A esporotricose é uma micose subcutânea causada por espécies do complexo Sporothrix schenckii que acomete seres humanos e animais, principalmente os gatos. Desde 1998 o Instituto Nacional de Infectologia Evandro Chagas/Fiocruz no Rio de Janeiro vem acompanhando uma epidemia dessa doença envolvendo seres humanos, cães e gatos, onde as principais formas de transmissão são arranhadura, mordedura e/ou contato com o exsudato das lesões cutâneas de gatos doentes. O diagnóstico definitivo da esporotricose felina é obtido a partir do isolamento do Sporothrix sp. em meios de cultura, entretanto, o resultado desse exame pode demorar até quatro semanas, o que em algumas situações pode retardar o início do tratamento antifúngico. Os exames citopatológico, histológico e imuno-histoquímico são opções viáveis e mais rápidas para o diagnóstico dessa micose em gatos, principalmente em situações quando não é possível realizar o isolamento fúngico. O diagnóstico precoce da esporotricose felina é importante na implementação rápida do tratamento antifúngico, melhorando o prognóstico na maioria dos casos. O objetivo deste estudo foi avaliar e comparar as diferentes técnicas utilizadas no diagnóstico da esporotricose felina antes e durante o tratamento antifúngico. Na primeira etapa do estudo foram comparados os exames citopatológico (coloração pelo método panótico rápido), histológico (impregnação pela prata de Grocott) e imuno-histoquímico de 184 gatos no diagnóstico da esporotricose sem tratamento antifúngico prévio utilizando o cultivo fúngico como teste padrão de referência. Estruturas leveduriformes foram observadas em 160 (87,0%) casos no exame citopatológico, 168 (91,3%) na histopatologia e 163 (88,3%) na imunohistoquímica. A associação das três técnicas elevou a sensibilidade do diagnóstico para 98,3%, o que enfatiza a necessidade de sua implementação como ferramentas de rotina, sobretudo quando a cultura fúngica não está disponível. Na etapa seguinte, a carga parasitária e o isolamento de Sporothrix sp. das lesões cutâneas de 74 gatos foram avaliados mensalmente antes e durante o tratamento com itraconazol por um período de 12 semanas. A mediana da carga fúngica observada antes do início do tratamento antifúngico foi maior (pMW = 0,013) nos gatos nos quais foi observada a persistência da lesão (Med=98,6) em relação aqueles em que houve cicatrização (Med=15,0). A redução da carga fúngica ocorreu em todas as lesões estudadas, assim como a redução da positividade da cultura fúngica e do exame citopatológico. Estes resultados sugerem uma redução no potencial zoonótico dos gatos, enfatizando a importância do tratamento precoce como medida de controle. Adicionalmente, o isolamento do fungo e a presença de estruturas leveduriformes nas lesões de gatos com esporotricose podem ser fatores preditores da falência terapêutica, indicando a necessidade da implementação de alternativas terapêuticas. / Sporotrichosis is subcutaneous mycose caused by species of fungus from the Sporothrix schenckii complex and affects humans and animals, especially cats. Since 1998, the Instituto Nacional de Infectologia Evandro Chagas/Fiocruz in Rio de Janeiro has been describing in Rio de Janeiro an epidemic of sporotrichosis, involving humans, cats and dogs, with most of cases related to transmission through scratches, bites or contact with lesions from infected cats. The definitive diagnosis is based on the isolation of the fungus in culture; however, the results may take up to four weeks and postpone treatment outset. The cytopathology, histopathology and immunohistochemistry should be considered as rapid and accessible alternatives for the diagnosis in cats, especially when the fungal culture is not available. The early diagnosis of feline sporotrichosis is desirable for the prompt beginning of the antifungal treatment, improving the prognosis in most of the cases. The aim of this study was to evaluate and compare different techniques for the diagnosis of feline sporotrichosis before and during the treatment with itraconazol. In the first part of the study, cytopathological (Quick Panoptic), histopathological (Grocott silver stain) and immunohistochemical examinations were compared regarding the diagnosis of sporotrichosis in 184 cats without previous treatment, by using fungal culture as a reference standard. The yeast-like cells were observed in 160 (87.0%) cases by cytopathological examination, in 168 (91.3%) by histopathology and in 163 (88.3%) by immunohistochemistry. The combination of the three methods led to the diagnosis of 98.3% of cases, pointing to the need of their implementation as regular tools, notably when fungal culture is not available. In the second part of the study, the fungal burden and the isolation of Sporothrix sp. in cutaneous lesions of 74 cats were monthly evaluated before and during the treatment with itraconazole for twelve weeks. The median of the fungal load detected before the outse of the antifungal treatment was higher (pMW=0.013) in cats in which there was a persistence of the cutaneous lesion (Med=98.6) in comparison to those in which healing of the lesion was observed (Med=15.0). The decrease of the fungal burden occurred in all the lesions in this study as well as the reduction of the positivity of the fungal culture and the cytopathological examination. These results suggest a reduction in the zoonotic potential of cats and emphasize the importance of the early treatment as a control measure. In addition, the isolation of the fungus and the presence of yeast-like cells in lesions of cats with sporotrichosis during the treatment can be a predictor of treatment failure and should alert for the need of alternative therapeutic regimens.

