Utilize este identificador para referenciar este registo: http://hdl.handle.net/10400.1/825
Título: Molecular analysis of HIV under the pressure of protease inhibitors
how to overcome existent problems
Autor: Nogueira, Ana Rita Godinho da Costa
Orientador: Gonçalves, João Braz
Canário, Adelino Vicente Mendonça
Palavras-chave: Teses
Biologia molecular
Data de Defesa: 2008
Resumo: The Human Immunodeficiency Virus is the causative agent that leads to AIDS. One of the enzymes that compose the viral particle is an aspartic protease that cleaves polyproteins during viral replication. This enzyme is necessary during the maturation process of the virions, and that is a preferential target for the design of new drug therapies, such as the protease inhibitors (PI). The aim of this thesis was to examine the effect of single amino acid substitutions in HIV-2 PR both in terms of resistance to PIs and in terms of drug free infectivity. For each mutant we have determined the level of its selective advantage relative to wild type virus over a range of drug concentrations. We have also characterized the HIV-2 proteases from patients under PI treatment genotipically and evaluated their phenotipically resistance to four selected PI by a recombinant viral assay (RV A), allowed us to study the mechanism of action of anti-HIV drugs. The results obtained from RV A may reflect a different pattern of HIV-2 resistance when compared with HIV-1 and in vitro studies can provide insight in what we expect in clinical trial. We have also developed a new functional cell based assay (CBA) for testing resistance that allows monitoring HIV-1 protease activity and susceptibility to PI in living cells without the need for culture infectious HIV. The signal obtained correlates with the intracellular activity of the protease and the optimized cleavage assay was used for testing current PIs to compare the IC50 values with virologic assays. The advantage of CBA is that we can have results within 48 hours, two times faster than a RV A, and can be used in reference laboratories as a powerful tool for identifying novel PIs. A third approach was made by screening an in vitro phage-display library of IgNAR variable domains against the target antigen HIV-1 protease to develop a novel monoclonal antibody against this enzyme. We used a single-chain antibody naïve library based in the variable domain of IgNAR cloned into pComb3X phagemid. Forty-eight possible anti-protease clones were selected and Phage- ELISA and ELISA tests showed the specific binders. The data obtained from screnning library of IgNAR have proved that the obtained antibodies were functional against the HIV-1 protease viral particle and that can be used in the future as a new gene therapy strategy with several potentialities in infectious diseases therapeutics
Descrição: Tese dout. , Ciências Biotecnológicas, 2008, Universidade do Algarve
URI: http://hdl.handle.net/10400.1/825
Designação: Doutoramento em Ciências Biotecnológicas
Aparece nas colecções:UA01-Teses

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