published_or_final_version / abstract / Pathology / Master / Master of Philosophy

Como em outros mosquitos, os trofócitos do corpo gorduroso de Cx. quinquefasciatus sintetizam vitelogenina (Vg), principal proteína armazenada pelo ovócito, formada por duas subunidades de 200 e 86 kDa. A ultraestrutura dos trofócitos revela o rápido desenvolvimento da maquinaria biossintética após a alimentação com sangue (aa) e a consecutiva degradação após as 48 h aa. Antes do repasto (AR), um conjunto de células indiferenciadas, limitado pelo epitélio folicular, conforma os folículos ovarianos. Após AR, o ovócito se destaca pelo acúmulo de lipídeos e Vg. O receptor de vitelogenina é encontrado somente nos ovários e análise por PCR em tempos mostrou aumento dos transcritos nos primeiros cinco dias após emersão e nas primeiras 48 h aa, durante a vitelogênese. O perfil transcricional de Vg mostrou um pico no terceiro dia de vida adulta e ao final do processo ovogênico / As in other mosquitoes, fat body trophocytes of Cx. quinquefasciatus synthesize vitellogenin (Vg), the major yolk protein stored by the oocyte, formed by two subunits of 200 and 86 kDa. The trophocytes ultrastructure reveals the rapid development of the biosynthetic machinery and the consecutive degradation around 48 h post blood meal (PBM). Before blood meal, a set of undifferentiated cells limited by follicular epithelium, conform the ovarian follicles. After blood meal, the oocyte is remarkable by accumulation of lipid inclusions and yolk granules. Vitellogenin receptors (rVitCx), are localized exclusively in the ovaries and real time PCR showed transcripts increase at the first five days after emergence (AE), and at the first 48 h PBM, during oogenesis. Vg transcripts profile showed a peak on the third day AE and at the end of the vitellogenic process

Introdução: Hexamerinas são proteínas de estocagem sintetizadas pelo corpo gorduroso de larvas de insetos e secretadas na hemolinfa, onde se acumulam. A função canônica das hexamerinas consiste em servir de reserva de aminoácidos e energia para a reconstrução de tecidos e órgãos durante a metamorfose. Este trabalho teve como objetivo a busca por evidências de funções alternativas das hexamerinas durante o ciclo de vida de abelhas A. mellifera. Resultados: Os perfis temporais de expressão das quatro hexamerinas (HEX 70a, HEX 70b, HEX 70c e HEX 110), verificados por meio de SDS-PAGE e western blot, corroboram sua função canônica na metamorfose. Consistente com esta função, as quatro hexamerinas foram localizadas no citoplasma das células do corpo gorduroso utilizando-se anticorpos específicos e microscopia confocal. No entanto, funções adicionais puderam ser inferidas com base nos seguintes resultados: (1) Foci das quatro hexamerinas foram localizados nos núcleos de algumas células do corpo gorduroso em metamorfose, levando à hipótese de que têm função anti-apoptótica durante este período crítico do desenvolvimento; (2) Além disso, HEX 70a e HEX 110 foram localizadas no citoplasma e núcleo de células ovarianas e testiculares, indicando função no desenvolvimento e maturação das gônadas; (3) A co-localização de um análogo de timidina (EdU) e HEX 70a nos núcleos das células dos ovaríolos, sugeriu fortemente uma função na proliferação celular. O knockdown de HEX 70a in vivo por meio de injeção de anticorpo específico prejudicou o crescimento dos ovaríolos de rainhas, reforçando a hipótese de função na proliferação celular, (4) interferiu na esclerotização da cutícula de operárias, indicando função na formação do exoesqueleto e (5) provocou a antecipação da ecdise adulta, provavelmente em resposta à ausência (ou diminuição) dos aminoácidos derivados das hexamerinas. Foram investigados também aspectos da regulação dos genes de hexamerinas. A manipulação experimental da dieta alimentar e dos títulos do hormônio juvenil (HJ) interferiram claramente na expressão dos genes de hexamerinas. A potencial ação reguladora do HJ foi reforçada pelos resultados de análises por bioinformática da região 5 UTR de cada gene de hexamerina (Martins et al., 2010) que revelaram potencial motivo de ligação à proteína Ultraspiracle (Usp), um membro do complexo receptor do HJ no DNA. Procedimentos para expressar as hexamerinas in vitro em sistema de bactérias e purificá-las estão em progresso visando a caracterização da estrutura e de interações entre as subunidades. Conclusão: Estes resultados ressaltam que as hexamerinas têm outras funções no ciclo de vida de A. mellifera, além da função já bem estabelecida de reserva de aminoácidos para a metamorfose. / Background: Insect hexamerins are storage proteins synthesized by the larval fat body and secreted into the hemolymph, where they accumulate. The canonical function of hexamerins is to provide amino acids and energy for the reconstruction of tissues and organs during pupal-to-adult development. The aim of the current study was