Proposição: Avaliar a associação entre as condições de saúde bucal, fatores comportamentais e sociodemográficos e o padrão citopatológico da mucosa bucal de homens adultos. Correlacionando o padrão citopatológico quantitativo e qualitativo com as variáveis bucais e sociodemográficas. Materiais e Métodos: A partir de dois esfregaços, um da borda da língua e outro do assoalho bucal de 117 homens com mais de 25 anos, foram quantificadas cem células de cada. As células foram coradas pelo método Papanicolaou modificado e classificadas em: Escamas, células superficiais com núcleo, células intermediárias e células parabasais. Variáveis sociodemográficas e comportamentais foram coletadas a partir de um questionário estruturado. Índice CPO-D e uso de próteses foram registradas a partir de um exame clínico intra-oral.As análises foram conduzidas utilizando o pacote estatístico Stata versão 10. O indivíduo foi considerado a unidade analítica. O nível de significância foi estabelecido em 5%. Resultados: No total das células na borda da língua, 75% eram intermediárias, 20% superficiais com núcleo e 5% escamas, sendo que células parabasais foram raramente observadas. Não foram observadas diferenças significativas para todas as variáveis em estudo em todos os tipos celulares, com exceção da ingestão de bebidas alcoólicas.Observou-se percentual significativamente maior de escamas e células superficiais com núcleo nos indivíduos que ingeriam de álcool comparados aos que não ingerem. Para as células intermediárias, o percentual foi significativamente menor nos indivíduos que bebem comparados aos que não bebem. Um padrão semelhante de distribuição celular foi observado no assoalho de boca. O consumo de álcool foi o único fator comportamental que influenciou significativamente no padrão citológico da mucosa bucal dos indivíduos analisados nos modelos multivariados. Conclusões: Foi encontrada associação entre um dos fatores comportamentais (o álcool) e o padrão citopatológico da mucosa bucal de homens adultos. As demais variáveis estudadas não apresentaram associação significativa com o padrão de descamação da mucosa bucal normal com a técnica utilizada. / Proposition: To evaluate the association between oral health status, socio- demographic and behavioral factors and standard cytology of the oral mucosa of adult men. Correlating the standard cytopathology quantitative and qualitative with oral and sociodemographic variables. Materials and Methods: From two smears, one edge of the tongue and other oral floor of 117 men over 25 years old, were quantified hundred cells each. Cells were stained with modified Papanicolaou method and classified into: Scales, superficial cells with nuclei, intermediate and parabasal cells. Sociodemographic and behavioral variables were collected from a structured questionnaire. DMFT index and use of prostheses were recorded from a clinical intra - oral.As analyzes were conducted using Stata version 10. The individual was considered the analytical unit. The level of significance was set at 5 %. Results: In total cells at the edge of the tongue 75% were intermediate, 20% core surface scales and 5%, and parabasal cells were rarely observed. No significant differences were observed for all study variables in all cell types, with the exception of beverage intake alcoólicas.Observou is significantly greater percentage of scales and superficial cells in the core subjects ingestores alcohol compared to those who do not drink. For intermediate cells, the percentage was significantly lower in individuals who drink compared to those who do not drink. A similar pattern of cell distribution was observed in the floor of mouth. Alcohol consumption was the only factor that significantly influenced the behavioral cytology of the oral mucosa of individuals analyzed in the multivariate models. Conclusions: An association between a behavioral factor (alcohol) and standard cytology of the oral mucosa of adult men. The other variables were not significantly associated with the default scaling of normal oral mucosa with the technique used.

Made available in DSpace on 2017-07-10T14:17:03Z (GMT). No. of bitstreams: 1 sostenez.pdf: 5444547 bytes, checksum: 0e57c73e9d81ba62bd8cce82698d43f6 (MD5) Previous issue date: 2013-10-17 / Bombyx mori nucleopolyhedrovirus (BmNPV) is an entomopathogenic and poliorganotrophic virus that infects Bombyx mori. BmNPV consists of a double strand DNA with two distinct phenotypes: the derived polyhedral virus (PDV), responsible for the primary infection in the insect s midgut; and the budded virus (BV), which disperses the infection in the hemocele, causing secondary or systemic infection. It is extremely virulent and when it infects silkworms, causes serious damages to the insect, usually leading to its death or harming the silk production, affecting all the productive chain, leading to economic losses. For a good silk production, a determinant factor is the functioning of the food duct, which is divided in foregut, midgut and hindgut, being the hindgut the place for water and mineral salts absorption and for the end of the digestive process. Several tissues have been established as virus targets, however, others have shown to be resilient to BmNPV. Given the importance of the hindgut, the present paper aimed to verify the susceptibility of its components, ileum, colon and rectum to an geographic isolated of BmNPV in Paraná, Brasil, the BmMNPV. In different post-inoculation days (dpi), the hindgut segment of 5th instar silkworms was dissected and processed for analysis in light microscopy, using conventional dyes and cytochemistry for viral detection and electronic scanning microscopy for the morphological details. The results revealed that the ileum, colon and rectum of the B. mori are constituted by simple epithelium, with alterations in cell morphology, covered