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Expressão imuno-histoquímica de Bcl-2, Ki-67 e caspase-3 ativa em líquen plano oral e leucoplasia com diferentes graus de displasia / Immunohistochemical expression of Bcl-2, Ki-67 and active caspase-3 in oral lichen planus and leukoplakia with different degrees of dysplasiaFernanda Mombrini Pigatti 26 August 2011 (has links)
O líquen plano oral (LPO) é uma doença inflamatória crônica de causa desconhecida, e seu potencial de malignização é um tema bastante controverso. Diversos estudos têm sugerido que pacientes portadores de líquen plano apresentam um maior risco de desenvolver câncer. Contudo, muitos autores acreditam que não haja dados suficientes que provem uma associação entre líquen plano e câncer. Para esses autores, a maioria dos casos que sofreram transformação maligna é decorrente de falhas no diagnóstico inicial da lesão. Portanto, objetivou-se avaliar a expressão imuno-histoquímica das proteínas relacionadas à apoptose e proliferação celular, respectivamente caspase-3 ativa, Bcl-2 e Ki-67 no LPO e em displasias epiteliais na tentativa de explicar a polêmica em relação ao potencial de transformação maligna do LPO e enfatizar a importância de um acompanhamento de longo prazo dos pacientes com esta doença. Com esse propósito, foram selecionadas 14 amostras de LPO, 14 amostras de leucoplasia com displasia epitelial, além de 09 amostras de mucosa bucal normal como controle. A avaliação da expressão de caspase-3 ativa, Bcl-2 e Ki-67 foi conduzida de acordo com a técnica da imunoperoxidase. A contagem das células imunomarcadas nas amostras de LPO, de leucoplasia com displasia epitelial e de mucosa bucal normal resultou em número destas células por mm2 nas diferentes regiões (camada basal, camada suprabasal e infiltrado inflamatório) para cada imunomarcador. A expressão de Bcl-2 em células epiteliais, da camada basal, ocorreu mais frequentemente no grupo da leucoplasia com displasia epitelial, com diferença estatística entre o grupo do LPO e o grupo controle. Verificou-se também, uma alta expressão da proteína Bcl-2 em células inflamatórias de lesões de LPO e de leucoplasia com displasia epitelial. A expressão do marcador Ki-67 foi superior em todos os níveis teciduais analisados nas lesões de LPO e de leucoplasia com displasia epitelial quando comparados com o grupo controle. Concluiu-se que a expressão mais elevada das proteínas Bcl-2 e Ki-67 nos casos de LPO e de leucoplasia com displasia epitelial, podem revelar a possibilidade da presença de alterações moleculares morfologicamente imperceptíveis. / The oral lichen planus (OLP) is a chronic inflammatory disease of unknown cause, and its malignant potential is a very controversial issue. Several studies have suggested that patients with lichen planus have a higher risk of developing cancer. However, many authors believe that there is insufficient evidence to prove an association between lichen planus and cancer. For these authors, most of the cases that had undergone malignant transformation is due to flaws in the lesion initial diagnosis. Therefore, the aim was to evaluate the immunohistochemical expression of proteins related to apoptosis and cell proliferation, respectively active caspase-3, Bcl-2 and Ki-67 in epithelial dysplasia and LPO in an attempt to explain the controversy regarding the potential malignant transformation of OLP and emphasize the importance of a long-term monitoring of patients with this disease. For this purpose, we selected 14 samples of OLP, 14 samples of leukoplakia with epithelial dysplasia, and 09 samples of normal oral mucosa as controls. The evaluation of the expression of active caspase-3, Bcl-2 and Ki-67 was conducted in accordance with the immunoperoxidase technique. The immunostained cells counting in samples of OLP, epithelial dysplasia in leukoplakia and normal oral mucosa resulted in a number of these cells per mm2 in the different regions (basal layer, suprabasal layer and inflammatory infiltrate) for each immunostained. The expression of Bcl-2 in epithelial cells, the basal layer, occurred more frequently in the group of leukoplakia with epithelial dysplasia, with statistical difference between the group of OLP and the control group. There was a high expression of Bcl-2 protein in inflammatory cells in OLP lesions and leukoplakia with epithelial dysplasia. The expression of the marker Ki-67 was superior in all analyzed tissue levels in OLP lesions and leukoplakia with epithelial dysplasia when compared to the control group. It was concluded that the highest expression of Bcl-2 protein and Ki-67 in cases of OLP and leukoplakia with epithelial dysplasia, can reveal the possible presence of molecular changes morphologically imperceptible.
