tolerância à glicose. Os níveis séricos dos marcadores inflamatórios (leucócitos, ácido úrico, adiponectina, leptina e proteína c reativa) e do marcador de função endotelial avaliado também foram similares nos dois grupos. A velocidade de deslocamento das hemácias no basal e após oclusão foram significativamente menores nas pacientes de estudo (p=0,02), mas o tempo para atingir a VDHmax e os parâmetros relativos à morfologia e densidade capilar foram semelhantes. Não observamos correlação entre a velocidade de deslocamento das hemácias e níveis plasmáticos de endotelina-1, androgênios ou parâmetros de resistência insulínica. A velocidade de deslocamento das hemácias associou-se positivamente aos níveis plasmáticos de estradiol (r= 0,45, p<0,05) e negativamente aos de colesterol total e LDL colesterol (r= -0,52, p<0,05; r=-0,47, p<0,05, respectivamente). Em conclusão nossos resultados fornecem evidência adicional de dano precoce à função microvascular em mulheres portadoras de síndrome dos ovários policísticos. Através da capilaroscopia periungueal dinâmica, demonstramos que mulheres jovens com moderado hiperandrogenismo, sem obesidade, RI, hipertensão ou dislipidemia, já apresentam disfunção microvascular nutritiva, caracterizada por redução na velocidade de fluxo das hemácias no basal e após oclusão. Estes achados micro-circulatórios não foram acompanhados de elevações nos níveis plasmáticos de endotelina-1. / Polycystic ovary syndrome is a frequent and complex disorder, showing a great phenotypic variability, with predominance of signs of ovarian dysfunction and, more particularly, of hyperandrogenism and oligo-anovulatory cycles. This disorder shows a high prevalence of insulin resistance -related metabolic, inflammatory and vascular alterations, which may present at early stages. The main purpose of this study was to investigate the presence of microvascular alterations in young and non-obese women with polycystic ovary syndrome through periungueal videolaparoscopy and dosage of endothelin-1 serum levels. The secondary purpose was to verify further associations between vascular findings, serum androgen levels, and clinical, biochemical, metabolic and inflammatory parameters related to cardiovascular risk. We conducted an observational, transversal and controlled study to evaluate 12 women who, according to Rotterdam criteria, were diagnosed with polycystic ovary syndrome, and also nine healthy volunteers. Our selective process excluded from both groups women with smoking habits, as well as those who had made use of oral contraceptive, metformin or antilipemic drugs within three months prior to the beginning of the study. The age range (22,82,3 X 24,62,7), body mass index (22,53,4 X 23,73,1), and waist circumference (7510,1 X 77,38,1) were similar in both groups. Our patients with polycystic ovary syndrome presented clinical hyperandrogenism. Analysis of serum estradiol, total testosterone, androstenedione levels or testosterone free index disclosed no significant differences between the groups, although SHBG appeared to be expressively lower in the studied group (p=0.003). Fasting blood glucose test, insulin level, HOMA-IR and lipid profile showed normal results, and without difference between the groups. As disclosed by the oral glucose tolerance test, the group with PCOS did not present tolerance to either glucose or diabetes mellitus. When evaluated, the serum levels of inflammatory markers (leukocytes, uric acid, adinopectin, leptin and c-reactive protein) and of endothelial function marker (endothelin-1) were also similar in both groups. The red blood cell velocity at baseline and peak were significantly lower in patients with PCOS (p=0.02), although the timeframe to reach blood cell velocity at baseline and peak and the parameters referring to morphology and capillary density were similar between the groups. No association was observed between the speed of movement of red blood cells and plasma levels of endothelin-1, androgens or insulin resistance parameters. The velocity of movement of red blood cells was positively related to estradiol plasma levels (rho= 0.45, p<0.05) and negatively to the levels of total cholesterol and LDL cholesterol (rho= -0.52, p<0.05; rho=-0.47, p<0.05 respectively). Taken together, our results provide an additional evidence of early lesion to microvascular function in women with polycystic ovary syndrome. By using a simple procedure, namely the dynamic nailfoldvideocapillaroscopy, we demonstrated that young women with mild hyperandrogenism, who do not present obesity, insulin resistance, high blood pressure or dyslipidemia, already show a nutritional microvascular dysfunction, characterized by a reduced speed of red blood cells flow at basal level and after occlusion. Such microcirculatory findings were not accompanied by an increase of endothelin-1 plasma levels.

