In the previous granting period we have described perturbations in the biochemical pathways and function of human umbilical vein endothelial cells infected with Trypanosoma cruzi. In the present proposal we have extended these observations and have also included experiments to elucidate biochemical pathways that may be unique to this parasite and that any provide insight into infection-associated alterations of host cell signal transduction that ultimately lead to perturbation of microvascular perfusion. The experiments are divided into two sections. Infection-associated perturbations of endothelial cells will be investigated by determining the effect of the parasite on host cell gene expression as it relates to the compromise of microvascular perfusion. Secondly, we will continue to investigate how infection influences signal transduction systems of the host endothelial cell by examining single cell calcium mobilization. There is recent evidence that T. cruzi possess a complicated signal transduction system and we are therefore interested in how the signal transduction system of the parasite and host endothelial cell interact and perhaps modulate or modify the host-parasite relationship. Specifically, we plan to study how calcium and calmodulin may be involved in the parasite signal transduction system. In this regard, we will characterize the biochemical and functional properties of parasite calcium-dependent calmodulin binding proteins in all life cycle stages and in addition develop molecular probes in order to determine how calmodulin binding proteins function both in vitro and in intact cell systems. Finally, we will examine the metabolism of IP3 and phospholipase C in each life cycle stage.
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