by an intima in its luminal side, and that its organization is similar to that of other described insects. The cytological analysis of the ileum, colon and fore rectum revealed that its epithelial cells are not susceptible to BmMNPV in neither of the times analyzed. However, the posterior hindgut or anal duct showed itself to be susceptible to the virus after the 5º dpi, developing all the classic signs described for the infection with AlphaBV, as the presence of viroplasm, nuclear hypertrophy, polyhedra in formation and mature ones. At the end of the infectious cycle, occurs cell lysis with the liberation of viral polyhedra in the intestinal lumen and, consequently, to the external medium, coinciding with the death of the insect. Even with no infection in the other regions of the hindgut, the surrounding tissues have shown infected, affecting the normal functioning of this intestinal region, verified through changes in fecal pellet, which was less compact and changed format. These results will contribute in the establishment of the BmMNPV infectious cycle. Furthermore, the basic knowledge of viral behavior is important for the development of infection control, prevention and previous identification methods of this disease in the field, once it also makes possible the removal of infected silkworms, diminishing the horizontal transmission of the virus in the creation rooms, in a way to reduce the loss of cocoons to be used in the confection of silk yarn / Bombyx mori nucleopolyhedrovirus multiple (BmMNPV) é um vírus entomopatogênico e poliorganotrófico, constituído por DNA fita dupla e apresenta dois fenótipos distintos: o vírus poliédrico derivado, responsável pela infecção primária no intestino médio do inseto; e o vírus broto, que se dispersa na hemocele causando a infecção secundária ou sistêmica. É extremamente virulento e quando infecta lagartas do bicho-da-seda, causa sérios danos ao inseto, geralmente levando-o à morte ou prejudicando a produção da seda, comprometendo toda a sua cadeia produtiva e gerando prejuízos econômicos. Para uma boa produção de seda, um fator determinante é o funcionamento do canal alimentar, que nos insetos é dividido em anterior, médio e posterior, sendo o posterior, local de absorção de água e sais minerais e da finalização do processo digestório. Vários tecidos já foram estabelecidos como alvos do BmMNPV, entretanto outros se mostraram resistentes. Dada a importância do intestino posterior, o presente trabalho objetivou verificar a susceptibilidade de seus componentes, íleo, cólon e reto de lagartas de B. mori do 5° instar ao BmMNPV isolado geográfico do Paraná. Em diferentes dias pós-inoculação (dpi), o intestino posterior foi dissecado e processado para análise em microscopia de luz, utilizando colorações convencionais e citoquímica para detecção viral, e microscopia eletrônica de varredura, para os detalhes morfológicos. Os resultados revelaram que o íleo, cólon e reto de B. mori apresentou morfologia semelhante a de outros lepidópteros. A análise citopatológica do íleo, cólon e o reto anterior revelou que suas células epiteliais não são susceptíveis ao BmMNPV, em nenhum dos tempos analisados. Já o reto posterior ou canal anal, mostrou-se susceptível ao vírus a partir do 5º dpi, desenvolvendo todos os sinais clássicos descritos para a infecção com o AlphaBV, como presença de viroplasma, hipertrofia nuclear, poliedros em formação e maduros. No final do ciclo infeccioso, ocorre a lise celular com a liberação dos poliedros virais para luz intestinal e, consequentemente, para o meio externo, coincidindo com a morte do inseto. Mesmo não havendo infecção nas demais regiões do intestino posterior, os tecidos circunvizinhos se mostraram infectados, o que possivelmente afetou o funcionamento normal desta região, sendo visíveis as modificações na formação do pellet fecal, que se mostrou menos compacto e com alterações no formato. Os resultados obtidos irão contribuir no estabelecimento do ciclo infeccioso deste patógeno. Além disso, o conhecimento básico do comportamento viral é importante para o desenvolvimento de métodos de controle da infecção, prevenção e identificação prévia desta doença no campo, pois, possibilita ainda, a retirada de lagartas infectadas, diminuindo a transmissão horizontal do vírus nos barracões de criação, de forma a reduzir a perda de casulos, a serem utilizados na confecção dos fios de seda

Made available in DSpace on 2017-07-10T14:17:13Z (GMT). No. of bitstreams: 1 Dissertacao -16.pdf: 1891470 bytes, checksum: 1b28975beba2d1e4f43d3a0f9f79a9aa (MD5) Previous issue date: 2014-02-11 / Bombyx mori is an insect of the order Lepidoptera that is only found only in germplasm banks; it is used in scientific research and for commercial purposes. In the latter case, the silk cocoon, which is produced at the end of the 5th larval instar, is used in the production of various yarns and fabrics. This branch of Brazilian agribusiness, known as sericulture, is well developed in the state of Paraná, where it is a form of small-scale family farming. Several factors impact negatively on Brazilian sericulture, such as diseases during rearing, and B. mori is susceptible to a virus from the Baculoviridae family, namely, Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), genus Alphabaculovirus (AlphaBV), which infects the larvae and jeopardises commercial production of the cocoon, causing losses to farmers and industry. Studies have proved that BmMNPV is polyorganotropic and there are several target organs, such as the tracheal system; however, details of its cytopathology are not known. The tracheal system is responsible for the aeration of the tissues of the insect. Thus, the present study aimed to describe the cytopathology of the tracheas of hybrid larvae of B. Mori, infected experimentally with BmMNPV, and isolated geographically in the