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Pesquisa de Toxoplasma gondii em mamíferos marinhos do Brasil / Pesquisa de Toxoplasma gondii em mamíferos marinhos do BrasilSamira Costa da Silva 31 March 2016 (has links)
A toxoplasmose é causada pelo protozoário coccídio Toxoplasma gondii e consiste em um dos processos parasitários mais comuns entre os animais endotérmicos. A presença desse protozoário em cetáceos pode estar associada à exposição dos animais aos oocistos de T. gondii eliminados pelas fezes de felídeos e/ou contato com solo contaminado com o parasita, que tenham comprometido a água do mar a partir de drenagens fluviais e fluentes ou ainda pelo escoamento de água de navios. O objetivo desse trabalho foi investigar a ocorrência de T. gondii em cetáceos encalhados, ou provenientes de captura acidental, ou que vieram a óbito durante o processo de reabilitação ou cativeiro, ao longo da costa brasileira, valendo-se para tanto de avaliações histopatológicas e de técnicas imuno-histoquímicas (IHQ). Os principais órgãos examinados foram fígado, pulmão, linfonodos, baço e cérebro. Esse estudo avaliou tecidos de 186 exemplares de 21 espécies diferentes de cetáceos da costa brasileira entre 1988 e 2014, cujas amostras foram encaminhadas e encontram-se depositadas no Banco de Tecidos de Mamíferos Marinhos, do Laboratório de Patologia Comparada dos Animais Selvagens (LAPCOM) do Departamento de Patologia, FMVZ-USP. Dos mamíferos marinhos analisados, a ocorrência de T. gondii foi confirmada em 1,1% (2/186), justificada pela presença de pelo menos um cisto em pulmão e/ou fígado. Os animais positivos pertenciam a duas espécies diferentes, provenientes do sudeste do Brasil; um golfinho nariz-de-garrafa (Tursiops truncatus) encalhado vivo no Rio de Janeiro - RJ, e uma orca (Orcinus orca) proveniente de cativeiro. As principais observações histopatológicas encontradas foram hepatite necrotizante, broncopneumonia fibrinosa supurativa com presença de cistos compatíveis com T. gondii, pneumonia broncointersticial fibrinosa com carneificação, glomérulonefrite membranosa e linfadenite necrótica. Devido à severidade das lesões suspeita-se que esse protozoário teve um importante papel no encalhe/óbito desses dois indivíduos. Esse estudo acrescentou duas novas espécies de cetáceos àquelas já reportadas como suscetíveis à infecção pelo protozoário, mas nunca antes descritos no Brasil. Os resultados ratificaram a ocorrência da infecção por T. gondii em cetáceos da costa brasileira e a sua importância em mamíferos marinhos em cativeiro e de vida livre / Toxoplasmosis is caused by the coccidian Toxoplasma gondii - phylum Apicomplexa, which is one of the most common parasites affecting endothermic animals. The presence of T. gondii in cetaceans may be associated with feline feces and/or soil contaminated with the parasite, which may reach water bodies through run-off. This study investigated the occurrence of T. gondii in cetaceans along the Brazilian coast. Tissue samples of 186 individuals stranded along the Brazilian coast between 1988 and 2014, belonging to 21 species of cetaceans and currently part of the Marine Mammal Tissue Bank of the Laboratory of Comparative Pathology, Pathology Department, School of Veterinary Medicine and Animal Sciences, University of São Paulo. Immunohistochemistry (IHC) for T. gondii was performed using formalin-fixed, paraffin embedded tissue sections of liver, lung, lymph nodes, spleen and brain. Infected tissues of a Guiana dolphin (Sotalia guianensis) were used as positive controls. A total of 1.1 % (2/186) of the animals evaluated were positive, which was justified by the presence of least one cyst in one of the evaluated organs/tissues. These specimens belong to two different species of cetaceans: Killer whale (Orcinus orca) and bottlenose dolphin (Tursiops truncatus), both from southeastern Brazil. One individual stranded alive in Rio de Janeiro (T. truncatus); and one was kept in captivity (O. orca) in São Paulo state. The most noteworthy lesions observed through microscopy included necrotizing hepatitis, fibrinous suppurative bronchopneumonia with cysts compatible with T. gondii, bronchointersticial fibrinous pneumonia, membranous glomerulonephritis and necrotizing lymphadenitis. Due to the severity of lesions protozoan was suspected to have an important role in the stranding/death of the two wild individuals. This study added two new species of cetaceans to those already reported as susceptible to infection by T. gondii, never before described in Brazil. These results confirm the occurrence of T. gondii infection in cetaceans from the Brazilian coast and the importance of this parasite to marine mammals kept in captivity and free ranging dolphins
