AbstractsBiology & Animal Science

Tese de mestrado. Biologia (Biologia Humana e Ambiente). Universidade de Lisboa, Faculdade de Ciências, 2014 Premature termination codons (PTCs or nonsense codons) can arise from various types of mutations in germ or somatic cells. PTCs promote premature translational termination and the induction of nonsense-mediated mRNA decay (NMD). NMD is a surveillance system that prevents the synthesis of C-terminally truncated proteins toxic for the cell. The physiological importance of NMD is manifested by the fact that about one third of genetic disease-associated mutations generate PTCs. In recent years, a novel therapeutic approach called suppression therapy has been developed that utilizes low molecular weight compounds to induce the translation machinery to recode a PTC into a sense codon. Beta-thalassaemia (β-thalassaemia) is one of the most common genetic diseases worldwide, and nonsense mutations occurring in the beta-globin (β-globin) are among the most frequent mutations associated with β-thalassaemia. Some studies have shown that aminoglycosides, low molecular weight compounds, and non-aminoglycosides can suppress PTCs in cystic fibrosis and Duchenne’s muscular dystrophy, but it remains unclear whether β-thalassaemia would also be responsive to a similar drug treatment. Preliminary results obtained in our lab have shown that the aminoglycoside G418 can suppress a nonsense mutation at codon 39 of the human β-globin mRNA, although at low levels in cultured erythroid cells. To investigate if suppression therapy can restore enough β-globin protein to correct the disease manifestations of β-thalassaemia, we tested whether G418 is able to induce efficient levels of suppression in a dose-dependent and time-course manner in HeLa cells transfected with plasmids containing the human β-globin wild type gene (βWT) or the counterpart carrying a nonsense mutation at codon 39 (β39). Our results show a slight increase in the levels of mRNA of the β39 transcript which may indicate a possible effect by the drug. Seen as we were unable to test the effect of the drug on a protein level, in the future, it would be of great interest to evaluate its effect on protein expression levels. Algumas das mutações que ocorrem em células somáticas ou na linha germinal podem resultar na formação de codões de terminação prematura da tradução (CTPs ou codões nonsense). Um terço das mutações associadas a doenças genéticas resulta na produção de CTPs. Em geral, a introdução dum CTP num transcrito induz a terminação prematura da tradução e a activação do mecanismo de decaimento do mRNA mediado por mutações nonsense (nonsense-mediated mRNA decay; NMD). O NMD é um mecanismo de controlo de qualidade que impede a produção de proteínas truncadas na extremidade C-terminal tóxicas para a célula. No decorrer dos últimos anos, tem sido desenvolvida uma nova terapia, designada como terapia de supressão, que utiliza compostos de baixo peso molecular para induzir a maquinaria de tradução a alterar um CTP para um codão sense. O objectivo da terapia de…