state of Paraná. Fifth instar hybrid larvae were divided into two groups; one control, and the other inoculated. After ingestion, and on different days post-inoculation (dpi), from the 2nd to the 9th dpi, the larvae were anesthetized and dissected. Segments of organs such as the integument, muscle and silk gland, containing branches of the trachea, were collected and fixed in Karnovsky modified for transmission electron microscopy. On the 2st dpi, fresh hemolymph analysis was conducted in order to determine the susceptibility of the hemocytes. The results revealed that the hemocytes were infected from the 2nd dpi and the epithelial cells of the trachea were infected from the 4th dpi. The cytopathology of the tracheal cells showed hypertrophic nucleus, containing the viroplasm, the site of the synthesis of the nucleocapsids. Subsequently, the formation and development of the polyhedra occured, accentuating the nuclear hypertrophy and culminating in cell lysis. Virions were also observed, immersed in the basal lamina of the trachea, which appeared to be disorganized. Thus, the cytopathology of the trachea was consistent with the infection caused by AlphaBV, and the data that was obtained provides a better understanding of the infectious cycle of BmMNPV in the body of the insect. The time of infection, later for the hemocytes, and the presence of virions in the basal lamina of the trachea, indicated that this system is a secondary target for infection, and also that the hemolymph is an important dispersant of viral infection / Bombyx mori é um inseto da ordem Lepidoptera encontrado somente em bancos de germoplasma, sendo utilizado em pesquisas científicas e para fins comerciais. Neste caso, seu casulo de seda, construído ao final do 5º instar larval, é usado na produção de diversos fios e tecidos. Este ramo da agroindústria brasileira, conhecido como sericicultura, se apresenta bem desenvolvido no Estado do Paraná, estando incluído no programa de agricultura familiar. Vários são os fatores que exercem influência na sericicultura nacional, como as doenças, e B. mori é susceptível a um vírus da família Baculoviridae, o Bombyx mori multiple nucleopolyhedrovirus (BmMNPV), gênero Alphabaculovirus (AlphaBV). Ao infectar as lagartas o vírus compromete a produção comercial do casulo, causando prejuízos aos produtores rurais e a indústria. Estudos comprovam que o BmMNPV é poliorganotrófico e vários são os órgãos-alvos, como o sistema traqueal; entretanto, detalhes de sua citopatologia não são conhecidos. O sistema traqueal é responsável pela aeração dos tecidos do inseto e o presente estudo objetivou descrever a citopatologia das traqueias de lagartas híbridas de B. mori infectadas experimentalmente pelo BmMNPV, isolado geográfico do Paraná. Para tanto, lagartas híbridas de 5º instar foram divididas em dois grupos, controle e inoculado. Neste, o inóculo viral foi fornecido na alimentação e em diferentes dias pós-inoculação (dpi), do 2º ao 9º dpi, as lagartas foram anestesiadas e dissecadas; segmentos do tegumento, músculo e glândula da seda, contendo ramos da traqueia, foram coletados e fixados em Karnovsky modificado para a microscopia eletrônica de transmissão. No 2º dpi foi efetuada análise a fresco da hemolinfa, para averiguar a susceptibilidade dos hemócitos. Os resultados revelaram que os hemócitos se apresentaram infectados a partir do 2º dpi e as células epiteliais da traqueia a partir do 4° dpi. A citopatologia das células traqueais revelou núcleo hipertrófico, contendo o viroplasma, que é o local de síntese dos nucleocapsídeos. Posteriormente, houve a formação e o desenvolvimento dos poliedros, acentuando-se a hipertrofia nuclear e culminando com a citólise. Vírions também foram visualizados na lâmina basal da traqueia, que se apresentou desorganizada. Assim, a citopatologia da traqueia condiz com a infecção causada por AlphaBV, e as informações obtidas permitem um melhor entendimento do ciclo infeccioso do BmMNPV no corpo do inseto. O tempo de infecção, posterior ao dos hemócitos, e a presença de vírions na lâmina basal da traqueia, indicam que este sistema é alvo secundário e, ainda, que a hemolinfa se apresenta como um importante dispersor da infecção viral.

Yeung, Tak Wai. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2007. / Includes bibliographical references (leaves 181-197). / Abstracts in English and Chinese. / Abstract (English) --- p.i / Abstract (Chinese) --- p.iv / Acknowledgements --- p.vi / List of Publications --- p.vii / Presentation --- p.vii / List of Abbreviations --- p.viii / Abbreviations in Figures --- p.xiii / Abbreviations in Symbols --- p.xiv / List of Cell Lines Used in this Study --- p.xv / Table of Contents --- p.xvi / List of Figures --- p.xxv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Cellular Redox State and Cancer Biology --- p.1 / Chapter 1.2 --- Reactive Oxygen Species (ROS) --- p.1 / Chapter 1.3 --- Regulation of Cellular Redox State by Intrinsic and Extrinsic Antioxidant Systems --- p.5 / Chapter 1.3.1 --- Intrinsic Antioxidant System --- p.6 / Chapter 1.3.2 --- Extrinsic Antioxidant System --- p.8 / Chapter 1.4 --- Glutathione --- p.9 / Chapter 1.4.1 --- General Information of Glutathione --- p.9 / Chapter 1.4.2 --- Functions of Glutathione --- p.12 / Chapter 1.4.2.1 --- As an Antioxidant and Free Radical Scavenger --- p.12 / Chapter 1.4.2.2 --- As a Detoxifier --- p.13 / Chapter 1.4.2.3 --- As a Regulator of Cell Signaling --- p.14 / Chapter 1.4.3 --- Synthesis of Glutathione --- p.15 / Chapter 1.4.4 --- Catabolism of Glutathione --- p.15 / Chapter 1.4.5 --- Transport and Uptake of Glutathione --- p.16 / Chapter 1.4.6 --- Glutathione in Cancer Biology --- p.18 / Chapter 1.4.6.1 --- "Role of Glutathione in the Regulation of Carcinogenesis, Growth and Apoptosis of Cancer Cells" --- p.18 / Chapter 1.4.6.1.1 --- Role of Glutathione in Carcinogenesis --- p.18 / Chapter 1.4.6.1.2 --- Role of Glutathione in the Growth of Cancer Cells --- p.20 / Chapter 1.4.6.1.3 --- Role of Glutathione in Apoptosis of Cancer Cells --- p.21 / Chapter 1.4.6.2 --- Role of Glutathione in the Regulation of Metastasis --- p.23 / Chapter 1.4.6.3 --- Role of Glutathione in Cancer Resistance and Therapy --- p.24 / Chapter 1.4.6.3.1 --- Role of Glutathione in Cancer Resistance --- p.24 / Chapter 1.4.6.3.2 --- Role of Glutathione in Cancer Therapy --- p.24 / Chapter 1.5 --- Aims of the Present Study --- p.25 / Chapter Chapter 2 --- In Vitro Study of Bαicαlein and Baicalin on Glutathione Depletion --- p.28 / Chapter 2.1 --- Introduction --- p.28 / Chapter 2.1.1 --- Scutellaria bαicαlensis Georgi --- p.28 / Chapter 2.1.1.