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Alguns aspectos da imunopatogenia da uveíte na erliquiose canina de ocorrência natural e experimental: avaliação anatomopatológica e imunoistoquímica / Some of the immunopathogenic aspects of the uveitis in natural and experimental occurrence of canine ehrlichiosis: anatomopathological and immunohistochemical analysesValérie Le Du da Silva 23 June 2006 (has links)
Para elucidar alguns aspectos da imunopatogenia da uveíte na erliquiose canina, avaliaram-se, por meio da análise imunistoquímica, bulbos oculares de cães experimentalmente infectados por Ehrlichia canis (grupo 1- G1), naturalmente infectados por Ehrlichia canis (grupo 2-G2) e na co-infecção natural de E. canis e Babesia sp. (grupo 3). Parâmetros clínicos e hematológicos foram avaliados. Empregaram-se o dot-blot-Elisa e a reação de imunofluorescência indireta para E. canis e Babesia sp. respectivamente. Para a confirmação diagnóstica, utilizou-se a reação de cadeia de polimerase (PCR) para E.canis. A contagem imunofenotípica para os anticorpos CD3, CD4, CD8, Tal1B5 e MAC 387 não demonstrou diferença significativa nas diferentes regiões analisadas do bulbo ocular. Observou-se no G1, G2 e G3, em todas as regiões analisadas diferença significativa da contagem imunofenotípica de células CD8+ em relação às células CD4+. Evidenciou-se diferença significativa entre a contagem percentual de células IgG2+ e CD79?+ na região de corpo ciliar do G3 em relação ao G1. A região da íris do G3, em relação ao G2, demonstrou diferenças significativas para o anticorpo IgG1. Evidenciou-se nos três grupos, a existência de correlação linear entre as células CD3+ e CD8+ e entre as células IgG2+ e CD79?+ em diversas regiões do bulbo ocular. O infiltrado inflamatório mostrou-se mais intenso nas regiões de corpo ciliar e ângulo iridocorneal, moderado em limbo e íris e mínimo em coróide. A avaliação semiquantitativa por score da intensidade do infiltrado inflamatório mostrou-se mais intensa nos animais que apresentavam co-infecção, sugerindo uma resposta imune mais intensa nesses cães. Demonstrou-se que o infiltrado inflamatório era composto, predominantemente, por linfócitos T CD3+ e B CD79+?. A maior porcentagem de células T CD3+ era CD8+, caracterizando, portanto, uma resposta imune do tipo citotóxica. A presença de células B CD79+? fala a favor de produção local de anticorpos. Observaram-se células imunomarcadas por IgG2 e poucas células marcadas por IgG1, sugerindo uma polarização da resposta imune para o padrão Th1, sendo um possível mecanismo imune de lesão na uveíte. Observou-se alta expressão de moléculas de MHC-classe II, sugerindo uma resposta imune intensa nos tecidos oculares, contudo ineficiente devido a deficiência de células CD4+. / The immunopathogenicity of uveitis in the canine ehrlichiosis was studied by conducting anatomy and immunohistochemical analyses in the ocular globes of dogs experimentally (Group 1) and naturally (Group 2) infected with Ehrlichia canis, and naturally coinfected with Ehrlichia canis and Babesia sp. (Group 3). Clinical and hematological parameters were evaluated. Dot-blot Elisa and indirect immunofluorescent test (IFA) were used to analyze E. canis and Babesia sp., respectively. PCR assay confirmed the diagnosis of the disease caused by E. canis. The immunophenotypic analysis with the antibodies CD3, CD4, CD8, Tal1B5 and MAC 387 revealed no significant differences between the various ocular regions analyzed. Significant differences were observed between the immunophenotypic analysis of CD8+ and CD4+cells from all regions analyzed from G1, G2 and G3; between the percentile counts of IgG2+ and CD79?+ in the ciliary body of G3 dogs when compared to G1, and for the IgG1 antibody counts of the iris region from G3 when compared to G2. A linear correlation between CD3+ and CD8+ cells and between IgG2+ and CD79?+ cells from several regions of the ocular globe was found for all three groups. The cellular inflammatory infiltrate observed in the ocular tissue was severe in the regions of the ciliary bodies and iridocorneal angle, moderate in the limbus and iris, and only slightly present in the choroids. A semiquantitative analysis of the intensity of the inflammatory infiltrate of the ocular globes was more intense in G3, which suggested a greater response of the immune system in these animals. The inflammatory infiltrate was composed of mainly B CD79+? and T CD3+ lymphocytes, which had CD8+ at a high percentage, thus characterizing this as a cytotoxic immune response. Results indicated that B CD79+? cells favored the production of local antibodies. IgG2 and very few IgG1 immunolabeled cells were found. This result indicated a polarization of the immune response to the Th1 pattern, which could be the mechanism of the lesions in the uveitis. A large number of cells expressing the MHC-class II molecules were observed, suggesting an intense immune response in the ocular tissues, however ineffective due to the CD4+ cell´s deficient.
