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Doctoral Thesis
DOI
10.11606/T.42.2008.tde-27032009-103125
Document
Author
Full name
Carolina Maria Berra
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2008
Supervisor
Committee
Menck, Carlos Frederico Martins (President)
Hojo, Elza Tiemi Sakamoto
Lardner, Nadja Cristhina de Souza Pinto
Marques, Marilis do Valle
Soares Netto, Luis Eduardo
Title in Portuguese
Estudos de reparo de DNA por excisão de nucleotídeos em lesões oxidativas em células de mamíferos.
Keywords in Portuguese
Fotossensibilização
Lesões oxidativas
Reparação de DNA
Abstract in Portuguese
Para melhor entender o envolvimento do NER em lesões oxidativas averiguou-se os efeitos da foto-excitação do azul de metileno (MB) in vitro e in vivo. Verificou-se que MB e luz induzem muitos sítios sensíveis a enzima FPG em DNA plasmidial. Mostrou-se que MB é incorporado por fibroblastos humanos proficientes ou deficientes em NER (XP-A e XP-C) e gerou oxigênio singlete (1O2) intracelularmente. A foto-excitação do MB formou também 8-oxoG em núcleos de fibroblastos. Células mutantes XP-A e XP-C foram mais sensíveis ao tratamento e apresentaram mais danos oxidativos em seu DNA genômico do que células selvagens. Contudo, todas as células mostraram a mesma cinética de reparo das lesões formadas. Além disso, houve mais marcação de g-H2AX nas células mutantes após a foto-excitação do MB. Assim, os resultados sugerem que a excitação do MB gera danos oxidativos e quebras na molécula de DNA, tanto in vitro quanto in vivo e que as proteínas XPA e XPC podem ter algum papel na proteção desse estresse, além da sua sabida participação em reparo de lesões induzidas por luz UV.
Title in English
The involvement of nucleotide excision repair in DNA oxidative lesions in mammals cells.
Keywords in English
DNA repair
Oxidative lesions
Photo excitation
Abstract in English
In order to study the participation of NER mechanisms in the removal of oxidative DNA lesions, we examined the effects of photoactivated methylene blue (MB) in vitro and in vivo. Plasmid DNA presents more FPG sensitive sites when MB was photoactivated than non-photoactivated. MB can be incorporated by NER-proficient or deficient cells and is able to generate singlet oxygen (1O2) inside cells. We detected also the presence of 8-oxoG in genomic DNA after treatment. XP-A and XP-C deficient cells are more sensitive to MB treatment and were found to have more oxidative DNA lesions than proficient cells. Also, there are more g-H2AX foci in mutant cells after treatment. However, there is a clear similarity in lesions repair kinetic between proficient and deficient cells. Therefore, these results suggest that photoactivated MB generates oxidative damage and DNA strand breaks both vitro and in vivo assays and that XPA and XPC proteins may also have a role in the protection of cellular oxidative stress, in addition to its participation in repair of UV-induced DNA damage.
 
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Publishing Date
2009-04-06
 
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