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.28 / Chapter 2.1.1.2 --- As an Antioxidant and Free Radical Scavenger --- p.29 / Chapter 2.1.1.3 --- Long History for Treatment of Cancers with the Obscure Mechanism --- p.30 / Chapter 2.1.1.4 --- Major Components --- p.31 / Chapter 2.1.2 --- Baicalein and Baicalin --- p.32 / Chapter 2.1.2.1 --- General Clinical Applications to Treat or Prevent Diseases --- p.32 / Chapter 2.1.2.2 --- As an Antioxidant and Free Radical Scavenger --- p.33 / Chapter 2.1.3 --- Hypothesis: Baicalein and Baicalin Induce Cancer Cell Death Via Glutathione Depletion --- p.35 / Chapter 2.2 --- Materials and Methods --- p.36 / Chapter 2.2.1 --- Chemicals --- p.36 / Chapter 2.2.2 --- Buffers and Solutions --- p.36 / Chapter 2.2.3 --- Animals --- p.37 / Chapter 2.2.4 --- Preparation of Rat Brain Microsomes --- p.37 / Chapter 2.2.5 --- Glutathione Depletion Assay In Vitro and Thiol Depletion Assay in Rat Brain Microsomes --- p.38 / Chapter 2.2.6 --- Statistical Analysis --- p.39 / Chapter 2.3 --- Results --- p.40 / Chapter 2.3.1 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Glutathione --- p.42 / Chapter 2.3.2 --- Effects of Baicalein and Baicalin on Sulfhydryl Contents of Rat Brain Microsomes --- p.42 / Chapter 2.4 --- Discussion --- p.44 / Chapter Chapter 3 --- Effects of Baicalein and Baicalin on Proliferation of Different Human Cancer and Normal Cells --- p.45 / Chapter 3.1 --- Introduction-Importance of Developing A Novel Compound Inducing Cancer Cells to Cell Death with the Least Side Effects on Normal Cells --- p.45 / Chapter 3.2 --- Materials and Methods --- p.46 / Chapter 3.2.1 --- Instruments --- p.46 / Chapter 3.2.2 --- Chemicals and Cell Culture Reagents --- p.46 / Chapter 3.2.3 --- Buffers --- p.46 / Chapter 3.2.4 --- Cell Lines --- p.47 / Chapter 3.2.5 --- Cell Culture --- p.48 / Chapter 3.2.6 --- Determination of Cell Proliferation by MTT Assay --- p.49 / Chapter 3.3 --- Results --- p.51 / Chapter 3.3.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.51 / Chapter 3.3.2 --- Effects of Baicalein on Different Normal Cell Lines --- p.56 / Chapter 3.4 --- Discussion --- p.58 / Chapter 3.4.1 --- Anti-Proliferative Effects of Baicalein and Baicalin on Different Cancer Cell Lines --- p.58 / Chapter 3.4.2 --- Effects of Baicalein on Cell Proliferation on Different Human Normal Cell Lines --- p.60 / Chapter Chapter 4 --- Glutathione-Depleting Effects of Baicalein on Cell Proliferation of Different Cell Lines --- p.61 / Chapter 4.1 --- Introduction-Brain Tumors --- p.61 / Chapter 4.1.1 --- Types and Classifications of Brain Tumors --- p.61 / Chapter 4.1.2 --- "Incidence Time, Patient Survival Time and Rate for" --- p.65 / Chapter 4.1.3 --- Symptoms and Diagnostic Methods for Brain Tumors --- p.66 / Chapter 4.1.4 --- "Treatments, Side Effects and Difficulties of Treatments for Brain Tumors" --- p.67 / Chapter 4.1.5 --- Glutathione Levels in Brain Normal and Cancer Cells --- p.69 / Chapter 4.2 --- Materials and Methods --- p.70 / Chapter 4.2.1 --- Instruments --- p.70 / Chapter 4.2.2 --- Chemicals --- p.70 / Chapter 4.2.3 --- Buffers --- p.70 / Chapter 4.2.4 --- Determination of Cell Proliferation by MTT Assay --- p.70 / Chapter 4.2.5 --- Determination of Intracellular Glutathione Depletion by Fluorescent Dye CMAC --- p.71 / Chapter 4.2.6 --- Determination of Cellular Reduced Glutathione Levels by DTNB-Coupled Glutathione Reductase Recycling Assay --- p.73 / Chapter 4.3 --- Results --- p.75 / Chapter 4.3.1 --- Effects of Baicalein on Intracellular GSH Levels and Cell Proliferation for Different Cell Lines --- p.75 / Chapter 4.3.2 --- Basal Intracellular Glutathione in Different Cell Lines --- p.81 / Chapter 4.4 --- Discussion --- p.84 / Chapter 4.4.1 --- Intracellular Glutathione Depletion and Cell Death Induction Effects of Baicalein on Different Cell Lines --- p.84 / Chapter 4.4.2 --- Relationship between Basal Glutathione Levels and Drug Susceptibilities --- p.85 / Chapter Chapter 5 --- Effects of Baicalein on Apoptosis and Caspase Pathways --- p.88 / Chapter 5.1 --- Introduction-Modes of Cell Death --- p.88 / Chapter 5.1.1 --- Necrosis --- p.88 / Chapter 5.1.2 --- Apoptosis --- p.89 / Chapter 5.2 --- Materials and Methods --- p.92 / Chapter 5.2.1 --- Chemicals --- p.92 / Chapter 5.2.2 --- Buffers --- p.92 / Chapter 5.2.3 --- Determination of Change of Mitochondrial Membrane Potential by JC-1 --- p.93 / Chapter 5.2.4 --- Determination of Apoptosis by Annexin V-Propidium Iodide Staining --- p.94 / Chapter 5.2.5 --- Determination of Cell Cycle Arrest by Propidium Iodide Staining --- p.95 / Chapter 5.2.6 --- "Determination of Caspase-3, -8 and -9 Activities by Fluorescent-Labeled Peptides" --- p.96 / Chapter 5.2.7 --- Determination of DNA Fragmentation --- p.97 / Chapter 5.2.8 --- Terminal Deoxynucleotidyl Transferase Mediated dUTP End Labeling (TUNEL) Assay --- p.99 / Chapter 5.2.9 --- Flow Cytometry --- p.101 / Chapter 5.3 --- Results --- p.102 / Chapter 5.3.1 --- Effects of Baicalein on Mitochondrial Membrane Potential by JC-1 Staining --- p.102 / Chapter 5.3.2 --- Effects of Baicalein