Papel das glicoproteínas-p (PGPS) na resistência de helmintos à ivermectina: uma abordagem experimental

• Ano de defesa: 2015
• Autor(a) principal: Luiza Almeida de Figueiredo
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de Minas Gerais; UFMG
• Palavras-chave em Português: Parasitologia
• Link de acesso: http://hdl.handle.net/1843/BUBD-9WHF7N

Improper use of drugs for parasitic diseases control induces genetic selection of resistant parasites subpopulations. The P-glycoproteins (PGPS) are ABC transporters whose allelic variations and gene expression are involved in resistance to anthelmintic drugs. Caenorhabditis elegans is a free-living nematode that has high physiological, molecular and morphological similarity with nematode parasites. The genetic interference mediated by double-stranded RNA (dsRNA) is a valuable tool in the analysis of gene function in C.elegans. The project aimed to induce resistance using anthelmintic ivermectin in C.elegans, and evaluate the differences in gene expression and phenotyping applying Pgp silencing in wild-type and resistant worms. The resistant strain was obtained using increasing doses of ivermectin during successive passages in vitro. After 48 weeks of selection it was obtained a resistant strain (30 ng/ml of ivermectin). The RNAs were extracted from resistant and susceptible C.elegans and converted in cDNAs. It was designed primers for amplification of 12 Pgps gene sequences by PCR and qPCR. The analysis in gene expression changes were performed by qPCR comparing the resistant and wild-type strains. The results showed that only 16.66% of the genes are up-regulated in resistant group compared to the wild-type. Subsequently, these sequences were cloned into vectors and transformed in suitable bacterial strain. After that, the worms were feeding with this transformed bacterial and expression changes of these genes were analyzed by qPCR. It was obtained silencing of 99% for Pgp 4 and 94% for Pgp 12 in the resistant strains, in comparison to wild-type (97% and 93%, respectively). Phenotype assays evaluating motility and cell death after the silencing showed that Pgp 4 is related to the susceptibility of ivermectin and Pgp 12 related to resistant phenotype. In addition, it was performed a study comparing the distance of P-glycoproteins in nematodes showing that C. elegans Pgps are very close to those of various parasitic nematodes, allowing a possible extrapolation of data. This study contributed to the understanding of the mechanisms involved in anthelmintic resistance in nematodes.