Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T12:09:46Z No. of bitstreams: 2 Dissertação - Cláudia de Jesus Silva Carvalho - 2015.pdf: 1551477 bytes, checksum: 310677b09bfb5d363c8bb56f37d3f55b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-11-13T12:11:26Z (GMT) No. of bitstreams: 2 Dissertação - Cláudia de Jesus Silva Carvalho - 2015.pdf: 1551477 bytes, checksum: 310677b09bfb5d363c8bb56f37d3f55b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) / Made available in DSpace on 2015-11-13T12:11:26Z (GMT). No. of bitstreams: 2 Dissertação - Cláudia de Jesus Silva Carvalho - 2015.pdf: 1551477 bytes, checksum: 310677b09bfb5d363c8bb56f37d3f55b (MD5) license_rdf: 23148 bytes, checksum: 9da0b6dfac957114c6a7714714b86306 (MD5) Previous issue date: 2015-03-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Abacavir (ABC), zidovudine (AZT) and lamivudine (3TC) are nucleoside reversetranscriptase inhibitors (NRTIs) widely used as the main axis of highly active antiretroviral therapy (HAART). Despite the effectiveness of combinations of NRTI in viral suppression, there is evidence to show that the genotoxic potential of NRTIs can be increased when administered in combination. In this context, this study proposed to investigate the toxic and genotoxic potential ABC induced when administered isolated and in combination with AZT and/or 3TC, using the wing SMART in Drosophila melanogaster. This test simultaneously evaluates two events related to carcinogenesis, mutation and somatic recombination, and is based on the identification of the mutants hairs resulting from lesions that lead to loss of heterozygosity in two marker genes involved in the formation of the wings of hair: mwh and flr3. In toxicity tests, groups of 100 larvae of third instar from the standard cross were treated for 48 hours with different concentrations of drugs. From the results obtained,was established a range of concentrations presenting more than 70% of survivors flies to proceed the analysis of genotoxicity. Employing the conditional binomial test, were compared the spots of mutants hair frequencies present in trans-heterozygotes and heterozygous flies for chromosome TM3 of each treated group with the respective controls. The results of investigations indicated that ABC was toxic to larvae in concentrations greater than 2 mg/ml. The combinations which contained ABC showed toxic effects similar to isolated ABC. All treatments were able to induce genetic alterations related to mutation and somatic recombination. Among all combinations, AZT/3TC was the one that showed less toxic and genotoxic potential at the concentrations tested. The potential recombinogenic both ABC isolated or in combination was relatively high, with indices of over 83.7%. / O abacavir (ABC), a zidovudina (AZT) e a lamivudina (3TC) são os inibidores da transcriptase reversa análogos de nucleosídeos (NRTIs) mais usados como eixo principal da terapia antirretroviral altamente ativa (HAART). Apesar da eficácia das combinações de NRTIs na supressão viral, há comprovações que demonstram que o potencial genotóxico dos NRTIs pode ser potencializado quando administrado em combinação. Neste contexto, este trabalho se propôs a investigar o potencial tóxico e genotóxico induzido pelo ABC quando administrado isoladamente e em combinação com a AZT e/ou a 3TC, utilizando o teste para detecção de mutação e recombinação em células somáticas de Drosophila melanogaster (SMART). O SMART avalia simultaneamente dois eventos relacionados com a carcinogênese, a mutação e a recombinação somática, e baseia-se na identificação de pelos mutantes resultantes de lesões que levam a perda de heterozigose em dois genes marcadores para forma dos pelos das asas: mwh e flr3. No teste de toxicidade, grupos de 100 larvas de terceiro estágio, provenientes do cruzamento padrão, foram tratados por 48 horas com diferentes concentrações dos fármacos. Dos resultados obtidos, foi estabelecida uma faixa de concentrações apresentando mais do que 70% de sobreviventes para proceder as análises de genotoxicidade. Empregando o teste binomial condicional, foram comparadas as frequências de manchas de pelos mutantes presentes nos indivíduos trans-heterozigotos e heterozigotos para o cromossomo TM3 de cada grupo tratado com seus respectivos controles. Os resultados dessas investigações indicaram que o ABC foi tóxico para as larvas em concentrações superiores a 2 mg/mL. As combinações que continham ABC apresentaram efeitos tóxicos semelhantes ao ABC isolado. Todos os tratamentos foram capazes de induzir alterações genéticas relacionadas com mutação e recombinação somática. Dentre todas as combinações, AZT/3TC foi a que demonstrou menor potencial tóxico e genotóxico nas concentrações testadas. O potencial recombinogênico tanto de ABC isolado quanto em combinação foi relativamente alto com índices superiores a 83,7%.

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Viruses are a major threat to humans due to their unique adaptability, evolvability and  capability to control their hosts as parasites and genetic elements. HIV/AIDS is the third largest cause of death by infectious diseases in the world, and drug resistance due to the viral mutations is still the leading cause of treatment failure. The flaviviruses, such as Dengue virus (DEN) and Japanese encephalitis virus (JEV), represent other major cause of morbidity and mortality, and the areas where these viruses are endemic are spreading rapidly. No curative therapy for any flavivirus could be made available as yet. The first part of this thesis focuses on the HIV-1 drug resistance caused by mutations in a major HIV drug target, the HIV-1 reverse transcriptase (RT) as a response to the largest class of clinically used anti-retrovirals, the NRTIs. A robust proteochemometric model was created to analyse the complex mutation patterns in RT drug resistance. The model identified more than ten frequently-occurring mutations, each conferring at least two-fold decrease in susceptibility for one or several NRTIs. Using our prediction server (hivdrc.org), the model can be applied to propose optimum combination therapy for patients harbouring mutated HIV variants. The second part of the thesis encompasses studies on a promising drug target, the NS2B(H)-NS3pro, in two flaviviruses, namely the dengue virus (DEN) and Japanese encephalitis virus (JEV). Functional determinants of DEN NS2B(H)-NS3pro were identified by site-directed mutagenesis. Further, peptide inhibitors were designed using proteochemometrics (PCM) and statistical molecular design (SMD), synthesized and assayed on DEN proteases, which resulted in some novel peptides with low micromolar or sub-micromolar inhibitor activity. The very poorly characterised JEV NS2B(H)-NS3pro  was cloned, purified and the kinetic parameters of this attractive drug target were determined for a series of model substrates and inhibitor. The results identified the role in target-ligand interaction of different residues on specific positions in the target (NS2B(H)-NS3pro) and ligands (substrates/inhibitors). Overall, the findings in this thesis contribute to rational antiviral drug discovery and therapy.