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Establishment of calcium intracellular measurement methodology by genetically encoded indicators to evaluate processes of proliferation, differentiation and cell death


Intracellular Ca2+ is the most versatile second messenger. It participates in several cellular processes such as proliferation, differentiation, cell death, contraction, secretion, and even at fertilization and learning. For decades the Department of Biophysics, UNIFESP, has studied the regulatory role of this divalent ion in the process of muscle contraction. In recent years, our group has concentrated on the participation of Ca2+ ion in the proliferation, differentiation and cell death especially in hematopoietic stem cells. This project aims to implement a new fluorimetry technique for measurements of intracellular levels of Ca2+ by using genetically encoded indicators (pericam and cameleon) in specific intracellular targets such as nucleus, mitochondria, endoplasmic reticulum, and cytoplasm. These molecular/pharmacological tools have been provided by Prof. Dr. Maria Cano Ababa, from the Universidad Autonoma de Madrid with permission of Prof. Roger Tsien of the University of California. After the implementation of the intracellular Ca2+ measurements equipment, we intent to investigate the signaling aspects of Ca2+ in the proliferation of hematopoietic stem cells by IL-3 and GM-CSF. We also intent to explore the integration of the oscillatory mobilization of Ca2+, activation of Ca2+ sensitive proteins and the molecular mechanisms of proliferation. Unique and transients Ca2+ signals induced by extracellular ATP, which activates P2 receptors, with differentiation effects into primitive cells obtained from leukemic strains also will be also studied. Finally, also using these pharmacological tools to investigate the role of intracellular Ca2+ in the death process induced by gomesina, an antimicrobial peptide with antitumor activity in B16 melanoma cells. Thus, this project aims to implement a new experimental methodology of Ca2+ measurements in specific site. This methodology is unprecedented in Brazil and will allow us to go deeper into the mechanisms of Ca2+ into control of the proliferation, differentiation and cell death. (AU)

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