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Expressão de citoqueratinas, filagrina, involucrina, E-caderina e p63 em lesões de molusco contagioso / Cytokeratins, involucrin, filaggrin, E-cadherin and p63 expression in molluscum contagiosum lesionsCallegaro, Clarissa Freitas 19 February 2009 (has links)
Introdução: O molusco contagioso (MC) é um vírus da família Poxviridae que infecta os queratinócitos epidérmicos levando a hiperplasia e formação de inclusões eosinofílicas intracitoplasmáticas os corpos de molluscum. Poucos estudos analisam as alterações induzidas por este Mollucipox vírus nas estruturas que compõem o citoesqueleto e a adesão celular dos queratinócitos. Objetivos: Verificar o padrão de expressão de citoqueratinas, filagrina, involucrina, E-caderina e p63 pela técnica de imuno-histoquímica em lesões de MC e compará-las com a epiderme adjacente aparentemente normal (EAAN). Método: Através de técnica de imuno-histoquímica, estudou-se a expressão de K1, K10, K14, K16, filagrina, involucrina, E-caderina e p63 em lesões de MC de 41 pacientes imunocompetentes. Os padrões de expressão nas lesões de MC foram comparados com a EAAN. Resultados: A expressão de K1/K10 ocorreu como o habitual nas camadas suprabasais da epiderme. A marcação de K14 foi observada nas camadas epidérmicas basal e suprabasal nas lesões de MC e EAAN. A K16, que é expressa somente em processos hiperproliferativos, foi demonstrada na camada espinhosa tanto nos focos de MC como na EAAN. Filagrina e involucrina expressaram-se nas camadas granulosa, espinhosa e em alguns casos até mesmo na camada basal na epiderme infectada e EAAN. A E-caderina esteve presente até a porção inferior dos corpos de molusco enquanto na EAAN apresentou-se nas camadas basal e espinhosa. A expressão nuclear do p63 ocorreu nas camadas basal e espinhosa tanto no MC como EAAN. Conclusão: A infecção pelo Molluscipox vírus parece interferir no processo de diferenciação terminal dos queratinócitos. A expressão tardia de K14 e p63 na camada espinhosa, assim como a expressão precoce de filagrina e involucrina, associada ao estado hiperproliferativo demonstrado pela presença aberrante de K16, refletem um distúrbio no processo de maturação dos queratinócitos infectados. As alterações observadas na EAAN podem representar evento precoce no distúrbio de queratinização induzido pelo vírus na pele infectada. / Background: Molluscum contagiosum (MC) is a Molluscipox virus infection of the epidermal keratinocytes with hyperplasia and intracytoplasmic inclusions the molluscum bodies (MB). Few studies address cytokeratins (K) profile in MC, mainly focusing the terminal epidermal keratinization process. Methods: In order to verify K1, K10, K14, K16, involucrin, filaggrin, E-cadherin and p63 expression in MC, 41 lesions were subjected to immunohistochemical technique. The immunolabeling pattern of MC was compared to adjacent normal appearing epidermis (ANAE). Results: K1 and K10 were expressed in supra basal layers of MC and ANAE. K14 was expressed in basal and suprabasal layers in MC and also in ANAE. K16 was expressed in MC and ANAE, through all spinous layers. Involucrin and filaggrin were observed in granular, spinous and even in basal layer of ANAE and MC. E-cadherin was present up to the first layers of epidermis with MB while ANAE exhibited E-cadherin labeling at basal and spinous layers. Basal and spinous layers keratinocytes nuclei, in both MC and ANAE, express p63. Conclusion: Infection by Molluscipox virus may noticeably alter keratinocyte differentiation status and cell adhesion. The presence of K14 and p63 in spinous layer, as well as early expression of involucrin and filaggrin, associated to a hyperproliferative state disclosed by the presence of K16, may be a result of a disruption in keratinocytes maturation process. The changes observed at ANAE may represent early events in keratinization disturbance.