on Apoptosis and Necrosis by Annexin V-Propidium Iodide Staining --- p.104 / Chapter 5.3.3 --- Effects of Baicalein on Cell Cycle Arrest by Propidium Iodide Staining --- p.108 / Chapter 5.3.4 --- "Effects of Baicalein on Caspase-3, -8 and -9 Activities" --- p.110 / Chapter 5.3.5 --- Effeets of Baiealein on DNA Fragmentation --- p.115 / Chapter 5.3.6 --- Effects of Baicalein on TUNEL Assay --- p.117 / Chapter 5.4 --- Discussion --- p.120 / Chapter Chapter 6 --- Pro-Oxidant Role of Baicalein on Reactive Oxygen Species Generation --- p.122 / Chapter 6.1 --- Introduction --- p.122 / Chapter 6.2 --- Materials and Methods --- p.122 / Chapter 6.2.1 --- Chemicals --- p.122 / Chapter 6.2.2 --- Determination of Cellular Reactive Oxygen Species Generation by Fluorescent Dye cDCFDA --- p.123 / Chapter 6.2.3 --- Determination of Mitochondrial Reactive Oxygen Species Generation by Fluorescent Dye Rhl23 --- p.124 / Chapter 6.3 --- Results --- p.125 / Chapter 6.3.1 --- Effects of Baicalein on Cellular ROS Generation by Fluorescent Dye cDCFDA --- p.125 / Chapter 6.3.2 --- Effects of Baicalein on Mitochondrial ROS Generation by Fluorescent Dye Rhl23 --- p.129 / Chapter 6.4 --- Discussion --- p.132 / Chapter Chapter 7 --- The Anticancer Mechanistic Study of Baicalein --- p.133 / Chapter 7.1 --- Introduction --- p.133 / Chapter 7.2 --- Materials and Methods --- p.134 / Chapter 7.2.1 --- Chemicals --- p.134 / Chapter 7.2.2 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.134 / Chapter 7.2.3 --- Reversibility of Baicalein-Induced Cellular ROS Generation --- p.136 / Chapter 7.2.4 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.137 / Chapter 7.2.5 --- "Reversibility of Baicalein-Induced Caspase-3, -8 and -9 Activation by Co-Treatment of Different Antioxidants" --- p.138 / Chapter 7.3 --- Results --- p.139 / Chapter 7.3.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death by Different Antioxidant Treatments --- p.139 / Chapter 7.3.1.1 --- Pre-treatments --- p.139 / Chapter 7.3.1.2 --- Co-treatments --- p.141 / Chapter 7.3.1.3 --- Post-treatments --- p.144 / Chapter 7.3.2 --- Reversibility of Baicalein-Induced Cellular ROS Generation by Co-Treatment of Different Antioxidants --- p.147 / Chapter 7.3.3 --- Reversibility of Baicalein-Induced Apoptosis by Co-Treatment of Different Antioxidants and Caspase Inhibitors --- p.152 / Chapter 7.3.4 --- Reversibility of Baicalein-Induced Caspase-3 Activation by Co-Treatment of Different Antioxidants --- p.156 / Chapter 7.3.5 --- Reversibility of Baicalein-Induced Caspase-8 and -9 Activation by Co-Treatment of Different Antioxidants --- p.160 / Chapter 7.4 --- Discussion --- p.164 / Chapter 7.4.1 --- Reversibility of Baicalein-Induced GSH Depletion and Cell Death --- p.164 / Chapter 7.4.2 --- "Reversibility of Baicalein-Induced ROS Generation," --- p.167 / Chapter 7.5 --- Concluding Remarks --- p.168 / Chapter Chapter 8 --- General Discussion --- p.169 / Chapter 8.1 --- Drug Delivery to Brain --- p.169 / Chapter 8.2 --- Protective Roles of Baicalein on Brain Cells --- p.170 / Chapter 8.2.1 --- Actions Against Oxidative Stress --- p.170 / Chapter 8.2.2 --- Actions Against Other Neurotoxic Damages --- p.171 / Chapter 8.2.3 --- Actions Against Neuronal Diseases --- p.172 / Chapter 8.3 --- Anticancer Roles of Baicalein on Astrocytoma --- p.173 / Chapter 8.4 --- Implications on the Dual Roles of Baicalein: Antioxidant and Pro-oxidant --- p.175 / Chapter 8.5 --- Future Perspectives --- p.175 / Chapter 8.5.1 --- Effects of Baicalein on Antioxidant System --- p.175 / Chapter 8.5.2 --- Effects of Baicalein on GSH Synthesis --- p.176 / Chapter 8.5.3 --- In Vivo Studies on Cytotoxic Effects of Baicalein --- p.177 / Chapter 8.5.4 --- In Vivo Studies on Anti-Tumor Effects and In Vitro Studies on Anti-Metastasis Effects of Baicalein --- p.178 / Reference List --- p.181

Includes nine pages of amendments. / Bibliography: leaves 140-211. / 211, [200] leaves, [12] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Aims to determine the reason for the lack of ability of many myeloid leukaemic cell populations to stimulate allogeneic lymphocytes in mixed leucocyte culture (MLC), with a view to manipulating the immunogenicity of these cells for therapeutic purposes. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1995

[Truncated abstract] Bone is a living tissue and is maintained by the coordinate action of osteoblasts and osteoclasts. The intercellular communication between these two cells is the quintessential mechanism in bone remodelling. Unfortunately, the importance of this interaction is often neglected and its significance is only realised when disruption of this “cross-talk” results in debilitating bone diseases. Additionally, the number of known proteins that are involved in this “cross-talk”, especially those that are osteoclast-derived, and act specifically on osteoblasts, is limited. This discrepancy leads to the question: Can osteoclasts directly control the growth and function of osteoblastic cells by expressing specific proteins that bind directly to osteoblasts? If so, is it possible to use these proteins to control and, possibly, treat bone disease? The objective of this thesis is to identify and characterise osteoclast-derived factors that can modulate bone homeostasis, as well as contribute to the intercellular communication between osteoblasts and osteoclasts ... Collectively, the data in this thesis culminates in one important conclusion: the identification of a novel paracrine secretory factor that has the potential to directly induce the formation of bone. These findings represent the first ever characterisation of a protein that allows the osteoclasts to directly control the growth and function of osteoblasts. Due to the potential function of ODOF to induce bone formation, this protein may be used therapeutically to treat bone disease.

