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Doctoral Thesis
DOI
https://doi.org/10.11606/T.42.2008.tde-23092008-132202
Document
Author
Full name
Marcos Gonzaga dos Santos
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Winter, Lucile Maria Floeter (President)
Alves, Maria Julia Manso
Cruz, Angela Kaysel
Silber, Ariel Mariano
Tosi, Luiz Ricardo Orsini
Title in Portuguese
Papel funcional da fosfatidilserina de Leishmania (Leishmania) amazonensis na infecção de macrófagos.
Keywords in Portuguese
Biologia molecular
Genética molecular
Protozoologia
Abstract in Portuguese
A caracterização da síntese de fosfatidilserina (PS) em Leishmania (Leishmania) amazonensis mostrou a presença de uma única via de síntese de PS, pela ação da enzima fosfatidilserina sintetase II (PSS II). A seqüência que codifica essa enzima, presente em cópia única no genoma, apresentou uma identidade de 38% e uma similaridade de 55% com seu homólogo humano. Tentativas de se nocautear esse gene não foram bem sucedidas, indicando que o gene é essencial à sobrevivência do parasita. Ensaios de incorporação de fosfolipídios marcados mostraram que o parasita captura do meio fosfatidiletanolamina, substrato da PSS II, mas a incorporação de PS se dá em uma taxa muito baixa. Também foi feita a caracterização do transporte de serina pelo parasita, que mostrou a existência de um único transportador com pH ótimo de 7,5, dependente da aitvidade metabólica da célula, com um Km de 0,826 +/- 0,183 mM e Vmax de 355,37 +/- 19,41 pmol/min * 2 * 107 promastigotas, que se mantém ativo a até 45 oC.
Title in English
Functional role of Leishmania (Leishmania) amazonensis phosphatidylserine in macrophage infection.
Keywords in English
Molecular biology
Molecular genetics
Protozoology
Abstract in English
The characterization of phosphatidylserine (PS) synthesis in Leishmania (Leishmania) amazonensis revealed a single pathway that presented phosphatidylserine synthase II (PSS II) activity. The single copy gene that encoded this enzyme showed 38% of identity and 55% of homology to the human homolog. Attempts for the gene knockout were not successful, indicating that the gene is essential for the parasite survival. Incorporporation assays of labeled phospholipids showed that the parasite take phosphatidylethanolamine, substract for the PSS II, from the medium, but the rate of incorporation of PS occurred at a very low rate. The biochemical characterization of the serine incorporation by the parasite showed the existence of a single transport system, with an optimum pH of 7.5, which was dependent of metabolic activity of the cell, that showed a Km of 0.826 +/- 0.183 mM and Vmax of 355.37 +/- 19.41 pmol/min *2 * 107 promastigotes, that remained active up to 45oC.
 
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Publishing Date
2008-09-24
 
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