Orientador: Paulo Eduardo Martins Ribolla / Banca: Alexandre Afrânio Peixoto / Banca: Newton Goulart Madeira / Resumo: O fungo termo dimórfico, Paracoccidioides brasiliensis é o agente etiológico da Paracoccidioidomicose (PCM), a micose sistêmica mais prevalente da América Latina. Este fungo vem sendo frequentemente isolado de amostras clínicas, tecidos de tatu (Dasypus novemcinctus) e recentemente foi também isolado de cão. Este trabalho avaliou a transição de micélio para levedura (M-L), a termo tolerância e o perfil de virulência em nove isolados de P. brasiliensis (quatro de pacientes humanos, quatro de tatus e um de cão), bem como a sua relação com a seqüência parcial e expressão do gene hsp70 (Heat Shock Protein 70) através de Real Time RT-PCR. Tanto os dados morfológicos como moleculares se mostraram variáveis dentre os diferentes isolados. Alguns destes dados, como sequenciamento e morfologia leveduriforme corroboram com a divisão de nossos isolados nas duas espécies crípticas simpátricas previamente propostas por Matute et al (2006). Nossos resultados confirmam que a HSP70 pode ser um importante fator de virulência por estar associado à termo tolerância, mas sua expressão parece não ser diretamente associada a altos padrões de virulência. / Abstract: The thermo dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The previous phylogenetic species recognition proved the existence of, at least, three cryptic species in this pathogen. In this work we evaluated the mycelia to yeast (M-Y) transition, thermo tolerance and virulence profiles of nine isolates of P. brasiliensis, (including members of two of the three species) as well as its relation to the partial sequence and expression of hsp70 gene. It was observed a large phenotypic variability concerning the M-Y transition. The isolates Bt84 and T10 took more time to convert to the yeast form. These same isolates presented stretched yeast cells at 36°C, instead of the typical round cells. It was also observed arthroconidia production during the M-Y transition for some of the nine isolates studied. The hsp70 expression showed to be variable among our isolates. The partial sequencing of hsp70 gene resulted in a Neighbour Joining tree that divided our isolates in two main groups. Conclusions: Our data confirm that hsp70 gene might be an important virulence factor, associated with the thermo tolerance, but its expression does not seem to be directly related to high virulence profiles. We also presented some preliminary results about mycological characters that could be important candidates for morphologic markers for species recognition, as well as the partial sequencing of one member of the hsp70 gene family that allowed the separation of our isolates in two clusters, that correspond to the two sympatric cryptic species that occur in our PCM hyper endemic area (Botucatu, SP, Brazil). / Mestre

Made available in DSpace on 2014-06-11T19:26:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-04-07Bitstream added on 2014-06-13T19:53:57Z : No. of bitstreams: 1 angella_af_me_botib.pdf: 1771008 bytes, checksum: 49bffe6b87df00a6ffab3e65cd19bdaa (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O fungo termo dimórfico, Paracoccidioides brasiliensis é o agente etiológico da Paracoccidioidomicose (PCM), a micose sistêmica mais prevalente da América Latina. Este fungo vem sendo frequentemente isolado de amostras clínicas, tecidos de tatu (Dasypus novemcinctus) e recentemente foi também isolado de cão. Este trabalho avaliou a transição de micélio para levedura (M-L), a termo tolerância e o perfil de virulência em nove isolados de P. brasiliensis (quatro de pacientes humanos, quatro de tatus e um de cão), bem como a sua relação com a seqüência parcial e expressão do gene hsp70 (Heat Shock Protein 70) através de Real Time RT-PCR. Tanto os dados morfológicos como moleculares se mostraram variáveis dentre os diferentes isolados. Alguns destes dados, como sequenciamento e morfologia leveduriforme corroboram com a divisão de nossos isolados nas duas espécies crípticas simpátricas previamente propostas por Matute et al (2006). Nossos resultados confirmam que a HSP70 pode ser um importante fator de virulência por estar associado à termo tolerância, mas sua expressão parece não ser diretamente associada a altos padrões de virulência. / The thermo dimorphic fungus Paracoccidioides brasiliensis is the etiological agent of Paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. The previous phylogenetic species recognition proved the existence of, at least, three cryptic species in this pathogen. In this work we evaluated the mycelia to yeast (M-Y) transition, thermo tolerance and virulence profiles of nine isolates of P. brasiliensis, (including members of two of the three species) as well as its relation to the partial sequence and expression of hsp70 gene. It was observed a large phenotypic variability concerning the M-Y transition. The isolates Bt84 and T10 took more time to convert to the yeast form. These same isolates presented stretched yeast cells at 36°C, instead of the typical round cells. It was also observed arthroconidia production during the M-Y transition for some of the nine isolates studied. The hsp70 expression showed to be variable among our isolates. The partial sequencing of hsp70 gene resulted in a Neighbour Joining tree that divided our isolates in two main groups. Conclusions: Our data confirm that hsp70 gene might be an important virulence factor, associated with the thermo tolerance, but its expression does not seem to be directly related to high virulence profiles. We also presented some preliminary results about mycological characters that could be important candidates for morphologic markers for species recognition, as well as the partial sequencing of one member of the hsp70 gene family that allowed the separation of our isolates in two clusters, that correspond to the two sympatric cryptic species that occur in our PCM hyper endemic area (Botucatu, SP, Brazil).