to search for evidence of alternative roles for the hexamerins in the life cycle of the honey bee, A. mellifera. Results: The canonical role of insect hexamerins received support from our data on the temporal expression profiles of the four honey bee hexamerin subunits (HEX 70a, HEX 70b, HEX 70c and HEX 110), as verified by SDS-PAGE and western blot using hemolymph and fat body samples. Consistent with the canonical function, the four hexamerins were localized in the cytoplasm of fat body cells, during metamorphosis, by using specific antibodies and confocal laser-scanning microscopy. However, additional functions could be inferred by the following findings: (1) The four hexamerins were also localized in the nuclei of some fat body cells, thus tentatively suggesting an anti-apoptotic role during metamorphosis; (2) Furthermore, HEX 70a and HEX 110 were localized in the cytoplasm and nucleus of ovarian and testicular cells, pointing to a role in gonad development and maturation. Co-labeling of the thymidine analog EdU and HEX 70a in the ovariole cell nuclei, strongly suggested a role in cell proliferation; HEX 70a depletion via injection of the specific antibody in queen pupae impaired ovariole growth, thus strengthening our hypothesis on a role in cell proliferation, (3) HEX 70a depletion also impaired cuticle sclerotization, indicating a function in exoskeleton formation, and (4) led to a precocious adult ecdysis, perhaps in response to the lack (or decrease) in hexamerin-derived amino acids. We also investigated aspects of the regulation of hexamerin genes. The experimental manipulation of diet consumption and juvenile hormone (JH) titer clearly interfered in the expression of hexamerin genes. Regulation by JH was also supported by a previous bioinformatics analysis of the 5 UTR region of each hexamerin gene (Martins et al., 2010), which revealed a potential binding site for Ultraspiracle (Usp), a member of the JH receptor complex in the DNA. Experiments are in progress for in vitro expression and purification of the four hexamerins aiming to further characterize their structures and interactions. Conclusion: Taken together, these results imply in novel roles for hexamerins in the life cycle of A. mellifera in addition to their well-established role as amino acids sources for metamorphosis.

This dissertation examines the production and management of genetic risk for breast and ovarian cancer in the United States in the new era of multi-gene panel testing. Drawing on three years of ethnographic fieldwork and in-depth interviews with genetics health professionals and women with mutations, this project is the first social science study to examine how breast and ovarian cancer genetic risk is constructed and managed among women with variants of uncertain significance or moderate-risk mutations. Moving beyond an individual-level focus on women’s risk management decisions, this project instead explores how the structures, practices, and organization of genetic medicine constrain and enable those decisions. There are four key findings from this study. First, the adoption of panel testing has shifted the boundaries of risk, disease, and patienthood and contributed to a spectrum of medicalization of breast and ovarian cancer risk. Women with high-risk breast and ovarian cancer mutations are now typically viewed and treated like full patients with a "disease," while women with moderate-risk mutations occupy a liminal space of qualified patienthood. Second, the structures and organization of genetic medicine in the United States point women with breast and ovarian cancer mutations toward risk-reducing mastectomy and breast reconstruction and encourage choosing those surgical responses over breast surveillance or staying flat. Mastectomy has become the standard “treatment” for the “disease” of genetic risk for breast cancer, regardless of whether women have high- or moderate-risk mutations and despite more conservative recommendations in clinical guidelines. Third, the structures of genetic medicine and the contemporary gender order in the United States are mutually constituted and co-produced. Breast reconstruction and gynecologic surgery practices both emerge from and reinforce gendered social and cultural norms that prioritize women's appearance and their reproductive capacity over their embodied experiences and daily quality of life. Finally, the discourses and practices of genetic medicine leave many women un- or under-prepared for the duration and severity of the side effects and consequences associated with breast reconstruction and risk-reducing salpingo-oophorectomy. By closely examining the social and structural dimensions of how cancer genetic risk is produced and managed in the United States, this project illuminates how clinical practices that magnify and focus on reducing certain risks simultaneously obscure and generate exposure to others.

