Broad, Long Term Objectives: Develop and apply tools that control, perturb and interrogate functioning single cells. Develop these tools for understanding a tissue's response to oxidative stress. These tools are based on a phenomenon called 'electroporation' - the creation of tiny, transient pores in the cell membrane using an electric field.
Specific Aims : 1. Quantitative and qualitative understanding of single-cell electroporation. Determine the experimental parameters and cellular properties that control the membrane porosity, pore resealing dynamics, pore size, and cell viability of single cells. Test the hypothesis that the induced membrane porosity depends on certain cellular characteristics as well as the applied field. 2. Intracellular titration of receptors and measurement of enzyme activity. Combine microfluidic methods and single-cell electroporation in order to obtain thermodynamic and kinetic data on interactions between intracellular species, mainly enzymes and receptors, and externally applied, cell impermeant ligands and substrates. Provide means for elucidation of intracellular signaling network dynamics. 3. Focal electroporation and electrophoretic analysis in organotypic hippocampal cultures. The technical aim is the development of tools to alter biological processes and measure quantitatively the results of those alterations in tissue with good spatial resolution. Electroporate small (below 100 ?m) regions of rat organotypic hippocampal cultures to study the response to oxidative stress leading to better defense against stressors. Develop on-line capillary electrophoresis on a chip to determine the intracellular concentration of glutathione, a protective chemical in all cells, in brain tissue at the same spatial resolution. Health relatedness: Cellular processes responsible for response to drugs, cancer, and response to stress often rest on complex interacting networks of proteins, DNA and RNA. The tools described empower researchers to study in more depth and with more ease these key cellular events in a quantitative way. ? ? ?
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