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Avaliação citológica, histológica e imunoistoquímica do linfoma alimentar em felinos domésticos / Cytological, histopathological and immunohistochemical evaluation of feline alimentary lymphomaBarriga, Viviana Molero 10 July 2013 (has links)
O linfoma alimentar é a forma anatômica mais frequente nos felinos e é o tipo de neoplasia que mais acomete o intestino delgado nessa espécie. É caracterizado pela infiltração de células linfóides neoplásicas em órgãos do trato gastrointestinal, com ou sem comprometimento de linfonodos mesentéricos. Seu diagnóstico pode ser considerado desafiador em muitos casos, devido às manifestações clínicas muitas vezes inespecíficas e às limitações da citologia e histopatologia. A imunoistoquímica, embora ainda pouco difundida, pode ser utilizada para confirmar o diagnóstico de linfoma alimentar, bem como determinar o tipo celular predominante. Objetivando-se caracterizar o linfoma alimentar felino, foi realizado estudo utilizando-se 40 casos de gatos com diagnóstico de linfoma alimentar (grupo 1), confirmados por meio da imunoistoquímica, no qual se avaliou os aspectos epidemiológicos, clínicos, citológicos, histológicos e imunoistoquímicos. Também foram avaliados 20 casos de felinos com doença inflamatória intestinal (grupo 2), confirmados segundo exame imunoistoquímico, quanto aos aspectos epidemiológicos e clínicos. E com o intuito de verificar a acurácia diagnóstica relativa dos exames de citologia, histologia e imunoistoquímica, foram utilizados os resultados dessas análises de ambos os grupos. Foi observado entre os felinos com linfoma alimentar estudados uma média de idade de 10,8 anos e as manifestações clínicas de maior ocorrência foram emagrecimento (55%), êmese (40%) e alterações nas fezes (40%). Entre as alterações ultrassonográficas observadas, as mais prevalentes foram linfonodomegalia mesentérica (86,11%) e espessamento de alças intestinais (80,55%). A maioria dos felinos estudados apresentou sorologia negativa tanto para FIV quanto para FeLV e apenas 14,28% e 8,57% foram positivos para FIV e FeLV, respectivamente. A correlação entre citologia e imunoistoquímica apontou sensibilidade, especificidade, valor preditivo positivo e negativo e razão de verossimilhança positiva e negativa de 93,3%, 65%, 66,6%, 92,85%, 2,65 e 0,10, respectivamente. O coeficiente de concordância Kappa de Cohen e a acurácia diagnóstica relativa foram de 0,55 e 77,14%, respectivamente. A correlação entre histopatologia e imunoistoquímica revelou sensibilidade, especificidade, valor preditivo positivo e negativo e razão de verossimilhança positiva e negativa de 89,74%, 60%, 81,39%, 75%, 2,22 e 0,18, respectivamente. O coeficiente de concordância Kappa de Cohen e a acurácia diagnóstica relativa foram de 0,52 e 79,66%, respectivamente. A associação entre sorologia para FIV e FeLV e tipo celular do linfoma alimentar não pode ser observada. O presente estudo permitiu concluir que a combinação de citologia, histopatologia e imunoistoquímica é necessária para o correto diagnóstico de linfoma alimentar, e o tipo celular de maior ocorrência foi o de células T. / Alimentary lymphoma is the most common anatomical form of feline lymphoma and is the neoplasm which more affects small intestine of this specie. It is characterized by infiltration of the gastrointestinal tract with neoplastic lymphocytes, with or without mesenteric lymph nodes involvement. The diagnosis of this illness can be challenger in several cases because of unspecific clinical signs and cytological and histopathological limitations. Immunohistochemistry is required where these evaluations were not definitive in addition to establish the cell phenotype T or B lymphocyte. The aim of this study was to characterize feline alimentary lymphoma. For these purpose, 40 cats with a diagnosis of alimentary lymphoma (Group 1) by immunohistochemistry were evaluated clinical, epidemiological, cytological and histologically, in addition to immunophenotype of the neoplasm tissues. Relative accuracy was performed using the results of cytology, histopathology and immunohistochemistry evaluation of 20 cats with inflammatory bowel disease diagnosis. The average age of the cats with alimentary lymphoma was 10.8 years and weight loss (55%), vomiting (40%) and fecal alteration (40%) were the most common clinical signs. The most prevalent ultrasonography abnormalities were mesenteric lymph nodes enlargement (86.11%) and intestinal wall thickening (80.55%). The majority of alimentary