Navegando por Palavras-chave "Inibidores de proteases sinalização de cálcio"

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    Estudo da modulação da atividade proteolítica no plasmodium e em tecidos de camundongos infectados
    (Universidade Federal de São Paulo (UNIFESP), 2014-02-26) Gomes, Mayrim Machado [UNIFESP]; Gazarini, Marcos Leoni [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Malaria is a major public health problem, the parasitic infection with the highest incidence in the world. The parasite that causes this disease belongs to the genus Plasmodium. In 2012 were recorded about 207 million cases in subtropical and tropical areas of the world and it results in more than 600,000 deaths per year. It is well known that proteolytic enzymes of Plasmodium are fundamental in the development and survival of the parasite in the host. Many Plasmodium cellular events are regulated by calcium and protein phosphorylation. Both signaling mechanisms are important modulators of protease activity, in particular the cysteine proteases (calpains) study, wich activity is calcium dependent. In P. falciparum, this protease presents a single copy in the genome, being a potential target of inhibition. Proteolysis of isolated parasites (P. falciparum) was assessed using fluorogenic substrates in spectrofluorimeter. The results showed a predominance of cysteine proteases activity in parasite’s cytoplasm. The intracellular calcium increase from organelle (ER) and from the extracellular medium (opening from membrane channels) changed significantly the proteolysis of Z-Phe-Arg-AMC substrate and was greatly inhibited by ALLN (selective calpain inhibitor). Intracellular calcium concentration measured with Fluo-4 AM calcium indicator was in the range 1-2µM, which indicates it is a µ-calpain responsible for proteolytic activity in Plasmodium. The dependence of calpain for intraerythrocytic development was evaluated by using the ALLN in P. falciparum culture, wich resulted in significant reduction of infected erythrocytes. The role of cAMP and kinases in intracellular proteolytic activity of cysteine proteases were not observed by the addition of cAMP analogs (8Br-cAMP/N6-cAMP) or kinases inhibitors/activators (PKA and PKC). However, we cannot reject the involvement of these elements in proteolytic activity modulation in different stages of parasite cycle. In infected mice with P. chabaudi, we checked the activity of MMPs, ACE and ACE2 in different tissues, compared with normal mice. In tissues in which there were differences in activity, normal tissues showed higher metalloproteases activity, suggesting the involvement of endogenous tissue inhibitors in infected tissues. Kinins are vasoactive peptides generated from proteolytic cleavage of its precursors, kininogens, which are synthesized by the liver. Bradykinin (BK), Lys-BK and kallidin act in the constitutive B2 receptor, whereas des-Arg9-BK, Lys-des-Arg9-BK and des-arg10 - kallidin acts in induced B1 receptor. Immunofluorescence assays showed more intense staining of the B2 receptor in infected livers, compared to normal mice livers. Based on these results, this thesis contributes to clarify the parasite cytoplasmic proteolytic activity in P. falciparum indicating new potential targets for antimalarial drugs, and changes in liver, kidneys and lungs physiology involving kinin signaling during infection by Plasmodium chabaudi, enabling to understand malaria pathophysiology