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Probing The Extracellular Space: Development of Molecular Imaging Platforms and Investigations of Metal-Mediated Receptor Signaling
- O'Sullivan, Justin James
- Advisor(s): Heffern, Marie C.
Abstract
The work described in this dissertation builds upon previous work in chemical biology andmolecular imaging. This thesis will touch upon synthetic chemistry, molecular biology, analytical development, and molecular imaging. The works described in general are all centered around developing new tools for monitoring biological analytes with a focus on extracellular applications. Chapter One outlines the history of medical imaging in general and how molecular imaging evolved out of it. This includes information on whole body imaging techniques and how they have been adapted towards imaging biological analytes at the molecular level in living systems. This chapter details the modalities with which we are able to design new imaging probes and discusses the advantages and drawbacks of various modalities. Chapters Two through four of this dissertation focus on the use of bioluminescence as a powerful imaging platform and details how this natural phenomenon can be modified to produce analyte responsive light emission. Chapter Two describes a novel bioluminescent imaging probe that was developed using a marine luciferin/luciferase system. In particular, the synthesis of a copper(II)-reactive luciferin, picolinic-caged diphenylterazine (Pic-DTZ) is described and its implementation with the engineered Nanoluciferase. The design, synthesis, and characterization of the probe is discussed in detail including its biological relevance and applications in monitoring serum copper status as well as extracellular copper status in high-throughput cellular assays. Chapter Three outlines a discovery made while working on the copper responsive probe detailed in chapter two. The copper mediated oxidation of imidazopyrazinones and subsequent inactivation of bioluminescence is described. This work details the molecular mechanisms behind how marine luciferases are inhibited by copper. In Chapter Four the development of a second bioluminescent probe, boronate esther-caged diphenylterazine (Bor-DTZ) is described. This caged luciferin is hydrogen peroxide responsive and chapter three outlines its design, synthesis, and characterization. Applications in cell-based assays are also described using breast cancer cells stably expressing Nanoluciferase. Chapter Five of the dissertation describes work in collaboration with Dr. Randy Carney towards the development of Raman active tags for imaging and characterizing extracellular vesicles using Raman spectroscopy. In particular, the synthesis of Raman active polyynes are described as well as their conjugation to antibodies specific for membrane proteins that are potential biomarkers for ovarian cancer. Subsequent extracellular vesicle capture with the tagged antibodies and Raman spectra analysis are described in detail. The work shows the promise of Raman spectroscopy as a modality for imaging extracellular vesicles as well as for diagnosis of early-stage cancers. It also demonstrates the immense potential extracellular vesicles hold as biomarkers. Though not directly related to molecular imaging, Chapter Six describes investigations in peptide GPCR interactions in the extracellular space. The chapter details structural and biological assays of oxytocin and related analogs in both apo and metal bound forms and the subsequent effect on bioactivity. Specifically, zinc and copper bound forms of oxytocin and the analogs are studied using electronic absorption spectroscopy and circular dichroism. Receptor activation was monitored by analyzing MAPK activation. The work details how zinc and copper induce different structural changes in oxytocin and analogs and effect receptor activation and downstream signaling. Lastly, Appendix One details preliminary work on identifying new metal binding peptides using a combinatorial approach in collaboration with Dr. Kit Lam. The design of cysteine and histidine free libraries and their synthesis using the one-bead-one-compound method are described as well as their subsequent screening use live cells and a colorimetric chelator. Though this project is in preliminary stages the work described details a foundation to build open for high throughput screening and identification of unique peptides that are capable of interacting with metal ions.
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