<p>An <i>in vitro</i> method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [¹¹C]vorozole to the active site of the enzyme. [¹¹C]Vorozole displayed high, specific binding <i>in vitro</i> to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [¹¹C]vorozole binding correlated well to aromatase activity measured by [³H]water release from 1β[³H]androstenedione. </p><p>In human GC <i>in vitro</i>, low concentrations of 5α-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [³H]water release but had no effects on aromatase concentration measured by [¹¹C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. </p><p>In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [³H]water release, together with serum androstenedione and estradiol-17β, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [¹¹C]Vorozole binding characteristics changed more rapidly than <i>de novo</i> synthesis of the enzyme. [¹¹C]Vorozole binding K_dshowed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17β. Rapid changes in substrate affinity rather than changes in substrate concentration or <i>de novo</i> synthesis of the enzyme may thus be important for regulation of ovarian aromatase. </p><p>The [¹¹C]vorozole <i>in vivo</i> technique yields additional information compared with traditional in vitro techniques. </p>

<p>Primary Sjögren's syndrome (pSS) is a chronic inflammatory connective tissue disease of unknown etiology. The disease primarily involves salivary and lacrimal glands which results in oral and ocular dryness (sicca symptoms). A wide spectrum of extraglandular features from various organs may be seen. </p><p>In this thesis, the frequency of psychiatric symptoms in women with primary Sjögren's syndrome was studied and an attempt was made to assess how these symptoms might influence their well being and quality of life. The main finding was that the women with pSS suffered significantly more often from symptoms of anxiety and depression when compared with age matched, healthy females and female patients with rheumatoid arthritis. The physical and mental well-being of the patients with pSS was significantly reduced compared to patient controls. </p><p>The possible link of psychiatric symptoms to the altered function of the hypothalamic-pituitary-gonadal axis and adrenal androgen secretion was elucidated. Women with pSS have intact cortisol synthesis but reduced serum concentrations of dehydroepiandrosterone sulphate (DHEA-S) (p<0.05) and an increased cortisol/DHEA-S ratio (p<0.05), compared to healthy controls. These findings may reflect a constitutional or disease-meditated influence on adrenal steroid synthesis. Positive correlation was found between DHEA-S serum levels and quality of sexual life (p<0.01) and mental well-being (p<0.01) in women with pSS. </p>

Primary Sjögren's syndrome (pSS) is a chronic inflammatory connective tissue disease of unknown etiology. The disease primarily involves salivary and lacrimal glands which results in oral and ocular dryness (sicca symptoms). A wide spectrum of extraglandular features from various organs may be seen. In this thesis, the frequency of psychiatric symptoms in women with primary Sjögren's syndrome was studied and an attempt was made to assess how these symptoms might influence their well being and quality of life. The main finding was that the women with pSS suffered significantly more often from symptoms of anxiety and depression when compared with age matched, healthy females and female patients with rheumatoid arthritis. The physical and mental well-being of the patients with pSS was significantly reduced compared to patient controls. The possible link of psychiatric symptoms to the altered function of the hypothalamic-pituitary-gonadal axis and adrenal androgen secretion was elucidated. Women with pSS have intact cortisol synthesis but reduced serum concentrations of dehydroepiandrosterone sulphate (DHEA-S) (p&lt;0.05) and an increased cortisol/DHEA-S ratio (p&lt;0.05), compared to healthy controls. These findings may reflect a constitutional or disease-meditated influence on adrenal steroid synthesis. Positive correlation was found between DHEA-S serum levels and quality of sexual life (p&lt;0.01) and mental well-being (p&lt;0.01) in women with pSS.

An in vitro method was developed for measuring aromatase, based on binding of competitive aromatase inhibitor [11C]vorozole to the active site of the enzyme. [11C]Vorozole displayed high, specific binding in vitro to human placenta and human granulosa cells (GC), both fresh and frozen/thawed cells, provided correct procedures were used. High, specific binding was also observed in pig and rat ovaries, whereas binding in other tissues was unspecific and usually low. Aromatase concentrations measured by [11C]vorozole binding correlated well to aromatase activity measured by [3H]water release from 1β[3H]androstenedione. In human GC in vitro, low concentrations of 5α-dihydrotestosterone (DHT), but not of other androgens, stimulated aromatase activity measured by [3H]water release but had no effects on aromatase concentration measured by [11C]vorozole binding. DHT may interact with aromatase differently than other androgens, perhaps by changing aromatase affinity to precursor. In the rat estrous cycle, aromatase activity in ovarian homogenate, measured by [3H]water release, together with serum androstenedione and estradiol-17β, peaked between 6 and 13 h after onset of the light period of proestrus, the former activity being independent of radioactive substrate concentration. [11C]Vorozole binding characteristics changed more rapidly than de novo synthesis of the enzyme. [11C]Vorozole binding Kd showed close inverse correlation to aromatase activity in ovarian homogenate and to serum estradiol-17β. Rapid changes in substrate affinity rather than changes in substrate concentration or de novo synthesis of the enzyme may thus be important for regulation of ovarian aromatase. The [11C]vorozole in vivo technique yields additional information compared with traditional in vitro techniques.