Date of Award

Fall 2010

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Andrew Tsourkas

Abstract

The process of programmed cell death (PCD) is a prevalent area of scientific research and is characterized by a highly complex proteolytic cascade. PCD is involved in development, homeostasis and the immune response. Further, unregulated PCD has been implicated in various, often, devastating pathologies including cancer, autoimmune diseases and neurodegenerative disorders. The importance of PCD in human health and disease has led to the widespread utilization of genetically encoded reporters for the non-invasive imaging of PCD in vitro and in vivo; however, it is currently not well understood whether the reporters themselves are susceptible to inactivation and/or degradation during PCD. Molecular reporters that do exhibit an unexpected sensitivity to their environment could lead to ambiguous findings and/or inaccurate conclusions. Interestingly, we have found that the commonly used bioluminescent reporter protein, Firefly Luciferase (fLuc), exhibits a rapid loss in activity in cells undergoing PCD. In contrast, a variant of Renilla Luciferase, RLuc8, demonstrated quite stable activity under the same conditions. Following extensive inhibition analyses, it was determined that reactive oxygen species (ROS), particularly hydrogen peroxide (H2O2), play a large role in the disparity between fLuc and RLuc8 activity, in cells undergoing PCD. ROS are natural byproducts of oxygen metabolism that are normally regulated by antioxidants; if the balance between ROS and antioxidants becomes skewed, cells can enter a state of ‘oxidative stress.’ It has been reported that many cases of PCD are associated with elevated levels of ROS. Consistent with these reports, when fLuc and RLuc8 were intracellularly coexpressed, it was found that the bioluminescence ratio, RLuc8:fLuc, served as a useful metric to report on caspase-dependent and –independent PCD in vitro and in vivo in an ROS-mediated manner. It is envisioned that this ratiometric reporter could have widespread impact on research endeavors involving the aforementioned maladies, including therapeutic development and evaluation.

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