[Truncated abstract] Hepatic progenitor cells (HPC) are becoming increasingly recognized as facultative stem cells capable of regenerating the liver during chronic liver injury and also as targets of malignant transformation. Similar markers are expressed by hepatocellular carcinoma (HCC) and HPC, and a precursor-product relationship is well established. This thesis focuses on the ways in which the HPC population can be controlled under circumstances of chronic liver injury, and in this manner, reduce the risk of progression to HCC reduced. The major aim of Chapters 3 to 5 was to elucidate the effect of interferon α (IFNα) therapy on HPC. Chronic hepatitis C affects approximately 250 million individuals world wide. Approximately 80% of infections progress to chronicity, which places the individuals at greater risk of developing HCC. The gold standard of treatment of chronic hepatitis C is a combination of pegylated IFNα and ribavirin. ...The results were surprising. While IFNγ exerted a pro-apoptotic and antiproliferative effect on HPC in vitro, administration of IFNγ to CDE-fed mice for 14 days increased fibrosis, enhanced inflammatory infiltration and exacerbated the HPC response, with concurrent hepatocyte cell death. In addition, increased morbidity and mortality were observed in the IFNγ-treated mice compared to control. IFNγ treatment was found to prime the liver for the HPC response by recruiting inflammatory cells and altering the hepatic cytokine profile, both of which may facilitate an increased HPC response. Numbers of activated HSC were also increased in the IFNγ-treated, CDE-fed mice, correlating with the increased fibrosis seen in these animals. This data contradicts the current experimental use of IFNγ for treatment of fibrosis. Based on our results, we suggest that IFNγ promotes HPC proliferation in the CDE model, by encouraging inflammatory infiltration and hepatocyte damage and this initiates pro-fibrotic events. Concurrent proliferation of HPC and activated HSC further supports the view that there is a close relationship between the two cell types, and thus, a link between the HPC response and fibrosis. In conclusion, findings documented in this thesis suggest that administration of IFNα and IFNγ can contribute to shaping the HPC response. IFNα therapy may reduce HCC risk in chronic hepatitis C patients by bringing the HPC population under control. In contrast, IFNγ treatment can exacerbate the HPC response, liver fibrosis and parenchymal damage, illustrating the need to approach this method of fibrosis treatment with caution.

Proposição: Avaliar a associação entre as condições de saúde bucal, fatores comportamentais e sociodemográficos e o padrão citopatológico da mucosa bucal de homens adultos. Correlacionando o padrão citopatológico quantitativo e qualitativo com as variáveis bucais e sociodemográficas. Materiais e Métodos: A partir de dois esfregaços, um da borda da língua e outro do assoalho bucal de 117 homens com mais de 25 anos, foram quantificadas cem células de cada. As células foram coradas pelo método Papanicolaou modificado e classificadas em: Escamas, células superficiais com núcleo, células intermediárias e células parabasais. Variáveis sociodemográficas e comportamentais foram coletadas a partir de um questionário estruturado. Índice CPO-D e uso de próteses foram registradas a partir de um exame clínico intra-oral.As análises foram conduzidas utilizando o pacote estatístico Stata versão 10. O indivíduo foi considerado a unidade analítica. O nível de significância foi estabelecido em 5%. Resultados: No total das células na borda da língua, 75% eram intermediárias, 20% superficiais com núcleo e 5% escamas, sendo que células parabasais foram raramente observadas. Não foram observadas diferenças significativas para todas as variáveis em estudo em todos os tipos celulares, com exceção da ingestão de bebidas alcoólicas.Observou-se percentual significativamente maior de escamas e células superficiais com núcleo nos indivíduos que ingeriam de álcool comparados aos que não ingerem. Para as células intermediárias, o percentual foi significativamente menor nos indivíduos que bebem comparados aos que não bebem. Um padrão semelhante de distribuição celular foi observado no assoalho de boca. O consumo de álcool foi o único fator comportamental que influenciou significativamente no padrão citológico da mucosa bucal dos indivíduos analisados nos modelos multivariados. Conclusões: Foi encontrada associação entre um dos fatores comportamentais (o álcool) e o padrão citopatológico da mucosa bucal de homens adultos. As demais variáveis estudadas não apresentaram associação significativa com o padrão de descamação da mucosa bucal normal com a técnica utilizada. / Proposition: To evaluate the association between oral health status, socio- demographic and behavioral factors and standard cytology of the oral mucosa of adult men. Correlating the standard cytopathology quantitative and qualitative with oral and sociodemographic variables. Materials and Methods: From two smears, one edge of the tongue and other oral floor of 117 men over 25 years old, were quantified hundred cells each. Cells were stained with modified Papanicolaou method and classified into: Scales, superficial cells with nuclei, intermediate and parabasal cells. Sociodemographic and behavioral variables were collected from a structured questionnaire. DMFT index and use of prostheses were recorded from a clinical intra - oral.As analyzes were conducted using Stata version 10. The individual was considered the analytical unit. The level of significance was set at 5 %. Results: In total cells at the edge of the tongue 75% were intermediate, 20% core surface scales and 5%, and parabasal cells were rarely observed. No significant differences were observed for all study variables in all cell types, with the exception of beverage intake alcoólicas.Observou is significantly greater percentage of scales and superficial cells in the core subjects ingestores alcohol compared to those who do not drink. For intermediate cells, the percentage was significantly lower in individuals who drink compared to those who do not drink. A similar pattern of cell distribution was observed in the floor of mouth. Alcohol consumption was the only factor that significantly influenced the behavioral cytology of the oral mucosa of individuals analyzed in the multivariate models. Conclusions: An association between a behavioral factor (alcohol) and standard cytology of the oral mucosa of adult men. The other variables were not significantly associated with the default scaling of normal oral mucosa with the technique used.

