Navegando por Palavras-chave "Plasmodium Falciparum"
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- ItemSomente MetadadadosAvaliação 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 PauloIntroduction: 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.
- ItemSomente MetadadadosEfeito da superexpressão da m1 alanil-aminopeptidase em plasmodium falciparum e efeito do cálcio na atividade proteolítica de aminopeptidases(Universidade Federal de São Paulo (UNIFESP), 2018-03-21) Hoff, Carolina Caldas [UNIFESP]; Gazarini, Marcos Leoni [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)Malaria Was The Cause Of Nearly 500,000 Deaths Worldwide In 2016. The Etiologic Agent Plasmodium Falciparum Causes The Most Severe Form Of The Disease In Humans. The Creation Of Potent Antimalarial Drugs Is Hampered By The Fact That The Parasite Can Become Resistant To Drugs. In This Work, We Studied Alanyl-Aminopeptidase-1 Of Plasmodium Falciparum (Pfa-M1), Which Is Involved In The Final Stage Of Hemoglobin Degradation, An Essential Process For Parasite Growth. To Present Additional Information On The Function Of The Enzyme And Its Potential As A New Antimalarial Target, We Used A New Strain Of P. Falciparum Overexpressing Pfa-M1 (Suppfa-M1) To Study The Implications In Cell Morphology, Parasite And Proteolytic Activity. Our Results Demonstrate That The Parasite Is Resistant To The Aminopeptidase Inhibitor (Bestatin) With Higher Expression Of The Protein. We Observed A Decrease In The Area ("M2) Of The Suppfa-M1 Parasite And The Number Of Merozoites Per Schizont When Compared To The Control Strains, Indicati
- ItemSomente MetadadadosInteração De Peptídeos Miméticos Da Alça Da Falcipaína-2 Co A Hemoglobina(Universidade Federal de São Paulo (UNIFESP), 2017-03-10) Sudbrack, Tatiane De Paula [UNIFESP]; Daghastanli, Katia Regina Perez [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)The Plasmodium falciparum is responsible for the seriousness and cases of death due to malaria. During its life cycle inside of red blood cells, the protozoon depends on the hemoglobin (Hb) hydrolysis for its osmotic stability maintenance and amino acid income. Amongst the proteases present in the digestive vacuole of parasite, falcipains are the main tool for the degradation of Hb, and its inhibition causes the death of the parasite. The structure of Falcipain 2 has a motif containing 10 amino acid residues (EI-V-N-P-L-T-K-K-G), located between the catalytic residues H and N, which its deletion results in a FP-2 mutant with no proteolytic activity over Hb. This FP 2’s motif constitutes a turn which is possibly responsible for interaction with Hb and the facilitation of proteolysis. Therefore, studying the interaction of this synthetic motif peptide and its analogues with Hb could help understanding the role of this atypical region in the activity of this protease. The aim of the present work was to study the physical-chemistry aspects of the interaction between HbA and the synthetic peptides from the falcipain 2 motif containing the original D and L amino acids sequence and the inverted one, utilizing spectroscopy of UV-vis and CD, DSC and ITC techniques. In addition, it is known that people with falciform anemy and HbS are more frequently found in malaria endemic areas. It is believed that over the years, in malaria-endemic areas, the population carrying the falciform genotype, has been favored, perpetuating this genetic trace. In this sense, this work also presents studies regarding the interaction of the mimetic peptides from the FP 2 motif with HbS. The obtained results for the interaction of the peptide from the FP 2 motif and for the synthetic analogues suggest the role of the FP 2 motif is to interact with Hb and change de conformation of the alpha and beta chains and the tetrameric structure of HbA. The denaturation of Hb possibly enables the exposition of the hydrolysis sites of this protein facilitating its proteolysis. The results revealed that the peptide A, with de original amino acid sequence, presented a more favorable interaction with HbA, causing greater changes in the native hemoglobin structure. On the other hand, the results regarding HbS revealed the interaction of any peptide with this hemoglobin is disfavored, considering that larger peptide/HbS molar ratios were required in order to observe any structural changes in this hemoglobin. The smaller interaction affinity between HbS and the mimetic peptides from the FP-2 suggests the HbS could be correlated with the resistance of falciform anemy patients to malaria. Also, these studies strongly suggest that the role of this FP-2 motif is to rearrange HbA into a more relaxed conformation. These data corroborate the ones found for HbS, as this hemoglobin is found in the tensioned conformation, which disables the FP-2 to interact with hemoglobin, avoiding its proteolysis and, therefore, the survival of the parasite.