In the studies about ovarian cancer cells, basal iNOS expression in the chemosensitive OV2008 cells was significantly higher than in the chemoresistant C13* cells. Cisplatin further increased iNOS expression in OV2008 cells, but had no effect in C13* cells. Furthermore, cisplatin dramatically reduced the expression levels of eNOS and nNOS, but again only in OV2008 cells. The data suggest that failure of cisplatin to upregulate iNOS and downregulate eNOS and nNOS in C13* cells could be an etiological factor in chemoresistance. Addition of exogenous NO at high levels, using SNAP, significantly increased p53 protein levels and caused apoptosis in both cell types. Specific iNOS inhibitor (1400W) partially blocked the pro-apoptotic effects of cisplatin in OV2008 cells, suggesting involvement of iNOS in cisplatin-induced apoptosis. However, blocking of all three isoforms of NOS with NG-amino-L-arginine in C13* cells dramatically changed these cells from chemoresistant to chemosensitive, greatly potentiating the pro-apoptotic effects of cisplatin. / Inhibition of Src-kinase activity reduces DNA synthesis in ovarian cancer cells. In an in vitro experiment, Src phosphorylated PKG on a tyrosine residue and PKG, presumable via serine-phosphorylation of Src, enhanced Src auto(tyrosine)phosphorylation. In ovarian cancer cells, inhibition of basal PKG activity with DT-2 decreased both basal and EGF-stimulated Src kinase activation and DNA synthesis. The data suggest that PKG at basal activity, is necessary for both basal and growth factor-stimulated Src kinase activation and enhanced DNA synthesis in human ovarian cancer cells. / The novel role of sGC/cGMP/PKG pathway on stimulating cell proliferation, potentially via interaction with the Src kinase pathway in human ovarian cancer cells, was demonstrated. ODQ dramatically reduced DNA synthesis rates, suggesting that basal sGC activity and basal cGMP levels are needed for ovarian cancer cell proliferation. DT-2 also reduced cell proliferation, suggesting the direct involvement of PKG. ANP and BNP had no effect on cell proliferation, suggesting that further activation of cGMP/PKG pathway above basal levels does not further enhance cell proliferation. / The present study also demonstrated that elevating cGMP slightly above the basal levels further protects pancreatic islet cells against spontaneous onset of apoptosis. The results showed that natriuretic peptides (both ANP and BNP) and low-level NO (i.e. physiological levels) as supply by NO donor, S-nitroso-N-acetylpenicilamine (SNAP) further prevented spontaneous apoptosis in pancreatic islets after isolation, whereas NO at high concentrations (i.e. pathological levels) promoted apoptosis in pancreatic islet cells. The commonly-used PKG inhibitor KT5823 and the newly-developed specific PKG inhibitor DT-2 completely prevented anti-apoptosic effect of ANP, suggesting the direct involvement of PKG in protection against spontaneous apoptosis. / The present study demonstrated that basal activity of sGC/cGMP/PKG signaling pathway is essential for partially limiting spontaneous apoptosis in pancreatic islet cells. The sGC inhibitor ODQ caused induction of apoptosis, which was completely blocked by co-treatment with ANP or BNP, agents that elevate cGMP via pGC, bypassing the ODQ block. Co-treatment with 8-Br-cGMP, a direct activator of PKG also completely prevented ODQ-induced apoptosis in islets. / Leung Lai-han. / "July 2006." / Adviser: Ronald Ray Fiscus. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1483. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 175-191). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

卵巢是女性生殖系統中一個重要的器官，負責為受精提供卵子，以及合成生殖過程中所必需，同時也在其他生理過程中起重要作用的各種激素。大約有30%的不育源於卵巢的問題，包括無排卵，無月經，月經週期不規律和激素水平異常等。雄激素:雌激素比例過高，卵泡發育異常，無排卵等卵巢功能障礙常見於各種疾病中，例如多囊性卵巢綜合征(PCOS)--一種影響5~10%育齡婦女的內分泌疾病，以及囊性纖維化( CF)--一種由囊性纖維化跨膜電導調節器(CFTR) 基因突變引起的遺傳疾病。然而引起這些卵巢功能障礙的確切機制並不清楚。 / 雌激素是在卵泡雌激素(FSH) 的調節下，在卵巢顆粒細胞中通過芳香化臨的住激素轉化而生成的。在論文第一部分的研究中，我們旨在證明CFTR 在卵巢顆粒細胞中的表達，以及它參與雌激素生成的過程。實驗結果證實了CFTR 在小鼠和人顆粒細胞中的表達，同時表明CFTR 通過一種碳酸氫根離子(HC0₃⁻) 敏感的可溶性腺苦酸環化梅(sAC) ，放大FSH 所刺激的雌激素生成過程。實驗結果顯示，在原代小鼠顆粒細胞中， HC0₃⁻能夠增強FSH 所引起的CREB 磷酸化，芳香化晦表達，以及雌激素的生成，而在抑制CFTR 的情況下，或在CFTR 敲除/DeltaF508 突變小鼠的顆粒細胞中， HCO3-的放大作用顯著降低。CFTR 和芳香化醋的表達水準在人顆粒細胞中具有正相關性，進一步支持CFTR 對雌激素生成的調節作用。在PCOS 患者的顆粒細胞和大鼠PCOS 模型的卵巢中， CFTR 和芳香化醋的表達水準顯著下調。這些結果提示， CFTR 對雌激素生成調節這一機制的缺陷可能參與了CF 和PCOS 中卵巢功能障礙的發病機理。 / 卵泡發育很大程度上依賴於顆粒細胞的增殖'生存和凋亡，這些過程在PCOS 中都會出現異常。論文的第二部分冒在研究顆粒細胞的CFTR 在PCOS 的卵泡發育異常中的作用。