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Molecular Imaging Approaches to Understanding the Roles of Hydrogen Peroxide Biology in Stress and Development

  • Author(s): Dickinson, Bryan Craig
  • Advisor(s): Chang, Christopher J.
  • et al.
Abstract

The production of hydrogen peroxide (H2O2) in biological systems is associated with a variety of pathologies including neurodegenerative diseases, cancer, and the general process of aging. However, a growing body of evidence suggests that the reactivity of this particular reactive oxygen species (ROS) is also harnessed for physiological processes. Molecular imaging using fluorescence microscopy offers a valuable approach for deciphering the multifaceted roles of H2O2 in biological processes. The use of aryl boronates for the selective detection of H2O2 in biological systems is a validated approach to the development of H2O2-responsive fluorophores. This dissertation describes the design, synthesis, and characterization of an assortment of new boronate-based fluorescent probes for H2O2, as well as their application toward uncovering new roles for H2O2 in stress and development. Peroxyfluor-2, Peroxyfluor-3, Peroxy Yellow 1, and Peroxy Orange 1 are turn-on fluorescent probes that can detect physiological levels of H2O2 produced for cell signaling, as well as monitor multiple ROS simultaneously in single cells. Mitochondria Peroxy Yellow 1 is a bifunctional probe featuring a triphenylphosphonium group for mitochondrial targeting and a single boronate for H2O2 detection that allows for the detection of mitochondrial H2O2 associated with a Parkinson's diseases model. Nuclear Peroxy Emerald 1 is a nuclear-targeted probe that reveals Sirtuin-dependent changes in nuclear H2O2 metabolism in C. Elegans. Peroxyfluor-6 (PF6) is a bifunctional probe featuring acetoxymethylester-protected phenol and carboxylic acid functionalities for enhanced cellular uptake and retention. PF6 reveals endogenous H2O2 production within neural stem cells and molecular biological experiments expose a new role for H2O2 in growth signaling within this critical brain cell population in vitro and in vivo. Finally, Peroxy Yellow 1 Methyl-Ester, a probe designed for analysis by flow cytometry, reveals that Aquaporins 3 and 8, but not Aquaporin 1, can mediate H2O2 uptake across the plasma membrane of mammalian cells, and that Aquaporin 3 can facilitate the uptake of endogenous H2O2 relevant to cell signaling.

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