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Master's Dissertation
DOI
10.11606/D.75.2008.tde-24082009-165818
Document
Author
Full name
Leandro Aparecido Grange Deziderio
Institute/School/College
Knowledge Area
Date of Defense
Published
São Carlos, 2008
Supervisor
Committee
Borges, Júlio César (President)
Palma, Mario Sergio
Araújo, Heloisa Sobreiro Selistre de
Title in Portuguese
Extração das isoformas da proteína precursora do amilóide em plasma rico em plaquetas para testes proteômicos como biomarcador da doença de Alzheimer
Keywords in Portuguese
Alzheimer
biomarcador
proteina precursora do amilóide
Abstract in Portuguese
Este trabalho de mestrado teve como objetivo o desenvolvimento de uma metodologia analítica focada no preparo de amostra protéica. O objeto de estudo foram os fragmentos solúveis das isoformas da proteína precursora do amilóide (APPs) presentes no plasma rico em plaquetas. As APPs têm sido amplamente estudadas em diversos grupos de pesquisa no Brasil e em outros no mundo como possíveis biomarcadores para a doença de Alzheimer. O preparo de amostra é a etapa fundamental que influencia significativamente nos resultados seguintes, especialmente quando se trata de amostras protéicas que exigem maiores cuidados. Para a avaliação dos melhores preparos de amostra para as APPs, foi utilizado SDS-PAGE e eletrotransferências de proteínas por Western Blotting. A eficiência dos preparos foi avaliada baseando-se nos resultados de revelação com anticorpos específicos para APP e medidas de densitometria de bandas. Após a escolha do melhor preparo de amostra utilizando SDS-PAGE e Western Blotting, as isoformas da APP foram separadas por eletroforese bidimensional (2DE). Durante a etapa de preparo de amostra, os resultados inesperados de massa molecular, o que indicou possível biodegradação das APPs. A identificação da fonte de interferência foi realizada estudando as variáveis dos preparos de amostra. Com isso foi possível determinar a fonte de interferência, mas uma avaliação mais detalhada das isoformas (como utilização de espectrometria de massas) não foi possível.
Title in English
Extraction of amyloid precursor protein isoforms from blood plasma´s platelet for proteomic tests as Alzheimer disease biomarker
Keywords in English
Alzheimer
amyloid precursor protein
biomarker
Abstract in English
The goal of this Master's work was to develop an analytical methodology focused on protein sample preparation. The analyte studied were soluble amyloid precursor protein isoforms (APPs) which has been studied in many groups in Brazil and around the world as a possible biomarker for Alzheimer's disease. Sample preparation is a crucial step that influence significantly on next results, especially about biological samples which require more attention. For the best sample preparation for APPs, was used SDS-PAGE and protein electrotransference by Western Blotting techniques. The efficiency of the sample preparations was evaluated based on specific antibody reactions and densitometry measures of these interactions. After that, the APP isoforms were analyzed by two dimensional electrophoresis (2DE). During the sample preparation, were obtained unexpected molecular mass results, which indicated some APPs biodegradation. For the determination of the interference source, the variants steps of the sample preparation were analyzed. The sample preparation interference source was identified, but a more detailed study of the isoforms (by mass spectrometry) was not possible as well as the analysis of the identity of the possible fragmented isoforms.
 
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LeandroGDeziderio.pdf (3.10 Mbytes)
Publishing Date
2009-08-26
 
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