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Orientador: Agnaldo Lopes da Silva Filho / Coorientador: Andréa Teixeira de Carvalho / Resumo: O câncer epitelial de ovário representa um desafio para Oncologia Ginecológica, devido à sua natureza insidiosa e alta mortalidade. Há também uma compreensão limitada da etiologia da doença ao nível molecular, o que continua a dificultar o desenvolvimento de alvos terapêuticos. Estudos recentes de morfologia, imuno-histoquímica e de genética molecular têm levado ao desenvolvimento de um novo paradigma para a patogênese e a origem do câncer epitelial de ovário, baseado em um modelo dualista de carcinogênese que divide o câncer epitelial de ovário (CEO) em duas grandes categorias chamadas de tipos I e II. O objetivo deste estudo foi avaliar o padrão das citocinas e quimiocinas encontradas no tecido ovariano de mulheres saudáveis e mulheres com CEO tipo I e tipo II. Além disso, descrever a associação desses biomarcadores com os dados clínico-patológicos. Foram analisadas amostras de tecido ovariano e ascite obtidas de mulheres com CEO (n=26) e amostras de tecido ovariano e lavado peritoneal de mulheres sem evidências de malignidade (n=16 - grupo de controle). Nas amostras de tecido, a expressão gênica foi avaliada utilizando a metodologia de PCR quantitativo em tempo real (qPCR) para os genes IFNG, IL-10, TGFB1, CCL2, CCL3, CCL5, CXCL8, CXCL9 e CXCL10. A detecção dos níveis de citoquinas/quimioquinas nos fluidos peritoneais foi realizada através do método Cytometric Bead Array (CBA) os marcadores IL-12p70, TNF, IL-10, IL-6, IL-1--8, IL-17A, IFN-IL-4, IL-2, CCL2, CCL5, CXCL-9 e CXCL-10. No grupo das pacientes com CEO, 10 (38,5%) apresentavam estágios I/II e 16 (61,5%) estágios III/IV. Com relação ao tipo de tumor, de acordo com a nova classificação, 8 (30,8%) eram do tipo I e 18 (69,2 %) do tipo II. A citorredução ótima foi obtida em 15 (57,7%) das mulheres com CEO. Mulheres com CEO tipo II apresentaram maiores níveis séricos do marcador CA-125 quando comparado às do tipo I. Não houve óbito no grupo... / Abstract: The epithelial ovarian cancer represents a challenge to Gynecologic Oncology due to its insidious nature and high mortality. There is also a limited understanding of disease etiology at the molecular level, which continues to hamper targeted therapeutic development. Recent morphological, immunohistochemical, and molecular genetic studies have led to the development of a new paradigm for the pathogenesis and origin of epithelial ovarian cancer based on a dualistic model of carcinogenesis that divides epithelial ovarian cancer into 2 broad categories designated types I and II. The aim of this study was to evaluate the cytokines and chemokines pattern in ovarian tissue from healthy women and women with EOC type I and type II. Also, we described the association of these biomarkers with the clinicopathological data. Samples of ovarian tissue and ascite obtained from women with EOC (n=26), samples of ovarian tissue and peritoneal wash from women with no evidence of malignancy were analyzed (n=16 - control group). In the tissue samples, gene expression were evaluated by quantitative real time PCR (qPCR) IFNG, IL-10, TGFB1, CCL2, CCL3, CCL5, CXCL8, CXCL9 and CXCL10. The detection of cytokine/chemokine levels in peritoneal fluids was measured by cytometric bead array immunoassay (CBA), IL-12p70, TNF, IL-10, IL-6, IL-1--8, IL-17A, IFN--4, IL-2, CCL2, CCL5, CXCL-9 and CXCL-10. In the group of women with EOC, 10 (38.5 %) had stage I/II and 16 (61.5 %) were stage III/IV . Concerning tumor type, according to the new classification, 8 (30.8 %) were type I and type II were 18 ( 69.2 % ) . Optimal cytoreduction was achieved in 15 (57.7 %) women with EOC. The CA-125 showed higher serum levels in patients with EOC type II compared to type I. There were no deaths in women with type I tumor while 6 (33.3 %) of patients with type II tumor died . Increased expression of IL-10, CXCL8 and CXCL9 genes in the group of women / Doutor

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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.

