Influência do cobre no padrão de expressão de genes envolvidos na interação de Paracoccidioides brasiliensis com a matriz extracelular
Carregando...
Data
2010-06-25
Autores
Oliveira, Haroldo Cesar de [UNESP]
Título da Revista
ISSN da Revista
Título de Volume
Editor
Universidade Estadual Paulista (Unesp)
Resumo
Paracoccidioides brasiliensis (Pb) é o agente etiológico da paracoccidioidomicose, micose sistêmica de grande importância no Brasil, país que possui a maior concentração de áreas endêmicas para essa doença no mundo. Uma das estratégias possivelmente utilizadas pelo patógeno seria a expressão de genes envolvendo adaptação às condições do hospedeiro, que pode também estar relacionadas à captação de micronutrientes. A matriz extracelular (MEC) desempenha um papel importante na regulação da adesão celular, diferenciação, migração e proliferação das células. Este estudo propõe uma análise de transcritos e de proteínas expressas em condições de depleção de cobre, na presença de quatro matrizes extracelulares – laminina, fibronectina e colágeno I e IV, mimetizando as condições de infecção por P. brasiliensis por meio das técnicas de RDA (Representational Difference Analysis) e eletroforese bidimensional. Para isso, o isolado Pb01 foi cultivado por 3 horas no meio quimicamente definido (MVM), depletado de cobre (Cu) e a seguir colocado em contato com os quatro diferentes componentes da MEC e a adesão foi avaliada por citometria de fluxo. Um aumento significativo (p ≤ 0,05) na adesão frente a todos os componentes da MEC foi observado quando o fungo foi cultivado sem Cu. Então, o RNA e os extratos protéicos foram obtidos de Pb sem Cu e Pb sem Cu em contato com os diferentes componentes da MEC. Ensaios de RDA foram realizados para demonstrar os genes envolvidos neste processo. Duas hibridizações foram realizadas nas proporções de 1:10 e 1:100 de tester e driver, respectivamente, com um excesso de driver para remover as seqüências comuns em ambas condições. Os produtos diferencialmente expressos foram amplificados, resultando em padrões distintos que foram seqüenciados...
Paracoccidioides brasiliensis (Pb) is the etiologic agent of paracoccidioidomycosis, a systemic mycosis of great importance in Brazil, which has the highest concentration of endemic areas for this disease in the world. One of the strategies used by the pathogen may be the expression of proteins related to the adaptation to the host conditions, which may also be related to the uptake of micronutrients. Extracellular matrix (ECM) plays an important role in the regulation of cell adhesion, differentiation, migration and proliferation of cells. This study proposes an analysis of transcripts and proteins expressed in condition of copper depletion in the presence of four components of extracellular matrix - laminin, fibronectin and collagen I and IV, mimicking the conditions of infection by P. brasiliensis, using techniques of RDA (Representational Difference Analysis) and two-dimensional electrophoresis. For this, we cultured the Pb 01 strain at 3 hours in a chemically defined media (MVM) with depletion copper (Cu). After, the fungus was placed at contact with the four differents ECM components and the adhesion was evaluated by flow cytometry. A significant increase of binding (p ≤ 0,05) to all ECM components was observed when the fungal was cultured without Cu. So RNA and protein extracts were obtained of Pb without Cu, and Pb without Cu in contact with the differents ECM components. RDA assay was developed to demonstrate the genes involved in this process. Two hybridizations were performed in the proportions of 1:10 and 1:100 of tester and driver respectively, with an excess of driver to remove the common sequences in both conditions. The differentially expressed products were amplified, resulting in distinct patterns that were sequenced revealing genes involved in differents process like virulence (25%), protein synthesis... (Complete abstract click electronic access below)
Paracoccidioides brasiliensis (Pb) is the etiologic agent of paracoccidioidomycosis, a systemic mycosis of great importance in Brazil, which has the highest concentration of endemic areas for this disease in the world. One of the strategies used by the pathogen may be the expression of proteins related to the adaptation to the host conditions, which may also be related to the uptake of micronutrients. Extracellular matrix (ECM) plays an important role in the regulation of cell adhesion, differentiation, migration and proliferation of cells. This study proposes an analysis of transcripts and proteins expressed in condition of copper depletion in the presence of four components of extracellular matrix - laminin, fibronectin and collagen I and IV, mimicking the conditions of infection by P. brasiliensis, using techniques of RDA (Representational Difference Analysis) and two-dimensional electrophoresis. For this, we cultured the Pb 01 strain at 3 hours in a chemically defined media (MVM) with depletion copper (Cu). After, the fungus was placed at contact with the four differents ECM components and the adhesion was evaluated by flow cytometry. A significant increase of binding (p ≤ 0,05) to all ECM components was observed when the fungal was cultured without Cu. So RNA and protein extracts were obtained of Pb without Cu, and Pb without Cu in contact with the differents ECM components. RDA assay was developed to demonstrate the genes involved in this process. Two hybridizations were performed in the proportions of 1:10 and 1:100 of tester and driver respectively, with an excess of driver to remove the common sequences in both conditions. The differentially expressed products were amplified, resulting in distinct patterns that were sequenced revealing genes involved in differents process like virulence (25%), protein synthesis... (Complete abstract click electronic access below)
Descrição
Palavras-chave
Paracoccidioides brasiliensis, Paracoccidioidomicose, Copper depletion, Laminin, Fibronectin
Como citar
OLIVEIRA, Haroldo Cesar de. Influência do cobre no padrão de expressão de genes envolvidos na interação de Paracoccidioides brasiliensis com a matriz extracelular. 2010. 70 f. Dissertação (mestrado) - Universidade Estadual Paulista, Faculdade de Ciências Farmacêuticas, 2010.