Date of Award

Spring 1-1-2010

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry & Biochemistry

First Advisor

Amy E. Palmer

Second Advisor

Marcelo C. Sousa

Third Advisor

Natalie G. Ahn

Abstract

Ca2+ is a ubiquitous and highly regulated signaling second messenger in cells, involved in a number of processes and pathways. Due to the complexity of Ca2+ signaling and its organization within a cell, it is inherently difficult to study. A number of fluorescent tools have been developed throughout the years in order to overcome these complications. Such probes allow for imaging of rapid Ca2+ transients in real-time at the single cell level, enabling the separation of the spatio-temporal aspects of Ca2+ signals. As Ca2+ is essential to cellular regulation and function, it is unsurprising Ca2+ + homeostasis is often altered in disease. In the studies herein, we examined Ca2+ as it pertains to pathogenesis in order to further current understandings about how its dysregulation leads to disease progression. By employing a specifically targeted Ca2+ sensor, we monitored changes in intracellular store levels and release caused by mutations in the Alzheimer’s disease related protein presenilin-1. Our results suggest mutations have differential effects on cellular Ca2+ homeostasis and stress the importance of directly investigating compartmentalized Ca2+ as opposed to inferring intracellular levels based on release into the cytosol. In addition, Ca2+ transients caused by bacterial infection of host cells were studied to determine molecular mechanisms involved in Salmonella-induced Ca2+ fluxes, providing insights into how bacteria modulate Ca2+ pathways to facilitate their survival. Finally, in order to characterize Ca2+ regulation in mitochondria, we used a mitochondrial targeted Ca2+ sensor to determine the involvement of a newly identified protein in Ca2+ uptake into the organelle. The use of fluorescent tools to study Ca2+ sheds light on effects of disease on Ca2+ regulation and homeostasis, providing mechanistic details that may lead to therapeutics.

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