Submitted by MARIANA MALAVAZI DESTRO null (m_destro@hotmail.com) on 2016-07-07T16:27:57Z No. of bitstreams: 1 Dissertação para arquivar Unesp.pdf: 751238 bytes, checksum: ef14aad090e2f5e2974329d58301aef8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-07-11T14:37:52Z (GMT) No. of bitstreams: 1 destro_mm_me_araca.pdf: 751238 bytes, checksum: ef14aad090e2f5e2974329d58301aef8 (MD5) / Made available in DSpace on 2016-07-11T14:37:52Z (GMT). No. of bitstreams: 1 destro_mm_me_araca.pdf: 751238 bytes, checksum: ef14aad090e2f5e2974329d58301aef8 (MD5) Previous issue date: 2016-06-03 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A criptococose é uma infecção fúngica causada por levedura do gênero Cryptococcus. Dentre as principais micoses sistêmicas nas últimas décadas, a criptococose vem assumindo um relevante papel, com altos índices de morbidade e mortalidade principalmente em pacientes imunodeprimidos. O presente trabalho teve por objetivo verificar a ocorrência de Cryptococcus spp. em amostras ambientais na área urbana de Bauru-SP, Brasil, estabelecendo os focos ambientais, com a finalidade de compreender a eco-epidemiologia do microrganismo. Cinquenta amostras ambientais de dez locais representativos foram processadas e semeadas em ágar semente de Níger (NSA) e Sabouraud. As colônias com características macro e micromorfológicas compatíveis com Cryptococcus foram submetidas a provas bioquímicas, sendo que 12/50 (24%) das amostras foram positivas para o gênero Cryptococcus. A partir dos resultados obtidos, pode-se concluir que foram encontradas espécies causadoras de criptococose no município de Bauru, constituindo-se na existência de focos ambientais, com potencial patogênico para o homem e animais. A compreensão da eco-epidemiologia do Cryptococcus constitui em uma importante ferramenta na estratégia de medidas preventivas. / Cryptococcosis is a fungal infection caused by yeasts of the genus Cryptococcus. One of the main systemic mycoses in recent decades, has been assuming an important role, with high rates of morbidity and mortality especially in immunosuppressed patients. The present study aimed to verify the existence of Cryptococcus spp. from environmental samples in the urban area of Bauru-SP, Brazil, establishing the environmental focus, with the purpose of understand the eco-epidemiology of the micro-organism. Fifty environmental samples from ten representative sites were processed and seeded in agar Niger seed (NSA) and Sabouraud. The colonies with macro and micromorphological compatible with Cryptococcus underwent biochemical tests, and 12/50 (24%) of the samples were positive for the genus Cryptococcus. From the results obtained, it can be concluded that species that cause cryptococcosis were found in the city of Bauru-SP, Brazil, constituting the existence of outbreaks with potential environmental pathogenic for human and animals. The eco-epidemiological understanding of the Cryptococcus constitutes an important tool in the strategy of preventive measures.