Orientador: Carlos Alberto de Magalhães Lopes / Banca: Eduardo Bagagli / Banca: Hélio Langoni / Banca: Ida Maria Foschiani Dias Baptista / Banca: Patrícia Sammarco Rosa / Resumo: A hanseníase é, ainda hoje, um problema global de saúde pública. O Brasil e a Índia são os países com maior prevalência da doença. A existência de tatus Dasypus novemcinctus naturalmente infectados por Mycobacterium leprae foi relatada nos Estados Unidos, México, Argentina e no Brasil, no Estado do Espírito Santo. Micobactérias potencialmente patogênicas e algumas ambientais já foram isoladas destes animais. A identificação do bacilo é dificultada, particularmente pela sua incapacidade de crescimento in vitro. Portanto, o uso de ferramentas moleculares representa uma alternativa mais rápida e sensível para diagnóstico de micobacterioses. Neste trabalho foram feitas baciloscopia, cultura e PCR com primers específicos para seqüências repetitivas de M. leprae, a partir de DNA extraído de amostras de fígado, baço, linfonodos e pele de 21 tatus D. novemcinctus, Euphractus sexcintus e Cabassous tatouay provenientes da região Centro-Oeste do Estado de SP, dos quais 17 eram provenientes do Banco de DNA do Laboratório de Biologia de Fungos do Departamento de Microbiologia e Imunologia - IBB. Para a padronização da metodologia foram utilizados hamsters experimentalmente infectados por M. avium e o modelo experimental para hanseníase (D. novemcinctus) do Biotério de Experimentação Animal do Instituto Lauro de Souza Lima, Bauru-SP. Com exceção de dois tatus, cujas amostras fezes foram positivas pela PCR, nenhum tatu selvagem apresentou infecção natural por micobactérias. Somente o tatu inoculado com biópsia de paciente virchowiano foi positivo, tanto na PCR, quanto no seqüenciamento genômico, que revelou 100% de identidade com o M. leprae. Os resultados preliminares deste estudo, com base na metodologia empregada, sugerem que existe um indicativo de que os tatus selvagens podem apresentar papel de reservatório de M. leprae na cadeia epidemiológica da doença, na região estudada. / Abstract: Leprosy is still a worldwide public health problem. Brazil, in addition to India, show the highest prevalence rates of the disease. Natural infection of armadillos Dasypus novemcinctus with Mycobacterium leprae has been reported in the United States, Mexico, Argentina and Brazil, in the State of Espírito Santo. Potentially pathogenic and ambiental mycobacteria have been isolated from these animals. Identification of bacilli is difficult, particularly because its inability to grow in vitro. The use of molecular tools represent a fast and sensitive alternative method for diagnosis of mycobacteriosis. In the present study, the diagnostic methods used were bacilloscopy, culture and PCR using specific primers for M. leprae repetitive sequences. The PCR were performed using genomic DNA extracted from liver, spleen, lymphnodes and skin of 21 D. novemcinctus, Euphractus sexcintus and Cabassous tatouay armadillos from the Middle Western region of the State of São Paulo. From those, 17 samples were taken from the DNA Bank of the Fungal Biology Laboratory at Department of Microbiology and Immunology IB - Botucatu, UNESP. Standardization of the technique was done in M. avium experimentally infected hamsters and M. leprae experimentally infected D. novemcinctus tissue from the Laboratory Animal House at Instituto Lauro de Souza Lima, Bauru-SP. No one of the wild armadillos showed natural mycobacterial infection. Only the armadillo inoculated with material collected from untreated multibacillary leprosy patient was PCR positive. The genomic sequencing revealed 100% identity with M. leprae. According to these preliminary studies, based on the methodology, we may conclude that wild armadillos seem play a role on epidemiology of leprosy in the Middle Western region of the State of São Paulo. / Doutor