實驗結果表明， CFTR 在PCOS 大鼠的囊，性卵泡的顆粒細胞中表達降低，同時伴隨著PCNA 和Bcl-2 的下調，而Bax 和cleaved caspase-3則沒有變化，提示顆粒細胞的增殖和生存/抗凋亡能力降低。敲減或抑制顆粒細胞中的CFTR 導致細胞存活降低， PCNA 和Bcl-2 表達下調，以及細胞凋亡增加，提示CFTR 對顆粒細胞增殖和生存的調節作用。CFTR 通過HC0₃⁻/ sAC/PKA 信號通路，調節基礎及FSH 刺激引起的ERK I!2 磷酸化，及其下游的CyclinD2 和PCNA表達，從而促進顆樹圍胞的增殖。顆粒細胞CFTR 的下調可能通過抑制細胞增殖和降低細胞生存能力，參與了PCOS 中的囊性卵泡的形成過程。 / 綜上所述，本論文證明了CFTR 在卵巢顆粒細胞上的表達，並且參與調節顆粒細胞雌激素生成和細胞的增殖和生存。CFTR 的缺陷或表達下調可能是導致CF和PCOS 的卵巢功能障礙的發病機理。 / The ovary is the female reproductive organ, which produces female gametes, oocytes for fertilization and sex hormones essential to reproduction and important to a wide range of physiological and pathological events as well. About 30% of infertility cases arise from ovarian problems, including anovulation, amenorrhea, irregular menstrual cycle and abnormal hormone levels. Ovarian disorders, such as high androgen to estrogen ratio, abnormal folliculogenesis and anovulation, are often seen in diseases, including polycystic ovarian syndrome (PCOS) and cystic fibrosis (CF). The former is an endocrine disorder affecting 5~10% women of reproductive age, and the latter is a common genetic disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). However, the exact mechanisms underlying the ovarian disorders seen in these diseases are not well understood. / Estrogen biosynthesis is profoundly influenced by follicle-stimulating hormone (FSH) that regulates the conversion of androgen to estrogen in ovarian granulosa cells by the rate-limiting enzyme aromatase. The first part of the study aims to investigate the expression of CFTR in granulosa cells and its involvement in regulating estrogen production. The results demonstrate the expression of CFTR in both mouse and human granulosa cells, and provide evidence demonstrating a previously unsuspected role of CFTR in amplification of FSH-stimulated ovarian estrogen biosynthesis and the involvement of a HC0₃⁻ sensor, the soluble adenylyl cyclase (sAC) in this synthesis. FSH-stimulated CREB phosphorylation, aromatae expression, as well as estradiol production are enhanced by HC0₃⁻ and sAC, which could be significantly reduced by CFTR inhibition or in ovaries or granulosa cells of cftr knockout/deltaF508 mutant mice. The fact that CFTR expression is found positively correlated with aromatase expression in human granulosa cells supports its role in regulating estrogen production in humans. Reduced CFTR and aromatase expression is also found in polycystic ovarian syndrome (PCOS) rodent models and human patients. These findings suggest that defective CFTR-dependent regulation of estrogen production may underline the ovarian disorders seen in CF and PCOS. / Folliculogenesis largely depends on the proliferation, survival and apoptosis of granulosa cells in the follicles and alteration in which has been found in PCOS. The second part of the study aims to investigate the possible involvement of granulosa cell CFTR in the impaired folliculogenesis in PCOS. The results show that downregulation of CFTR is found in the cystic follicles, which is accompanied by reduced expression of PCNA and Bcl-2, but not Bax and cleaved caspase-3, in the ovaries of PCOS rat models, indicating reduced cell proliferation and survival/anti-apoptotic ability. Knockdown or inhibition of CFTR in granulosa cell culture results in reduced cell viability, downregulation of PCNA and Bcl-2 and increase of apoptosis, supporting a role of CFTR in regulating granulosa cell proliferation and survival. CFTR exerts its effect on granuloa cell proliferation by modulating basal and FSH-stimulated ERKl/2 phosphorylation and the expression of its downstream target CyclinD2 and PCNA through the HC0₃⁻/sAC/PKA pathway. These findings suggest that downregulation of CFTR may play a role in the formation of cystic follicles by inhibiting granulosa cell proliferation and reducing cell survival ability, therefore providing a possible mechanism for the abnormal folliculogenesis in PCOS. / In conclusion, the present study has demonstrated the expression of CFTR in the ovarian granulosa cell and its role in regulation of granulosa cell proliferation, survival and estrogen production. Defect of CFTR in CF and downregulation of CFTR in PCOS may contribute to the abnonnal honnone profile and impaired folliculogenesis in both disease conditions. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Chen, Hui. / "October 2011." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-137). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / ABSTRACT --- p.i / 摘要 --- p.iv / ACKNOWLEDGEMENT --- p.vi / LIST OF PUBLICATIONS --- p.viii / ABBREVIATIONS --- p.xiii / LIST OF FIGURES AND TABLES --- p.xvi / Chapter 1 --- CHAPTER I: Introduction --- p.1 / Chapter 1.1 --- The ovary --- p.1 / Chapter 1.1.1 --- Structure and function of the ovary --- p.1 / Chapter 1.1.2 --- Follicle development --- p.5 / Chapter 1.1.3 --- Ovulation and luteinization --- p.7 / Chapter 1.1.4 --- Ovarian hormone biosynthesis --- p.10 / Chapter 1.2 --- Diseases with ovarian dysfunction --- p.14 / Chapter 1.2.1 --- Polycystic ovarian syndrome (PCOS) --- p.14 / Chapter 1.2.1.1 --- Introduction to PCOS --- p.14 / Chapter 1.2.1.2 --- Diagnostic criteria --- p.14 / Chapter 1.2.1.3 --- Abnormal hormone profile in PCOS --- p.16 / Chapter 1.2.1.4 --- Abnormal folliculogenesis in PCOS --- p.18 / Chapter 1.2.1.5 --- Etiology --- p.22 / Chapter 1.2.2 --- Cystic Fibrosis (CF) --- p.24 / Chapter 1.2.2.1 --- Introduction to CF --- p.24 / Chapter 1.2.2.2 --- Cause and pathogenesis of CF --- p.25 / Chapter 1.2.2.3 --- Ovarian disorder in CF --- p.27 / Chapter 1.3 --- CFTR in reproduction --- p.29 / Chapter 1.3.1 --- Introduction to CFTR --- p.29 / Chapter 1.3.2 --- Channel function --- p.30 / Chapter 1.3.3 --- Protein regulator function --- p.32 / Chapter 1.3.4 --- Regulation of CFTR expression --- p.34 / Chapter 1.3.5 --- Role of CFTR in reproduction --- p.35 / Chapter 1.3.6 --- CFTR in the ovary --- p.39 / Chapter 1.4 --- General hypothesis and aims --- p.39 / Chapter 1.4.1 --- General hypothesis --- p.39 / Chapter 1.4.2 --- Aims of the study --- p.40 / Chapter 2 --- CHAPTER II: General Methods --- p.42 / Chapter 2.1 --- Meterials --- p.42 / Chapter 2.1.1 --- Animals --- p.42 / Chapter 2.1.2 --- Chemicals and reagents --- p.42 / Chapter 2.1.3 --- Antibodies --- p.44 / Chapter 2.1.4 --- Primers --- p.45 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Determination of estrous cycle --- p.45 / Chapter 2.2.2 --- Granulosa cell culture --- p.46 / Chapter 2.2.3 --- PCGS rat model --- p.47 / Chapter 2.2.4 --- Collection of human granulosa cells --- p.47 / Chapter 2.2.5 --- Reverse transcription-polymerase chain reaction (RT-PCR) --- p.48 / Chapter 2.2.6 --- Western blot --- p.50 / Chapter 2.2.7 --- Histological studies --- p.53 / Chapter 2.2.8 --- siRNA transfection --- p.55 / Chapter 2.2.9 --- Intracellular pH measurement --- p.56 / Chapter 2.2.10 --- Whole-cell patch clamp recording --- p.57 / Chapter 2.2.11 --- Statistics --- p.57 / Chapter 3 --- CHAPTER III: Result I - The Role of CFTR in FSH-stimulated Estrogen Production: Implication in Cystic Fibrosis and PCGS --- p.59 / Chapter 3.1 --- Summary --- p.59 / Chapter 3.2 --- Introduction --- p.60 / Chapter 3.3 --- Methods --- p.63 / Chapter 3.3.1 --- Intracellular cAMP assay --- p.63 / Chapter 3.3.2 --- Nuclei isolation and nuclear cAMP measurement --- p.63 / Chapter 3.3.3 --- CREB phosphorylation assay --- p.64 / Chapter 3.3.4 --- Estradiol enzyme immunoassay --- p.64 / Chapter 3.4 --- Results --- p.64 / Chapter 3.4.1 --- Functional expression of CFTR in granulosa cells --- p.64 / Chapter 3.4.2 --- Expression and localization of sAC in granulosa cells and its involvement in BC03f CFTR-dependent cAMP production --- p.66 / Chapter 3.4.3 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated CREB phosphorylation --- p.67 / Chapter 3.4.4 --- Effect of CFTR and HC0₃⁻ on basal and FSB-stimulated aromatase expression and estradiol production --- p.68 / Chapter 3.4.5 --- Impaired CREB phosphorylation aromatase expression and estradiol production by granulosa cells from CFTR-deficient mice --- p.70 / Chapter 3.4.6 --- Reduced CFTR and aromatase expression in human PCOS granulosa cells and rat PCOS ovaries --- p.71 / Chapter 3.5 --- Discussion --- p.87 / Chapter 4 --- CHAPTER IV: Result II - The Role of CFTR in Granulosa Cell Proliferation and survival in PCOS --- p.91 / Chapter 4.1 --- Summary --- p.91 / Chapter 4.2 --- Introduction --- p.92 / Chapter 4.3 --- Methods --- p.95 / Chapter 4.3.1 --- Cell viability assay (MTT and MTS assay) --- p.95 / Chapter 4.3.2 --- ERKI/2 phosphorylation assay --- p.95 / Chapter 4.4 --- Results --- p.96 / Chapter 4.4.1 --- Reduced CFTR expression in PCOS rat models --- p.96 / Chapter 4.4.2 --- Downregulation of genes related to proliferation and survival in PCOS --- p.96 / Chapter 4.4.3 --- CFTR affect viability of granulosa cells --- p.97 / Chapter 4.4.4 --- CFTR regulate cell cycle protein and promote proliferation via HC0₃⁻/sAC/PKA and ERK pathway --- p.98 / Chapter 4.4.5 --- CFTR regulates apoptosis-related protein expression --- p.100 / Chapter 4.5 --- Discussion --- p.114 / Chapter 5 --- CHAPTER V: General Discussion --- p.119 / Chapter 5.1 --- Role of CFTR in ovarian function --- p.119 / Chapter 5.2 --- Role of CFTR/HC0₃⁻/sAC in modulating FSH signaling in the ovary --- p.120 / Chapter 5.3 --- CFTR/HC0₃⁻/sAC as a general modulator in receptor-mediated signaling cascades --- p.122 / Chapter 5.4 --- Concluding remarks --- p.123 / REFERENCES --- p.124 / APPENDICES --- p.138

