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    Caracterização da TCCYP19 e o estudo do seu papel durante a infecção celular causada pelo Trypanosoma cruzi
    (Universidade Federal de São Paulo (UNIFESP), 2020-12-18) Santos, Gregory Pedroso Dos [UNIFESP]; Schenkman, Sergio [UNIFESP]; Universidade Federal de São Paulo
    Chagas disease caused by the protozoan parasite Trypanosoma cruzi is characterized by an extensive inflammatory process in the organs and sites most affected by the infection. To understand the evasion mechanisms developed by the parasite in response to attempts to control the infection by the host organism - several studies have being published describing an intriguing relationship between reactive oxygen species (ROS) produced by infected cells, and their ability to induce or increase in the proliferation of T. cruzi. So far, the mechanisms involved in these processes are still unknown. However, these studies suggested that parasites have developed a mechanism to deal with oxidative stress, which nullifies the antimicrobial capacity, or which is capable of converting these oxidative environments to their benefit. In this thesis, we studied a chaperone of 19 kDa, expressed by T. cruzi, entitled cyclophilin 19 (TcCyp19), that has high similarity to cyclophilin A (CypA), expressed by mammals. CypA acts in the activation of the cellular immune and inflammatory response, as well as in the translocation of the p47phox subunit - responsible for activation of the NADPH oxidase enzyme complex, which leads to the production reactive oxygen species. We found that TcCyp19 is expressed in all T. cruzi stages, being more abundant in the epimastigote form of the parasite. Through adaptations carried out by genetic engineering, we have generated parasites capable of expressing TcCyp19 fused to a HA tag. This approach allowed us to detect its secretion by the intracellular amastigote form in the cytosol of the host cell . Furthermore, we observed that modulations in the expression of TcCyp19, both in the parasite and through the expression carried out directly in the host cell, increased the intracellular multiplication capacity of the parasite concomitant with TcCyp19 release in the cytosol of infected cells. We also noticed a parallel increase in the production of ROS, during infections and when rat myoblast L6 cells expressed TcCyp19. In contrast, parasites depleted of TcCyp19 gene showed a substantial reduction in the intracellular amastigotes proliferation, similar to what was observed for wild type parasites treated with antioxidant enzymes or with a cyclosporine A (a specific inhibitor of cyclophilins). The knockout parasites also generated a significant reduction in the production of ROS in infected cells. Both reduced proliferation and low ROS production were reestablished by the add-back parasites or when infecting L6 cells that express the TcCyp19. Mechanistically, the production of ROS generated by TcCyp19 appears to be related to the activation of the NADPH oxidase by the translocation of the p47phox subunit to the cell surface as previously found for CypA. In fact, part of the TcCyp19 colocalize of stretches of p47phox aligned with microfilaments in L6 cells and concentrate in the leading edges of the cells in regions proposed to contain active NADPH oxidase. We conclude that TcCyp19 is a key molecule responsible for inducing increased production of ROS, which in turn can influence proliferation of parasites and guarantee the prevalence of infection.