PPG - Bioprodutos e Bioprocessos

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https://hdl.handle.net/11600/61205

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Navegando PPG - Bioprodutos e Bioprocessos por Orientador(es) "Azevedo, Mauro Ferreira de [UNIFESP]"

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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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    Investigação das moléculas envolvidas na permeabilização das membranas e na regulação do processo de egresso de plasmodium falciparum na hemácia infectada
    (Universidade Federal de São Paulo (UNIFESP), 2019-07-31) Melo, Hamille Rocha [UNIFESP]; Azevedo, Mauro Ferreira de [UNIFESP]; http://lattes.cnpq.br/1657599115711431; http://lattes.cnpq.br/6091182573007243; Universidade Federal de São Paulo (UNIFESP)
    Introduction: In spite of the recent advances towards its control, malaria is still a burden to society, killing mainly children under five years of age. While successful treatment with total cure is available, drug resistance threatens patients care in the future and therefore, new antimalarial medicines are required. A potential target for chemotherapeutic intervention is host cell egress, a tightly regulated process that happens after parasites grow and multiply inside the human erythrocytes. Parasite enzymes are known to act in concert dismantling parasitophorous vacuole (PV) and red blood cell (RBC) membranes, but the hierarchy of their action is not completely understood. Objectives: The aims of this study were to establish a method that can be applied to identify compounds that affect egress and to investigate the sequence of the biochemical events involved. Methods: In order to synchronize parasites in two key moments that precede egress, P. falciparum 3D7 parasites were engineered to express endogenous calcium dependent protein kinase 5 (CDPK5) or protein kinase G (PKG) in fusion with a destabilization domain (DD). Expression of each DD tagged kinase is dependent on the ligand Shld-1 and its removal reversibly block egress. Expression of the sensitive reporter nanoluc (NLuc) either secreted do the PV or exported to the RBC was applied to quantify egress. Results: As expected, removal of Shld-1 nearly completely blocked egress in both transgenic lines and when Shld-1 was reintroduced to cultures previously synchronized with sorbitol and heparin at 4548 hours post invasion, Nluc activity in the supernatant increased in CDPK5-DD and PKG-DD cultures after 1 and 2 hours respectively. Microscopic and FACS analysis confirmed the increase in reporter activity correlated to egress. About a couple dozen compounds were evaluated. Egress of CDPK5-DD and PKG-DD were affected differently by a few compounds, demonstrating the method can be applied identify parasite enzymes and/or signalling pathways that act after PKG and before CDPK5. Conclusion: Remarkably, data provided by this study suggest a calpain and a few kinases such as PKA act temporally between PKG and CDPK5. Application of this protocol might allow the identification of many more compounds that affect egress and elucidating when their targets are required respectively to PKG and CDPK5 activation.