Czupryna, Julie
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Publication The Development, Characterization and Implementation of a Reactive Oxygen Species-Responsive Ratiometric Bioluminescent Reporter and Its Use As A Sensor for Programmed Cell Death(2010-12-22) Czupryna, JulieThe 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.Publication Superparamagnetic Iron Oxide Nanoparticle Probes for Molecular Imaging(2006-01-01) Thorek, Daniel L. J; Chen, Antony K; Czupryna, Julie; Tsourkas, AndrewThe field of molecular imaging has recently seen rapid advances in the development of novel contrast agents and the implementation of insightful approaches to monitor biological processes non-invasively. In particular, superparamagnetic iron oxide nanoparticles (SPIO) have demonstrated their utility as an important tool for enhancing magnetic resonance contrast, allowing researchers to monitor not only anatomical changes, but physiological and molecular changes as well. Applications have ranged from detecting inflammatory diseases via the accumulation of non-targeted SPIO in infiltrating macrophages to the specific identification of cell surface markers expressed on tumors. In this article, we attempt to illustrate the broad utility of SPIO in molecular imaging, including some of the recent developments, such as the transformation of SPIO into an activatable probe termed the magnetic relaxation switch.