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Master's Dissertation
Full name
Jordana Cristina Oliveira
Knowledge Area
Date of Defense
São Paulo, 2014
Uliana, Silvia Reni Bortolin (President)
Monte Neto, Rubens Lima do
Véliz, Mauro Javier Cortez
Title in Portuguese
Desenvolvimento de estratégias alternativas para teste de fármacos: obtenção e caracterização de linhagens mutantes estáveis de Leishmania expressando luciferase.
Keywords in Portuguese
Genes repórteres
Abstract in Portuguese
A leishmaniose é causada por protozoários do gênero Leishmania e no Brasil, as principais espécies causadoras da leishmaniose cutânea são Leishmania (V.) braziliensis e Leishmania (L.) amazonenses. O tratamento da leishmaniose apresenta diversas dificuldades, portanto é fundamental a descoberta de novos fármacos ativos, podendo ser detectada em células cultivadas in vitro e também em animais íntegros, através da técnica de bioimageamento. Neste trabalho, propusemo-nos a produzir linhagens de L. (V.) braziliensis e L. (L) amazonenses expressoras de luciferase e caracterizar o comportamento das linhagens mutantes em testes de sensibilidade a fármacos e de infecção in vitro e in vivo. Foi confirmada a emissão de luz pelas linhagens mutantes das duas espécies de Leishmania, em promastigotas e amastigotas. O comportamento das linhagens mutantes obtidas em relação a curvas de crescimento, sensibilidade aos fármacos tamoxifeno e anfoterina B em promastigotas, perfil de infectividade e sobrevivência em macrófagos e sensibilidade de amastigotas à anfotericina B foi comparado ao comportamento das linhagens parentais, não sendo observadas diferenças significativas. Camundongos BALB/c infectados com a linhagem expressora de luciferase de L. (L.) amazonenses desenvolveram lesões comparáveis aos animais infectados com a cepa selvagem, sendo possível quantificar a carga parasitária nesses animais por bioimageamento. Os resultados obtidos neste trabalho indicam que os parasitas mutantes expressores de luciferase obtidos podem ser utilizados em testes de sensibilidade a fármacos tanto in vitro como in vivo, representando um avanço metodológico nessa área de pesquisa.
Title in English
Development of alternative strategies for drug testing: obtainment and characterization of stable mutant strains of Leishmania expressing luciferase.
Keywords in English
Reporter genes
Abstract in English
Leishmaniasis is caused by protozoan parasites in Brazil, the main causative species of cutaneous leishmaniasis are Leishmania (Viannia) braziliensis and Leishmania (Leishmania) amazonenses. The treatment of leishmaniasis presents several difficulties, and the discovery of new active drugs for the treatment of leishmaniasis is therefore fundamental. The enzyme luciferase is a reporter widely used in screening tests for new drugs. This enzyme catalyzes the oxidation of luciferase in the presence of ATP emitting light that can be detected in cultured cells in vitro as well as in intact animals, using the technique of bioimaging. In this work, we sought to produce strains of L. (V.) braziliensis and L. (L) amazonenses expressing luciferase and characterize the behavior of these mutant strains in drug susceptibility tests and in in vitro and in vivo infections. Production of light was detected in mutants of both species, in all life cycle stages. Mutant strains were compared to their corresponding parental lines as to their growth pattern, infectivity and survival profile in macrophages and sensitivity to amphotericin B and tamoxifen. No significant differences were observed for these parameters. BALB/c mice infected with the luciferase expressing line of L. (L.) amazonenses developed lesions comparable to those in animals infected with the wild-type strain. The parasite load in these animals was quantified through bioimaging. The results obtained of this study indicate that the mutant parasites expressing luciferase can be used for drug susceptibility testing in vitro and in vivo, representing a methodological advance in this area of research.
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