A esporotricose é uma micose subcutânea causada por espécies do complexo Sporothrix schenckii que acomete seres humanos e animais, principalmente os gatos. Desde 1998 o Instituto Nacional de Infectologia Evandro Chagas/Fiocruz no Rio de Janeiro vem acompanhando uma epidemia dessa doença envolvendo seres humanos, cães e gatos, onde as principais formas de transmissão são arranhadura, mordedura e/ou contato com o exsudato das lesões cutâneas de gatos doentes. O diagnóstico definitivo da esporotricose felina é obtido a partir do isolamento do Sporothrix sp. em meios de cultura, entretanto, o resultado desse exame pode demorar até quatro semanas, o que em algumas situações pode retardar o início do tratamento antifúngico. Os exames citopatológico, histológico e imuno-histoquímico são opções viáveis e mais rápidas para o diagnóstico dessa micose em gatos, principalmente em situações quando não é possível realizar o isolamento fúngico. O diagnóstico precoce da esporotricose felina é importante na implementação rápida do tratamento antifúngico, melhorando o prognóstico na maioria dos casos. O objetivo deste estudo foi avaliar e comparar as diferentes técnicas utilizadas no diagnóstico da esporotricose felina antes e durante o tratamento antifúngico. Na primeira etapa do estudo foram comparados os exames citopatológico (coloração pelo método panótico rápido), histológico (impregnação pela prata de Grocott) e imuno-histoquímico de 184 gatos no diagnóstico da esporotricose sem tratamento antifúngico prévio utilizando o cultivo fúngico como teste padrão de referência. Estruturas leveduriformes foram observadas em 160 (87,0%) casos no exame citopatológico, 168 (91,3%) na histopatologia e 163 (88,3%) na imunohistoquímica. A associação das três técnicas elevou a sensibilidade do diagnóstico para 98,3%, o que enfatiza a necessidade de sua implementação como ferramentas de rotina, sobretudo quando a cultura fúngica não está disponível. Na etapa seguinte, a carga parasitária e o isolamento de Sporothrix sp. das lesões cutâneas de 74 gatos foram avaliados mensalmente antes e durante o tratamento com itraconazol por um período de 12 semanas. A mediana da carga fúngica observada antes do início do tratamento antifúngico foi maior (pMW = 0,013) nos gatos nos quais foi observada a persistência da lesão (Med=98,6) em relação aqueles em que houve cicatrização (Med=15,0). A redução da carga fúngica ocorreu em todas as lesões estudadas, assim como a redução da positividade da cultura fúngica e do exame citopatológico. Estes resultados sugerem uma redução no potencial zoonótico dos gatos, enfatizando a importância do tratamento precoce como medida de controle. Adicionalmente, o isolamento do fungo e a presença de estruturas leveduriformes nas lesões de gatos com esporotricose podem ser fatores preditores da falência terapêutica, indicando a necessidade da implementação de alternativas terapêuticas. / Sporotrichosis is subcutaneous mycose caused by species of fungus from the Sporothrix schenckii complex and affects humans and animals, especially cats. Since 1998, the Instituto Nacional de Infectologia Evandro Chagas/Fiocruz in Rio de Janeiro has been describing in Rio de Janeiro an epidemic of sporotrichosis, involving humans, cats and dogs, with most of cases related to transmission through scratches, bites or contact with lesions from infected cats. The definitive diagnosis is based on the isolation of the fungus in culture; however, the results may take up to four weeks and postpone treatment outset. The cytopathology, histopathology and immunohistochemistry should be considered as rapid and accessible alternatives for the diagnosis in cats, especially when the fungal culture is not available. The early diagnosis of feline sporotrichosis is desirable for the prompt beginning of the antifungal treatment, improving the prognosis in most of the cases. The aim of this study was to evaluate and compare different techniques for the diagnosis of feline sporotrichosis before and during the treatment with itraconazol. In the first part of the study, cytopathological (Quick Panoptic), histopathological (Grocott silver stain) and immunohistochemical examinations were compared regarding the diagnosis of sporotrichosis in 184 cats without previous treatment, by using fungal culture as a reference standard. The yeast-like cells were observed in 160 (87.0%) cases by cytopathological examination, in 168 (91.3%) by histopathology and in 163 (88.3%) by immunohistochemistry. The combination of the three methods led to the diagnosis of 98.3% of cases, pointing to the need of their implementation as regular tools, notably when fungal culture is not available. In the second part of the study, the fungal burden and the isolation of Sporothrix sp. in cutaneous lesions of 74 cats were monthly evaluated before and during the treatment with itraconazole for twelve weeks. The median of the fungal load detected before the outse of the antifungal treatment was higher (pMW=0.013) in cats in which there was a persistence of the cutaneous lesion (Med=98.6) in comparison to those in which healing of the lesion was observed (Med=15.0). The decrease of the fungal burden occurred in all the lesions in this study as well as the reduction of the positivity of the fungal culture and the cytopathological examination. These results suggest a reduction in the zoonotic potential of cats and emphasize the importance of the early treatment as a control measure. In addition, the isolation of the fungus and the presence of yeast-like cells in lesions of cats with sporotrichosis during the treatment can be a predictor of treatment failure and should alert for the need of alternative therapeutic regimens.