Banca de DEFESA: ORTÊNCIA DE OLIVEIRA SOUSA

Uma banca de DEFESA de MESTRADO foi cadastrada pelo programa.
DISCENTE : ORTÊNCIA DE OLIVEIRA SOUSA
DATA : 06/12/2021
HORA: 14:00
LOCAL: Sala Virtual Remota
TÍTULO:

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PALAVRAS-CHAVES:

Snake toxins. Phospholipase A2. Antiviral. Therapy. Dengue Virus

PÁGINAS: 82
GRANDE ÁREA: Ciências Biológicas
ÁREA: Biologia Geral
RESUMO:

Bioactive molecules, such as peptides, proteins and enzymes are found in the biodiversity showing therapeutic potential in neglected diseases. Snake venom and its fractions are of great interest due to its virucidal potential demonstrated in in vitro studies. The Dengue Virus (DENV) is the most important arbovirus that infect humans - with 400 million cases every year, and with an increasing number of severe cases with dengue hemorrhagic fever or dengue shock syndrome. Although there is no specific medication to treat dengue infection, snake venoms may represent a potential source of antiviral activity. In this perspective, the present study aims to evaluate the effects of different toxins isolated from the Bothrops diporus snake venom on DENV. For the study, cell cultures of the C6/36 and VERO lineages were established for viral cultivation and propagation, from the isolation of DENV from the serum of patients previously characterized by molecular biology. The detection of virus serotype maintained in culture was performed by RT-qPCR using specific oligonucleotides, and viral titration performed by flow cytometry, with confirmation of cell infection by indirect immunofluorescence and the performance of the kinetics of viral infection of DENV serotype 1 (DENV1). The results obtained for viability tests in VERO cells of the different toxins tested for standardization demonstrated that the Crotoxin in 1h and 24h showed viability greater than ± 60% and when incubated for 5 days, the concentration of 50 µg / mL affected 50% of cell viability. The BthTX-II incubated for 4 days, showed viability greater than ± 50%. While PLA2 BthTX-II from Bothrops jararacussu incubated for four days showed viability greater than ± 50%. When tested the B. diporus venom and toxins – the target biomolecules of this study – on the cell cytotoxic assay, the venom (50µg/mL) showed CC50; and PLA2-Asp (12.5 - 50 µg / mL) affected cell viability in the percentage of ± 50 - 65% respectively. PLA2-Lys has been shown to affect 50% of cells at a concentration of 50 µg/mL. The viral serotypes 1 and 3 were detected after the 2nd viral culture of RT-qPCR. Among the serotypes evaluated, when performing DENV1 replication kinetics in C6/36, the virus showed a gradual increase in viral particles which were stabilized from the 5th to 6th day of incubation being detected by indirect immunofluorescence. In conclusion, after propagation stages it was possible to identify and detect by molecular and cellular methods only DENV1. In cytotoxicity analysis, which is one of the first tests of a drug candidate and performed in mammalian cells, the CC50% presented PLA2s ranged between 25-50 µg/mL. The preliminary results obtained allows the continuation of the study, which consists of evaluating the antiviral activity of snake toxins and venoms. Since PLA2s showed antiviral potential, more researches on the same class of biomolecules contributes to a better understanding of the mechanism of action.

MEMBROS DA BANCA:
Externo à Instituição - LIVIA CARICIO MARTINS - FIOCRUZ
Presidente - 739.335.462-49 - SORAYA DOS SANTOS PEREIRA - UNIR
Externo à Instituição - STELLA REGINA ZAMUNER - USP