University of Macau / Institute of Chinese Medical Sciences

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

The objectivie of this study was to describe the splanchnology and topography of the female reproductive organs of the African lion. The reproductive organs of three embalmed cadavers from three-year-old known aged nulliparous lionesses weighing between 120 kg and 140 kg were studied. Two fresh carcasses from another study were used for some of the topographical photos since these rendered better quality images. The project was approved by the Animal Use and Care Committee and Research Committee of the University of Pretoria (protocol number V038-09). The topography and splanchnology of the reproductive organs were studied and described in situ and after removal. The kidneys were located far caudally in relation to the thirteenth ribs with the left kidney further caudal. The suspensory ligament was very well developed. It originated in a fan-like manner from the dorso-lateral abdominal wall lateral to the kidney extending up to a few centimetres cranial to the kidney. The proper ligament of the ovary was well developed and consisted of several clearly distinguishable bands. The broad ligament resembled that of the cat with the exeption of the cranial part of the mesovarium being very well developed. The round ligament was well developed and inserted on the medial femoral fascia. It therefore did not extend to the vulva as in other canine and feline species. The left ovary was longer, wider and heavier than its right counterpart and the ovaries were relatively small in relation to body weight. The ovarian bursa had a short mesosalpinx that did not cover any part of the ovary and the fimbriae extended the entire length of the ovary. The urethral tuberculum as well as the urethral crest was very well developed. The left uterine horn was longer than the right and the tip of the uterine horn was located dorsal to the proper ligament. The uterine tube was prominently convoluted, situated entirely on the lateral aspect of the ovary and was found to open directly into the tip of the uterine horn and not onto a papilla. The female reproductive organs of the African lion resemble that of the domestic cat and dog with some major differences especially to the size and development of certain structures. The clinical relevance of these differences has to be debated and some thoughts might include the following: 1) does the suspensory and proper ligaments of the gravid uterus contract to suspend the entire uterus closer to the body wall during hunting since the lioness is the primary hunter in the pride? 2) does the lioness have to roll on her back and extend her back legs after mating to facilitate sperm entering the dorsally located tip of the uterine horn while the round ligament stabilises the terminal part of the uterine horn, and does this have a bearing on the fact that multiple copulations are required over a prolonged period for conception by lions? 3) do more ovulations take place from the bigger left ovary and? 4) are more conceptusses borne by the longer left uterine horn? The anatomical information obtained during this study was subsequently applied in a surgical study on sixteen lionesses using laparoscopy to perform laparoscopic ovariectomy and salpingectomy. The availability of these two laparoscopic procedures subsequently led to a wider interest to its application in population control of lions in the smaller national parks of South Africa. / Dissertation (MMedVet)--University of Pretoria, 2012. / gm2014 / Companion Animal Clinical Studies / unrestricted

<p> This research was designed to gain a better understanding of the relationships between nutrition, aging and oogenesis. Cohorts of 1800 inbrod, adult house flies were maintained on various undefined and chemically defined diets. From these flies samples for histology were removed periodically, and the rest were analyzed for their survival and their ability to develop and lay viable eggs. The survival of the females was more affected by nutrition and other factors than that of the males. In females fed sugar water, oogenesis was arrested at an early stage, but survival was lower than on a milk diet. From adult emergence until the completion of one or more ovarian cycles, the larval and adult fatbodies and the ovary were compared as to their histology, histochemistry and cytology. / Thesis / Master of Science (MSc)

No description available.

Systems pathology attempts to introduce more holistic approaches towards pathology and attempts to integrate clinicopathological information with “-omics” technology. This doctorate researches two examples of a systems approach for pathology: (1) a personalized patient output prediction for ovarian cancer and (2) an analytical approach differentiates between individual and collective tumour invasion. During the personalized patient output prediction for ovarian cancer study, clinicopathological measurements and proteomic biomarkers are analysed with a set of newly engineered bioinformatic tools. These tools are based upon feature selection, survival analysis with Cox proportional hazards regression, and a novel Monte Carlo approach. Clinical and pathological data proves to have highly significant information content, as expected; however, molecular data has little information content alone, and is only significant when selected most-informative variables are placed in the context of the patient's clinical and pathological measures. Furthermore, classifiers based on support vector machines (SVMs) that predict one-year PFS and three-year OS with high accuracy, show how the addition of carefully selected molecular measures to clinical and pathological knowledge can enable personalized prognosis predictions. Finally, the high-performance of these classifiers are validated on an additional data set. A second study, an analytical approach differentiates between individual and collective tumour invasion, analyses a set of morphological measures. These morphological measurements are collected with a newly developed process using automated imaging analysis for data collection in combination with a Bayesian network analysis to probabilistically connect morphological variables with tumour invasion modes. Between an individual and collective invasion mode, cell-cell contact is the most discriminating morphological feature. Smaller invading groups were typified by smoother cellular surfaces than those invading collectively in larger groups. Interestingly, elongation was evident in all invading cell groups and was not a specific feature of single cell invasion as a surrogate of epithelialmesenchymal transition. In conclusion, the combination of automated imaging analysis and Bayesian network analysis provides an insight into morphological variables associated with transition of cancer cells between invasion modes. We show that only two morphologically distinct modes of invasion exist. The two studies performed in this thesis illustrate the potential of a systems approach for pathology and illustrate the need of quantitative approaches in order to reveal the system behind pathology.