A síndrome dos ovários policísticos (SOP) é o distúrbio endócrino-metabólico mais frequente na menacme, com prevalência de 7 a 10 %, contribuindo com o aumento do risco cardiovascular e/ou diabetes mellitus tipo II nessas mulheres. OBJETIVOS: Identificar as características epidemiológicas e os diferentes fenótipos da SOP, a prevalência da síndrome metabólica encontrada em cada fenótipo e os fatores associados ao risco metabólico dessas pacientes. CASUÍSTICA E MÉTODO: Estudo observacional com 566 mulheres entre 14 e 39 anos portadoras de SOP, segundo o consenso de Rotterdam. O risco metabólico foi avaliado pela análise descritiva com intervalo de confiança de 95%. As variáveis quantitativas foram testadas pelo método de Shapiro-Wilk e teste não paramétrico de Mann-Whitney. Para a análise multivariada usou-se a razão de prevalências entre as diversas variáveis independentes e o desfecho risco metabólico. Identificamos os fatores associados ao risco metabólico empregando a regressão de Cox com variância robusta. RESULTADOS: Das 566 pacientes, 27,9% tinham entre 20 e 24 anos; 84,5% eram afrodescendentes; 90,6% apresentavam irregularidade menstrual; 91,8% hirsutismo; 77,7% ovários aumentados e/ou policísticos; 15,7% com pelo menos um filho; IMC elevado em 66,5%; CA superior a 88 em 51%; pressão arterial sistólica e diastólica elevadas em 38,9% e 20% das pacientes respectivamente; 7,7% intolerância a carboidratos, 40,8% de HDL-colesterol reduzido, 8,8% de triglicerídeos elevados. Encontramos risco metabólico em 21%, com predomínio dos fenótipos E (28,4%), B (25%) e A (22%). Antecedentes familiares de diabete, hipertensão arterial, câncer ginecológico e câncer não ginecológico não contribuíram, com significância estatística, para o aumento de eventos metabólicos. O acréscimo de um ano na idade elevou o risco em 5%. A cada subida de uma unidade no IMC foram adicionados 8%. A presença de hirsutismo triplicou o risco. Pacientes com pelo menos um filho apresentaram duas vezes mais síndrome metabólica do que as sem filhos. CONCLUSÕES: Foi observada maior frequência de síndrome metabólica entre os fenótipos que apresentam em comum oligoanovulação e hirsutismo (E, B e A). Em pacientes com SOP a idade, a paridade, a presença de hirsutismo e obesidade foram os fatores independentemente relacionados ao aumento do risco metabólico / Polycystic ovary syndrome (PCOS) is an endocrine and metabolic disorder that is more frequent in premenopausal, affecting 7 to 10% of women, contributing to the increase of cardiovascular and/or type II diabetes mellitus risk. OBJECTIVES: To identify the epidemiological characteristics and different phenotypes of PCOS, the prevalence of metabolic syndrome found in each phenotype and metabolic risk factors associated with these patients. PATIENTS AND METHODS: Observational study of 566 women between 14 and 39 years with PCOS according to the Rotterdam criteria. The metabolic risk was assessed by descriptive analysis with a confidence interval of 95%. Quantitative variables were tested by using Shapiro-Wilk method and nonparametric Mann-Whitney test. For the multivariate analysis the prevalence ratio between several independent variables and the outcome metabolic risk were used. Factors associated with the metabolic risk were identified by using Cox regression with a robust variance. RESULTS: Of 566 patients, 27.9% were between 20 and 24 years, 84.5% were of African descents; 90.6% had oligoanovulation; 91.8% hirsutism; 77.7% enlarged ovaries and/or polycystic, 15.7% with at least one child in high BMI 66.5%, CA 88 exceeding 51%; systolic and diastolic blood pressure elevated by 38.9% and 20% of patients, respectively, 7.7% carbohydrate intolerance, 40.8% HDL-cholesterol changed, 8.8% triglyceride levels. Metabolic risk found in 21%, with a predominance of E phenotypes (28.4%), B (25%) and A (22.1%). Family history of diabetes, hypertension, gynecological cancer and gynecological cancer does not contribute with statistical significance for increased metabolic events. The one-year increase in age raised the risk by 5%. Every increase of one unit in BMI 8% were added. Presence of hirsutism tripled the risk. Patients with at least one child were twice as metabolic syndrome than those without children. CONCLUSIONS: A higher frequency of metabolic syndrome phenotypes that have in common oligoanovulation and hirsutism (E, B and A) were observed. Independently associated factors with the metabolic risk in PCOS patients were age, parity, hirsutism and obesity

