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Development of Molecular Probes for Biomedical Imaging of Cancer and Neurological Disease

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Development of Molecular Probes for Biomedical Imaging of Cancer and Neurological Disease TITLE PAGE DEVELOPMENT OF MOLECULAR PROBES FOR BIOMEDICAL IMAGING OF CANCER AND NEUROLOGICAL DISEASE by ALLISON G. CONDIE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Thesis Advisor: Yanming Wang Co-advisor: Stanton L. Gerson Department of Chemistry CASE WESTERN RESERVE UNIVERSITY August, 2015 COMMITTEE APPROVAL CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis/dissertation of Allison G. Condie candidate for the degree of Doctor of Philosophy.* Committee Chair Robert G. Salomon Committee Member Yanming Wang Committee Member Stanton Gerson Committee Member Clemens Burda Committee Member Mary Barkley Date of Defense May 1, 2015 *We also certify that written approval has been obtained for any proprietary material contained therein. Page | ii TABLE OF CONTENTS Title Page ............................................................................................................................. i Committee Approval ........................................................................................................... ii Table of Contents ............................................................................................................... iii List of Tables ..................................................................................................................... ix List of Figures ..................................................................................................................... x List of Schemes ................................................................................................................ xxi Acknowledgements ......................................................................................................... xxii Symbols and Abbreviations ........................................................................................... xxiii Abstract ......................................................................................................................... xxvii Chapter 1. Introduction ....................................................................................................... 1 1.1 Review of molecular imaging ........................................................................... 1 1.1.1 Overview of molecular imaging ........................................................... 1 1.1.2 Magnetic resonance imaging ................................................................ 1 1.1.3 Positron emission tomography .............................................................. 3 1.1.4 Optical imaging ..................................................................................... 5 1.2 Review of myelin imaging ................................................................................ 8 1.2.1 Motivation and basis for disease ........................................................... 8 1.2.2 Histological or in vitro imaging agents ................................................. 9 Page | iii 1.2.3 In vivo imaging agents ........................................................................ 10 1.3 Review of cancer imaging .............................................................................. 11 1.3.1 Motivation and basis for disease ......................................................... 11 1.3.2 DNA damage and repair ..................................................................... 12 1.3.3 BER and AP sites ................................................................................ 13 1.3.4 Existing agents for imaging and detection .......................................... 15 Chapter 2. Myelin Imaging with CIC .............................................................................. 16 2.1 Hypothesis and methodology .......................................................................... 16 2.2 CIC synthesis and fluorescent characterization .............................................. 17 2.3 In vitro and ex vivo histology ......................................................................... 21 2.4 In vivo visualization of CNS nerves ............................................................... 24 2.5 Conclusions ..................................................................................................... 30 2.6 Materials and methods .................................................................................... 31 2.6.1 General methods ................................................................................. 31 2.6.2 Synthesis ............................................................................................. 32 2.6.3 Fluorescence characterization ............................................................. 38 2.6.4 General animal methods ..................................................................... 40 2.6.5 Tissue staining .................................................................................... 40 2.6.6 Microscopy ......................................................................................... 42 Chapter 3. Probes for Cancer Imaging Through AP Site Detection ................................ 44 Page | iv 3.1 Hypothesis and methodology .......................................................................... 44 3.2 Synthesis of compounds for AP site detection ............................................... 45 3.3 Fluorescent characterization of probes ........................................................... 51 3.4 Discussion and conclusions ............................................................................ 53 3.5 Materials and methods .................................................................................... 54 3.5.1 General methods ................................................................................. 54 3.5.2 Synthesis ............................................................................................. 54 3.5.3 Fluorescence quantum yield measurements ........................................ 72 Chapter 4. Fluorescently-tagged DNA Oligomer SSB Assay for Probe Evaluation ....... 73 4.1 Hypothesis and methodology .......................................................................... 73 4.2 Evaluation of potential UDG inhibition .......................................................... 76 4.3 Evaluation of potential APE inhibition ........................................................... 78 4.4 Compound screening by dose-response .......................................................... 81 4.5 Comparison to ARP and MX .......................................................................... 82 4.6 Identification of a transient band .................................................................... 83 4.7 Evaluation of probe-AP site lesion repair: endonuclease survey ................... 86 4.8 Discussion and conclusions ............................................................................ 88 4.9 Materials and methods .................................................................................... 89 4.9.1 General methods ................................................................................. 89 4.9.2 Reactions to establish assay validity ................................................... 91 Page | v 4.9.3 Evaluation of AP site binding probes ................................................. 94 4.9.4 Evaluation of BER .............................................................................. 95 Chapter 5. Genomic DNA Binding Assay for Probe Development ................................ 97 5.1 Hypotheses and methodology ......................................................................... 97 5.2 Blocking with MX ........................................................................................ 100 5.3 Binding time course evaluation .................................................................... 101 5.4 Competition with MX and ARP ................................................................... 102 5.5 Measurement of cellular response to DNA damaging drugs ........................ 104 5.5.1 Measurement of FUDR response ...................................................... 104 5.5.2 Measurement of MMS response ....................................................... 107 5.6 Discussion and conclusions .......................................................................... 108 5.7 Materials and methods .................................................................................. 109 5.7.1 General methods ............................................................................... 109 5.7.2 Heat/acid treated genomic DNA ....................................................... 109 5.7.3 FUDR and MMS treatment of DLD1 cells ....................................... 112 Chapter 6. Microscopy and in Vivo Imaging of Cancer ................................................ 115 6.1 Microscopy ................................................................................................... 115 6.1.1 Hypothesis and methodology ............................................................ 115 6.1.2 Optimization of fixative .................................................................... 116 6.1.3 Nonspecific binding with FEt2 ......................................................... 117 Page | vi 6.1.4 FUDR dose response with Cy5MX .................................................. 119 6.1.5 Potential pitfalls in microscopy ........................................................ 121 6.2 In vivo imaging in mouse xenograft models ................................................
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