• JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
  • JoomlaWorks Simple Image Rotator
 
  Bookmark and Share
 
 
Tese de Doutorado
DOI
10.11606/T.42.2008.tde-09092008-173354
Documento
Autor
Nome completo
Sara Maria Zago Gomes
Unidade da USP
Área do Conhecimento
Data de Defesa
Imprenta
São Paulo, 2008
Orientador
Banca examinadora
Bevilacqua, Estela Maris Andrade Forell (Presidente)
Gagioti, Sonia Maria
Lopes, Lucia Rossetti
Oliveira, Sergio Ferreira de
Verlengia, Rozangela
Título em português
Expressão e atividade da NAD(P)H-oxidase de membrana nas células trofoblásticas de camundongos.
Palavras-chave em português
Ânion - superóxido
Células trofoblásticas
Fagocitose
gp91 - phox
NADP(H) - oxidase
p67 - phox
Resumo em português
As células trofoblásticas provenientes de cones ectoplacentários na fase de pós-implantação expressam as subunidades do complexo NAD(P)H-oxidase de membrana p22-phox, gp91-phox, p47-phox, p40-phox, p67-phox e Rac1 previamente descritas em fagócitos e envolvidas com a atividade fagocitária destas células. Ocorre modulação da expressão das subunidades do complexo enzimático NAD(P)H-oxidase sob o estímulo com PMA, agente capaz de fosforilar a subunidade p47-phox e ativar esta enzima em fagócitos profissionais. Nossos ensaios experimentais indicam que as células trofoblásticas são capazes de gerar espécies reativas de oxigênio dependente da atividade do complexo NAD(P)H-oxidase de membrana de baixa intensidade quando não estimuladas e de forma muito mais intensa quando estimuladas pelo PMA. As características de expressão e atividade do complexo enzimático NAD(P)H-oxidase de membrana encontradas neste estudo sugerem que esta enzima é semelhante a encontrada em outros fagócitos e, desta forma possa talvez estar também envolvida com processos de defesa da interface materno-fetal.
Título em inglês
Expression and activity of the membrane NADP(H) - oxidase in the mouse trophoblast cells.
Palavras-chave em inglês
Anion superoxide
go 91 - phox
NADP(H) - oxidase
p67 - phox
Phagocytosis
Trophoblast cell
Resumo em inglês
In macrophages and neutrophils, the activation of the phagocytosis is defined by the acquisition of competence for microbicide, tumoricide and cytolytic functions resultant of generation and release of oxygen reactive species, besides the phagocytic process per se. Like these phagocytes, trophoblast cells in many species are also phagocytic. This cellular population involves completely the embryo and exhibits different and specific characteristics along the gestation. In rodents and primates, these cells are strategically positioned between maternal and fetal circulation and exhibit invasive and phagocytic activity, respectively responsible for anchorage of the embryo into the endometrium and uptake of an adequate nutritional supply for the embryo development. In rodents, these activities present a maximum degree during the implantation period, gradually declining, as the placenta develops. In the presence of strange particles at the maternal-placental interface, however, this process can be reactivated and, in this case, may be related to defense's mechanisms. Previous studies performed in our laboratory showed the potential of the trophoblast in producing and releasing reactive species of oxygen/nitrogen, in a very similar manner to that observed in macrophages and neutrophils. The production of such molecules is associated to different enzymes, but the localization of hydrogen peroxide on trophoblast cell surface has suggested a NAD(P)H-oxidase activity. NAD(P)H-oxidase is formed by the cytochrome b558 associated to the cellular membrane (subunits p22-phox + gp91-phox), the cytosolic subunits p47-phox, p67-phox and p40phox and, the GTPases Rac1 and Rac2 in an electron generator system that uses NADH or NADPH as substratum. Once activated, the enzymatic complex is responsible for the electron inflow to the molecular oxygen, yields superoxide anion. Thus, based on the literature and results previously obtained by our group, this study analyzed the protein and gene expression of the NAD(P)H-oxidase complex subunits respectively by immunolocalization and Westernblotting and rt-PCR, in the mouse trophoblast stimulated with PMA. Rt-PCR semi-quantitative analyses showed increase expression of the subunits p22-phox, gp91-phox, p47-phox, p67-phox, p40phox and Rac1 in PMA-treated in comparison with non treated ectoplacental cones. The expression of the subunits gp91-phox, p47-phox and p67-phox were confirmed by Western blotting and, like gene expression also increased in the presence of PMA. These subunits were mostly located in the trophoblast giant cell population, associated to the phagocytic process at the maternal-placental interface. Increased expression of such subunits may be related to an increase in the NAD(P)H-oxidase activity. To analyze this possibility and to determine the role played by NAD(P)H-oxidase activity in the reactive oxygen species produced by trophoblast cells, cellular assays were performed using the oxyethidium fluorescence, a product of dihydroethidium oxidation by superoxide anion. Thus, under PMA stimulus and antimycin A that blocks the mitochondrial NAD(P)H-oxidase activity and, apocynin and allopurinol, respectively blocking the membrane NAD(P)H-oxidase and xhantine oxidase and, still, using specific superoxide and hydrogen peroxide scavengers (superoxide dismutase enzyme and catalase) we showed the generation of reactive species of oxygen-NAD(P)H-oxidase dependent by trophoblast cells, mostly when stimulated. These results come to add important information about the potential of the trophoblast in producing reactive species at the maternal-fetal interface and, open a new investigation interest on the NADPH-oxidase regulatory processes and its involvement in defense functions of the embryo in both healthy and pathological processes that can determine the failure of the gestation.
 
AVISO - A consulta a este documento fica condicionada na aceitação das seguintes condições de uso:
Este trabalho é somente para uso privado de atividades de pesquisa e ensino. Não é autorizada sua reprodução para quaisquer fins lucrativos. Esta reserva de direitos abrange a todos os dados do documento bem como seu conteúdo. Na utilização ou citação de partes do documento é obrigatório mencionar nome da pessoa autora do trabalho.
Data de Publicação
2008-09-11
 
AVISO: Saiba o que são os trabalhos decorrentes clicando aqui.
Todos os direitos da tese/dissertação são de seus autores
Centro de Informática de São Carlos
Biblioteca Digital de Teses e Dissertações da USP. Copyright © 2001-2018. Todos os direitos reservados.