In this dissertation, we developed cell stiffness as a biomarker in ovarian cancer for the purpose of grading metastatic potential. By measuring single cell stiffness with atomic force microscopy and quantifying in vitro invasiveness of healthy and cancerous ovarian cells, we demonstrated that cancerous ovarian cells have reduced stiffness compared to the healthy ones and invasive ovarian cancer cells are more deformable than noninvasive ovarian cancer cells. The difference in cell stiffness between two genetically similar cell lines was attributed to actin-mediated cytoskeletal remodeling as revealed by comparative gene expression profile analysis, and was further confirmed by fluorescent visualization of actin cytoskeletal structures. The actin cytoskeletons were innovatively quantified and correlates with cell stiffness distributions, further implicating actin-mediated cytoskeletal remodeling in stiffness alteration from the perspective of structure-property relationship. The correlation between stiffness and metastatic potential was also demonstrated in pancreatic cancer cell line AsPC-1, which shows reduced invasivess and increased stiffness upon treatment with N-acetyl-L-cysteine (NAC), a well known antioxidant, reactive oxygen species (ROS), scavenger and glutathione precursor. The correlation between cell stiffness and metastatic potential as demonstrated in ovarian and pancreatic cancer cells indicated that mechanical stiffness may be a useful biomarker to evaluate the relative metastatic potential of ovarian and perhaps other types of cancer cells, and might be useful clinically with the development of rapid biomechanical assaying techniques. We have also investigated the stiffness evolution through progression of the cell cycle for the healthy ovarian phenotype and the invasive cancer ovarian phenotype, and found that the healthy phenotype at G1 phase are significantly stiffer than other single cells except the invasive phenotype at late mitosis; other groups are not significantly different from each other. We have also investigated intracellular heterogeneity and mechanical nonlinearity in single cells. To this end, we developed a methodology to analyze the deformation-dependent mechanical nonlinearity using a pointwise Hertzian method, and tested the method on ultrathin polydimethylsiloxane (PDMS) films which underwent extremely large strains (greater than 50%). Mechanical stiffening due to large strain and geometrical confinement were observed. The onset of nonlinearity or mechanical stiffening occurs at 45% of the film thickness, the geometry induced stiffening causes an increase in stiffness which shows a strong power law dependence on film thickness. By applying the pointwise Hertzian method on stiffness measurements with AFM that were collected on living cells, we also investigated the nonlinear and heterogeneous mechanics of single cells, since attachment of cells to stiff substrate during indentation may impact their mechanical responses. Even under natural biological conditions, cells confined in narrow spaces may experience heightened mechanical stiffness. Through indentation-dependent force mapping, analysis of the local cell stiffness demonstrated spatial variation. The results indicated that the mechanical properties of single cells are highly nonlinear and are dependent upon the subcellular features under the applied force as well as the dimensions of the cellular material. We identified single cell stiffness as a potential biomarker of the metastatic potential in ovarian cancer, and quantified the effect of geometrical confinement on cell mechanics. The results presented in this dissertation not only made contributions to the development of accurate, non-invasive clinical methods to estimate metastatic potential of ovarian and perhaps other types of cancer, but also shed light on the intracellular mechanical information by developing new techniques to quantify the effect of geometry on cell mechanics.

