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A disfunção mitocondrial e a agregação proteica são dois fenómenos que têm vindo a ser relacionados com as doenças neurodegenerativas e ambas estão interligadas com o stress oxidativo celular. A disfunção mitocondrial relaciona-se com o dano nos ácidos nucleicos e a agregação proteica com erros no folding proteico. No entanto, ainda não se definiu a agregação como uma causa ou consequência das doenças neurodegenerativas. Assim sendo, estadissertaçãodemestradotevecomoprincipaisobjetivosanalisarainfluênciadediferentes concentrações de peróxido de hidrogénio, um agente oxidante produzido pelas nossas células, na agregação proteica e no material genético, bem como o papel da vitamina E na reversão do efeito do H2O2. Para isso, amostras celulares de Saccharomyces cerevisiae foram recolhidas a meio da fase exponencial de crescimento. A análise dos agregados insolúveis foi realizada através da técnica eletroforética SDS-PAGE seguida de lise proteica e a quebra do DNA foi analisada através de eletroforese de agarose 1,5% seguida de RAPD-PCR. A exposição ao agente oxidante ao longo do tempo potenciou a agregação proteica, nomeadamente da ovalbumina. No entanto, o agente antioxidante não demonstrou qualquer tipo de reversão do efeito oxidante, parecendo apresentar um efeito pró-oxidante. Apesar de se observarem diferenças na agregação proteica ao longo do tempo, não se observaram quebras no material genético.
Mitochondrial dysfunction and protein aggregation are two phenomena that have been correlated with neurodegenerative diseases and are both interlinked with cellular oxidative stress. Mitochondrial dysfunction is associated witn nucleic acid damage and protein aggregation with mistakes in protein folding. However, aggragtion has not yet been defind as a cause or consequence of neurodegenerative diseases. Therefore, this thesis' main objectives were to analyze the influence of different concentrations of hydrogen peroxide, an oxidant agent produced by our cells, in protein aggregation and genetic material as well as the role of vitamin. E in reversing the effect of H2O2. For that purpose, Saccharomyces cerevisiae cellular samples were collected throughout the exponential growth phase. Analysis of insoluble aggregates was performed using the electrophoretic technique SDS-PAGE followed byprotein lysis and DNA breakage bands through 1,5% agarose electrophoresis followed by RAPD-PCR. Exposure to the oxidizing agent over time has enhanced protein aggregation, namely ovalbumin. however, the anti-oxidant did not appear to reverse the oxidizing effect and seems to have a pro-oxidant effect. Although differences in protein aggregation were observed over time, there were no breaks in the genetic material.
Mitochondrial dysfunction and protein aggregation are two phenomena that have been correlated with neurodegenerative diseases and are both interlinked with cellular oxidative stress. Mitochondrial dysfunction is associated witn nucleic acid damage and protein aggregation with mistakes in protein folding. However, aggragtion has not yet been defind as a cause or consequence of neurodegenerative diseases. Therefore, this thesis' main objectives were to analyze the influence of different concentrations of hydrogen peroxide, an oxidant agent produced by our cells, in protein aggregation and genetic material as well as the role of vitamin. E in reversing the effect of H2O2. For that purpose, Saccharomyces cerevisiae cellular samples were collected throughout the exponential growth phase. Analysis of insoluble aggregates was performed using the electrophoretic technique SDS-PAGE followed byprotein lysis and DNA breakage bands through 1,5% agarose electrophoresis followed by RAPD-PCR. Exposure to the oxidizing agent over time has enhanced protein aggregation, namely ovalbumin. however, the anti-oxidant did not appear to reverse the oxidizing effect and seems to have a pro-oxidant effect. Although differences in protein aggregation were observed over time, there were no breaks in the genetic material.
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Keywords
Doenças neurodegenerativas Danos do DNA Agregação proteica Vitamina E Stress oxidativo Efeito antioxidante