lymphoma cats were FIV and FeLV negative and 14.28% and 8.57% were FIV and FeLV positives, respectively. The cytology and immunohistochemistry correlation presents sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio tests of 93.3%, 65%, 66.6%, 92.85%, 2.65 and 0.10, respectively. The Cohen´s kappa test and the relative accuracy were 0.55 and 77.14%, respectively. The histopathology and immunohistochemistry correlation presents sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratio tests of 89.74%, 60%, 81.39%, 75%, 2.22 and 0.18, respectively. The Cohen´s kappa test and the relative accuracy were 0.52 and 79.66%, respectively. Association of FIV and FeLV result tests and alimentary lymphoma phenotype was not observed. In conclusion, the results of the present study suggest that cytology, histology and immunohistochemistry combination is necessary for a correct diagnosis, and T cell alimentary lymphoma is the most common phenotype.
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Avaliação da cultura de células-tronco do epitélio olfatório de cães sem raça definida (Canis familiaris Linnaeus, 1758) / Evaluation of stem cell culture of olfactory epithelium from Mongrel dogs (Canis familiaris Linnaeus, 1758)Alves, Flávio Ribeiro 12 February 2009 (has links)
As células provenientes do epitélio olfatório apresentam capacidade regenerativa durante toda a vida, embora este mecanismo ainda não esteja completamente elucidado. O potencial de diferenciação de células-tronco provenientes do epitélio olfatório de cães sem raça definida (Canis familiaris Linnaeus, 1758) foi avaliado utilizando-se 12 cães adultos e 12 cães com 60 dias de vida intra-uterina, oriundos do Hospital Veterinário da FMVZ-USP. Após coletado, o epitélio olfatório foi submetido a protocolo histológico padrão para hematoxilina-eosina, azul de toluidina e PAS. O material fixado em glutaraldeído 2,5% foi processado para observação sob microscopia eletrônica de transmissão. O índice de proliferação nuclear foi medido para detecção de PCNA e Ki67 nuclear. Células foram expandidas em cultura em meio D-MEM/F-12 acrescido de Soro Fetal Bovino hyclone (CO2-95% à 37ºC) para caracterização e diferenciação celular (osteogênica, adipogência e neurogênica). O cultivo das células provenientes do epitélio olfatório de cães com 60 dias de vida intra-uterina mostrou maior evolução em cultura quando comparados aos animais adultos, sendo as primeiras utilizadas para execução dos protocolos de caracterização e diferenciação. As células em cultivo apresentaram expressão CD29 positiva e marcação positiva para OCT-4, citoqueratina 18 (Ck18) e vimentina. A diferenciação osteogênica demonstrou, ao final de 21 dias, células com morfologia típica, caracterizadas pela coloração de Alizarin Red e Von Kossa. A diferenciação adipogênica mostrou pouco número de células, apresentando grânulos adipogênicos, corados por Oil Red, contudo sem morfologia típica. A diferenciação neurogênica demonstrou células que expressaram marcação para GFAP, Neurofilamentos, Oligodendrócitos e βtubulina III. As células isoladas a partir do epitélio olfatório de cães com 60 dias de vida intra-uterina evidenciaram populações de células-tronco, determinadas pela expressão de marcadores específicos e diferenciação em outros tipos de tecido, devendo-se considerar este epitélio como uma fonte potencial para aquisição de células, particularmente progenitores neuronais, que possam somar aos estudos e seu uso em terapia celular. / Olfactory cells demonstrate regenerative capacity along life, although, its mechanism is still obscure. We evaluated the differentiation capacity of olfactory stem cell of mongrel dogs (Canis familiaris Linnaeus, 1758) in 12 adult dogs and 12 fetuses at term (60 days of pregnancy) from the Veterinary Hospital of FMVZ-USP. Following the sampling, the epithelia were submitted to standard histological process and stained with HE, toluidine blue and PAS. Samples fixed with 2.5% glutaraldehyde solution were processed for transmission electron microscopy. Proliferative index were obtained with the detection of PCNA and Ki67. Cells were kept in culture in D-MEM/F-12 medium supplemented with hyclone bovine fetal serum (CO2-95% at 37ºC) for characterization and cell differentiation (osteogenesis, adipogenesis, and neurogenesis). Cell culture of fetuses at term demonstrated higher evolution in comparison to adult animals, being submitted to the protocols of characterization and differentiation. Cell culture demonstrated positive reaction for CD29, OCT-4, Ck18 (citokeratin) and vimentin. Osteogenic differentiation demonstrated, 21 days, typical morphological cells characterized by Alizarin Red and Von Kossa staining. Adipogenic differentiation demonstrated less number of cells containing granules, stained with Oil Red, although being not typical shape. Neurogenic differentiation demonstrated positive staining for GFAP, Neurofilament, Oligodendrocyte and III β-tubulin. Cells isolated from epithelia of fetuses at term demonstrated population of stem cells determined by the expression of specific-staining and differentiation into other type of cells, which lead us to consider the olfactory epithelia as a source of potential stem cells particularly for neurogenic differentiation to be applied for further studies in cell therapy.
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Stress and GABAA receptor regulationSkilbeck, Kelly Johanne January 2009 (has links)
Doctor of Philosophy (PhD) / GABAA receptors are implicated in the pathology of psychiatric disorders such as schizophrenia and depression. They are rapidly affected by stress in a sex-dependent fashion, suggesting that GABAA receptors may be relevant to understanding the association between stress and psychiatric disorders. Thus, this thesis examined how GABAA receptors are affected in both male and female mice exposed to stress in adulthood (Chapter 2), early-life (Chapter 3-5) and a combination of both early-life and adulthood stress (Chapter 6). 2. The effects of acute adulthood stress (3 minute warm swim stress) on GABAA receptor binding in the brains of male and female mice were examined using quantitative receptor autoradiography. The total number of GABAA receptor [3H]GABA binding sites was increased following swim stress in specific forebrain cortical regions of female mice swum individually or in a group, but decreased in male mice when swum in a group only. These findings confirm and extend previous studies, identifying the cortical regions involved in rapid stress-induced changes in GABAA receptors. 3. Post-natal handling models in rodents comparing control (brief handling sessions; EH) with no intervention stress conditions (NH), indicate that the NH condition results in an anxious adulthood phenotype and this was confirmed in the present thesis using the elevated plus-maze behavioural test. Using this model the effects of early-life stress on adulthood GABAA receptors were then examined. 4. Regional densities of GABAA receptor α1 and α2 subunit proteins were observed in the adult brain of male and female mice using immunoperoxidase histochemistry. NH males showed a loss of the α2 subunit from the thalamus and the lower layers (IV-VI) of the primary somatosensory cortex, whilst NH females showed a reduction of α2 but an increase in α1 protein in the lower layers of the primary somatosensory cortex only. These regionally specific alterations in the α1:α2 subunit ratio suggest that early-life stress disrupts the developmental α subunit switch, which occurs in a regionally-dependent fashion over the first two weeks of rodent life. 5. Double-labelling immunofluorescence and confocal microscopy were used to examine the effects of sex and early-life stress on GABAA receptor synaptic clustering. Regardless of sex, mice exposed to early-life stress (NH) showed reduced colocalisation of the GABAA receptor α2 subunit with the synaptic marker protein gephyrin relative to the control condition (EH). This suggests that early-life stress impairs adulthood inhibitory synaptic strength and is consistent with the increased anxiety of the stressed relative to control mice. 6. Finally, the effects of early-life stress on adulthood swim stress-induced changes in GABAA receptor binding were examined using quantitative receptor autoradiography in forebrain cortical regions. Findings showed that the effect of adulthood stress on the total number of GABAA receptor binding sites for [3H]GABA in forebrain cortical regions was altered by early-life stress in both male and female mice, suggesting that the rapid adulthood stress response of GABAA receptors is affected by early-life experience. 7. Together these results show that GABAA receptors are sensitive to subtle changes in the environment in both early-life and adulthood and that these neurochemical responses to stress in adulthood are sex-dependent. The short and long-term stress-sensitivity of the GABAergic system implicates GABAA receptors in the non-genetic aetiology of psychiatric illnesses in which sex and stress are important factors.