Τα stem κύτταρα είναι ένας υποπληθυσμός κυττάρων με δύο κύριες ιδιότητες: αυτοανανέωση και διαφοροποίηση. Τα stem κύτταρα διαμένουν σε ένα εξειδικευμένο μικροπεριβάλλον, την φωλεά, η οποία παίζει σημαντικό ρόλο στη διατήρηση της ισορροπίας μεταξύ της αυτοανανέωσης και της διαφοροποίησης. Τελευταία δεδομένα εισηγούνται ότι ο καρκίνος αναπτύσσεται από ένα υποσύνολο κυττάρων με ιδιότητες ανάλογες αυτών των φυσιολογικών stem κυττάρων. Τα κύτταρα αυτά αποκαλούνται καρκινικά stem κύτταρα. Η θεωρία των καρκίνικών stem κυττάρων υποστηρίζει ότι τα καρκινικά stem κύτταρα εγκαινιάζουν και συντηρούν την ανάπτυξη και εξέλιξη του όγκου, ευθύνονται για την κυτταρική ετερογένεια των καρκίνικών κυττάρων του όγκου, είναι υπεύθυνα για τις μεταστάσεις και παραμένουν στους ασθενείς παρά τη χρήση των συμβατικών χημειοθεραπευτικών παραγόντων. Πρόσφατα δεδομένα πιστοποιούν την ύπαρξη καρκινικών stem κυττάρων στην ωοθήκη. / Stem cells are a subpopulation of cells with two key properties: self-renewal and differentiation. Stem cells reside in a specialized microenvironment, i.e. niche, which plays an important role in the balance between self-renewal and differentiation. Recent data suggest that cancer develops from a subset of cells with properties similar to those of normal stem cells. These cells are called cancer stem cells. Cancer stem cell hypothesis suggest that cancer stem cells initiate and preserve the growth of tumor, they are responsible for cellular heterogeneity and metastasis of tumor and they are, finally, drug-resistant.Latest data suggest the presence of cancer stem cells in the ovary.

Como em outros mosquitos, os trofócitos do corpo gorduroso de Cx. quinquefasciatus sintetizam vitelogenina (Vg), principal proteína armazenada pelo ovócito, formada por duas subunidades de 200 e 86 kDa. A ultraestrutura dos trofócitos revela o rápido desenvolvimento da maquinaria biossintética após a alimentação com sangue (aa) e a consecutiva degradação após as 48 h aa. Antes do repasto (AR), um conjunto de células indiferenciadas, limitado pelo epitélio folicular, conforma os folículos ovarianos. Após AR, o ovócito se destaca pelo acúmulo de lipídeos e Vg. O receptor de vitelogenina é encontrado somente nos ovários e análise por PCR em tempos mostrou aumento dos transcritos nos primeiros cinco dias após emersão e nas primeiras 48 h aa, durante a vitelogênese. O perfil transcricional de Vg mostrou um pico no terceiro dia de vida adulta e ao final do processo ovogênico / As in other mosquitoes, fat body trophocytes of Cx. quinquefasciatus synthesize vitellogenin (Vg), the major yolk protein stored by the oocyte, formed by two subunits of 200 and 86 kDa. The trophocytes ultrastructure reveals the rapid development of the biosynthetic machinery and the consecutive degradation around 48 h post blood meal (PBM). Before blood meal, a set of undifferentiated cells limited by follicular epithelium, conform the ovarian follicles. After blood meal, the oocyte is remarkable by accumulation of lipid inclusions and yolk granules. Vitellogenin receptors (rVitCx), are localized exclusively in the ovaries and real time PCR showed transcripts increase at the first five days after emergence (AE), and at the first 48 h PBM, during oogenesis. Vg transcripts profile showed a peak on the third day AE and at the end of the vitellogenic process