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Doctoral Thesis
DOI
10.11606/T.42.2008.tde-31032009-170358
Document
Author
Full name
Robson Sartorello
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Garcia, Celia Regina da Silva (President)
Barcinski, Marcello Andre
Silva, Aline Maria da
Smaili, Soraya Soubhi
Wunderlich, Gerhard
Title in Portuguese
Aspectos de transdução de sinal em Plasmodium.
Keywords in Portuguese
Plasmodium
Biologia celular
Cálcio
Malária
Proteínas
Transdução de sinal celular
Abstract in Portuguese
A colina quinase, 1ª enzima de uma via que forma 45% da membrana de P. falciparum foi clonada, expressa e purificada, e um anticorpo policlonal desenvolvido. Estudos de transporte da enzima demonstraram que a PfChok é transportada para o citossol por uma via independente do inibidor Brefeldina A. Obteve-se informação da estrutura secundária e uma modelagem da estrutura terciária da proteína, tendo sido localizados sítios de fosforilação para proteínas quinases. Através de uma nova abordagem em genômica funcional de Plasmodium, o gene da proteína adaptadora PfRACK foi códon otimizado e inserido em células de mamíferos. Estudos de dinâmica de Ca2+ em microscopia confocal indicaram que sua sinalização nas células estudadas é inibida na presença de PfRACK, dependente da interação direta com receptores de IP3. A enzima localiza-se distintivamente em relação à RACK1 endógena. Nossos resultados abrem a possibilidade de novos estudos, ao estabelecer a transfecção de genes do parasita para estudos de mecanismos de transdução de sinal na interação Plasmodium-hospedeiro.
Title in English
Plasmodium transduction signals aspects.
Keywords in English
Plasmodium
Calcium
Cell biology
Cell signal transduction
Malaria
Proteins
Abstract in English
Choline Kinase, the first enzyme of a pathway responsible for up to 45% of Plasmodium falciparum parasite membrane, was cloned, expressed and purified, and a policlonal antibody was developed to follow its localization along the intraerythrocytic cycle. The enzyme is transported to parasite´s cytosol, through a pathway independent of the Endoplasmic Reticulum to Golgi apparatus. Information about the secondary structure was obtained, as well a model of the tertiary structure, where several kinases phosphorylation sites were identified. The PfRACK gene was codon-optimized and transfected in mammalian cells. Dynamic Ca2+ studies by confocal microscope have revealed that Ca2+ signaling of the transfected cells was inhibited by PfRACK, dependent on direct interaction with InsP3 receptors. The protein co-localizes distinctly of the endogenous RACK1. Our results open new possibilities of malaria functional genomics, by establishing the transfection of parasites genes into mammalian cells with the aim to study transduction signals mechanisms of the Plasmodium-host interaction.
 
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Publishing Date
2009-04-06
 
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