A recombinat Chinese Hamster Ovary (rCHO) cell line designated as CHO SEAP was utilized in this investigation to optimize protein production. Two bench top stirred-tank bioreactors, namely a pitched-blade and a packed-bed basket bioreactor, were utilized for a comparative study to determine which bioreactor would produce the best results in terms of protein production. The objective of this research project was to provide basic data that shows cells cultured in a packed-bed basket bioreactor in perfusion mode will generate more protein product than cells in batch mode suspension culture with a pitched-blade bioreactor. The packed-bed bioreactor creates a homeostatic environment similar to the environment found in vivo, where waste products are constantly removed and fresh nutrients are replenished. Closed batch cultures do not provide a homeostatic environment. In batch culture systems, nutrients are depleted and waste products accumulate. The results from this experiment could help investigators involved in protein and/or vaccine production facilities select the appropriate bioreactor and mode of operation to optimize cell productivity for generation of a specific protein product. CHO cells have been used for the production of vaccines, recombinant therapeutic proteins, and monoclonal antibodies, and these cells are now the cell line of choice in the biopharmaceutical industry. Traditional vaccine production methods in egg embryos are slow and outdated, whereas roller bottle-based cell culture techniques are time consuming and have limited scalability. These limitations justify the need for development of stirred tank bioreactors. Cells cultured in a packed-bed bioreactor are not exposed to hydrodynamic forces, as is the case with pitched-blade bioreactors, allowing for maximum growth and protein expression. This mode of operation involves the constant removal of media depleted of nutrients and the addition of fresh media with more nutrients to keep the cells growing. Long run times decrease the constant need for re-seeding cells and re-establishing seed cultures, thus, reducing setup time and labor dramatically. Secreted products are automatically separated from cells in perfusion, eliminating filtration and membrane fouling. A detailed description of both modes of operation are discussed in this thesis.

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No description available.

This study was designed to characterize the distribution of angiotensin II (AII) binding sites in the hamster brain. Brain sections were incubated with [125I][sar1, ile8]-angiotensin II in the absence and presence of angiotensin II receptor subtype selective compounds, losartan (AAT, subtype) and PD123177 (AT2 subtype). Binding was quantified by densoritometric autoradiograms and localized by comparison with adjacent thionin stained sections. The distribution of AII binding sites was similar to that found in the rat, with some exceptions. [125I][sar1, ile8]-angiotensin II binding was not evident in the subthalamic nucleus and thalamic regions, inferior olive, suprachiasmatic nucleus, and piriform cortex of the hamster, regions of prominent binding in the rat brain. However, intense binding was observed in the interpeduncular nucleus and the medial habenula of the hamster, nuclei void of binding in the rat brain. Competition with receptor subtype selective compounds revealed a similar AII receptor subtype profile in brain regions where binding is evident in both species. One notable exception is the medial geniculate nucleus, predominately AT1 binding sites in the hamster but AT2 in the rat. Generally, the AII binding site distribution in the hamster brain parallels that of the other species studied, particularly in brain regions associated with cardiovascular and dipsogenic functions. Functional correlates for AII binding sites have not been elucidated in the majority of brain regions and species mismatches might provide clues in this regard.

No description available.

No abstract provided. / Thesis / Doctor of Philosophy (PhD) / Scope and contents: Seven adenoviruses, including oncogenic and nononcogenic serotypes from human and simian hosts, were utilized to investigate their effects upon the chromosomes of human and Syrian hamster cells. Human cells support adenovirus multiplication while hamster cells do not support replication of infectious adenovirus. The chromosome damage induced by adenoviruses in abortive infection of hamster cells was compared with respect to the effect of virus dose upon the incidence and the types of chromosome aberrations. The effect of different adenoviruses upon the amount and types of chromosome damage was also examined. The effect of adenovirus infection upon DNA synthesis of human and hamster cells was examined, and the relevance of adenovirus-induced chromosome aberrations to the etiology of human cancers is discussed.

No description available.

No description available.

No description available.