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An investigation into the role of climbing fibres in cerebellar functionCerminara, Nadia L. (Nadia Lisa), 1975- January 2002 (has links)
Abstract not available
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Determining cross-reactivity between human and mouse tissue using mono-specific antibodiesMonazzami, Avin January 2007 (has links)
<p>ABSTRACT</p><p>The Swedish Human Proteome Resource (HPR) is a project about mapping of human genes and proteins. It aims to describe the function and distribution of all human genes and corresponding proteins, using in-house produced antibodies and tissue microarrays (TMA) for enzyme based immunohistochemistry. The mono-specific antibodies are used for immunostaining of human tissue. Specific predicted antigens named Protein Epitope Signature Tag (PrEST) are needed to produce mono-specific antibodies. PrEST are produced using recombinant technology from predicted genes and used as immunogens to produce (mono-specific) antibodies in rabbits. In this study, 84 mono-specific antibodies with known specificity to human proteins were used for immunohistochemical analysis of mouse tissues to determine the cross reactivity between mouse and human.</p><p>For 6 of the 84 antibodies the results differed between mouse and human tissue. A comparison between the PrEST used with the mouse proteome using database search programs showed homologies that were less than 100% in these six cases.</p><p>Thus, future studies on cross reactivity will focus on how to increase the accuracy in its prediction at the in silico stage of the experiment.</p>
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Characterization of Male Breast Cancer : From Molecule to Clinical OutcomeNilsson, Cecilia January 2012 (has links)
The aim of this thesis was to investigate different aspects of male breast cancer (MBC), and to compare these with findings in female breast cancer (FBC). In paper I, a population–based study was performed to investigate possible differences in treatment and outcome between MBC and FBC patients. MBC and FBC presented with a similar distribution of stage. Although no differences in primary treatment strategy were demonstrated, MBC patients had significantly poorer overall and relative survival, indicating a more aggressive disease. Paper II aimed to assess the value of clinicopathological factors and molecular subtypes in MBC. One hundred and ninety-seven MBC tumors were characterized using immunohistochemistry (IHC) and the findings were correlated to outcome. Lymph node positivity, larger tumor size and ER-negativity were independent risk factors for breast cancer death. Tumor grade, HER2, Ki 67 or IHC classification into molecular subtypes did not demonstrate any prognostic information. In paper III, the same patient material as in paper II was used for evaluation of proliferation markers. High levels of cyclin A and cyclin B expression and an elevated mitotic count were predictive of breast cancer death. Ki-67 was re-evaluated using different cut-offs, but no prognostic value could be demonstrated. Contrarily, overexpression of cyclin D1 was associated with a lower risk of breast cancer death. In papers IV-V, the molecular background of MBC tumors was investigated. Global GEX analyses were performed and two novel subgroups of MBC tumors were identified; luminal M1 and luminal M2. When comparing the degree of similarity with the “intrinsic” subtypes in FBC tumors, more than half of the MBC tumors remained unclassified. Comparative genomic hybridization was used to investigate DNA aberrations. Two MBC subgroups were identified, of which one did not resemble any of the female subgroups. In both studies on the molecular level, a majority of patients were classified into the subgroup with a more aggressive tumor behavior. In conclusion, MBC seems to be a unique tumor entity. The established molecular subtypes in FBC are not applicable in MBC. Other prognostic profiles, specific for MBC, need to be identified.
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