Navegando por Palavras-chave "Ensaio Fenotípico"

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    Avaliação da combinação de ferramentas de expressão gênica condicional em Plasmodium falciparum para estudos de genômica funcional e screening de potenciais antimaláricos
    (Universidade Federal de São Paulo (UNIFESP), 2020-09-04) Santos, Caroline Lima dos [UNIFESP]; Azevedo, Mauro Ferreira de [UNIFESP]; http://lattes.cnpq.br/1657599115711431; http://lattes.cnpq.br/6563769993198823; Universidade Federal de São Paulo
    Introduction: Despite the advances, malaria remains a major public health problem, leading to the death of thousands of people every year, mainly children under 5 years old. The study of essential genes for the parasite's life cycle is important for the development and validation of new antimalarials. Using reverse genetics, it has been possible to characterize important genes for the development of P. falciparum. It is common to use conditional gene expression to carry out these studies, but there’s still an expression leak that prevents a wider use. Methods for detecting the action of antimalarial drugs currently used take a long time to generate results, some of which are of low sensitivity and practicality, requiring the development of a fast method, of low cost and capable of being used on large scale. Objectives: To enhance conditional gene expression systems based on the combination of existing tools and to evaluate their application in assays for the detection of fast-acting antimalarials and in studies of essential genes. Methods: In order to evaluate the performance of conditional gene expression systems, plasmids were constructed with different combinations of these, being fused with Nluc-GFP, to assess regulation. Synchronized parasites in ring or trophozoite stages were grown in different combinations of the ligands used to regulate the systems and the results of gene expression determined by bioluminescence and fluorescence. For future functional analyzes of essential genes, strains were generated with some plasmids integrated in the genomic loci of interest. Results: The fusion of the conditional gene expression systems enhanced the induction capacity by up to 41x, with the 5D fusion being faster to induce in rings and the ribozyme glmS in trophozoites. Using the Nluc- 5D-glmS strain, which contains the 3 systems combined, as a method of detecting the action of antimalarials, it was possible to detect a 50% decay activity by the action of chloroquine in just 4 hours and to differentiate slow-acting antimalarials from fast-acting ones. In preliminary experiments, it was possible to observe a phenotypic effect in the parasites due to the modulation of the expression of the kinases CDPK1 and CDPK5 using some of the combinations of systems generated. Conclusion: Notably, when constructing a plasmid with different domains in fusion, its regulatory potential is multiplied and by using the parasites that express this construction it is possible to identify the action of drugs quickly, with high sensitivity and at low cost. You can also use them to conditionally knock out essential genes such as CDPK1 and CDPK5.
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    Descoberta de novos antivirais para Flavivírus a partir de uma abordagem fenotípica
    (Universidade Federal de São Paulo (UNIFESP), 2019-05-30) Pilger, Denise Regina Bairros De [UNIFESP]; Freitas Junior, Lucio Holanda Gondim De [UNIFESP]; http://lattes.cnpq.br/2136191319465692; http://lattes.cnpq.br/1034340400100837; http://lattes.cnpq.br/1034340400100837; Universidade Federal de São Paulo (UNIFESP)
    The Flavivirus genus comprises more than 70 pathogenic viruses with different pathologies and a significant public health impact in different regions of the world, with potential to emerge in non-endemic regions. Dengue virus (DENV), yellow fever (YFV) and Zika (ZIKV) are some of the most important human pathogenic flaviviruses with similar symptoms ranging from mild fever and malaise to hemorrhage, shock and failure of multiple organs. Despite the existence of protective vaccines for both viruses, their use is contraindicated for immunocompromised individuals, pregnant women and children. The only treatment is the mitigation of symptoms associated with diseases, with analgesics, antipyretics and replacement of body fluid. Specific antiviral chemotherapies could reduce the morbidity and mortality associated with these infections. In addition, during an outbreak, a specific drug could be administered at the onset of any symptoms associated with infections. Therefore, there is an urgent need to establish a more objective, sensitive and reproducible biological assay to search new antiviral therapies. Thus, an image-based phenotypic assay was developed and optimized for the screening of three commercial compound libraries (NIH Clinical Collection, National Institute of Health, LOPAC1280, Sigma, and Pathogen Box, MMV) against a panel of viruses of the genus Flavivirus. We highlight the results obtained through: (i) NIH library, from which we identified two compounds against dengue virus serotype 2 with high selectivity (> 10); (ii) LOPAC1280, from which we identified three compounds with promising activity against the yellow fever virus. In addition, the activity spectrum of these compounds was evaluated against other viruses of the same genus, as well as their ability to induce resistance in yellow fever virus, and how they interact with the viral life cycle. (iii) Pathogen Box library was screened in parallel with six different viruses to stablish an activity profile of the compounds. As a result, 133 compounds were selected, some of which have already been described as having antiviral activity, validating the assay as a viable tool for the discovery of candidates with potential antiviral activity. In summary, the methodology described in this work represents an important contribution to the initial processes of antiviral development. With these experiments it was possible to find new potent and effective compounds for YFV, which provides a solid contribution to the early stages of the discovery of new drugs for diseases caused by flavivirus.