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Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans

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Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans Development and Application of Analytical Techniques for Evaluating Function in Pancreatic Islets of Langerhans by Cynthia Marie Cipolla A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Chemistry) in the University of Michigan 2015 Doctoral Committee: Professor Robert T. Kennedy, Chair Professor Raoul Kopelman Professor Mark E. Meyerhoff Professor Leslie S. Satin © Cynthia M. Cipolla 2015 DEDICATION To my family and friends for their love and support. ii ACKNOWLEDGEMENTS I would first and foremost like to thank my advisor, Dr. Robert Kennedy, for his support and guidance throughout my graduate career and for his contributions to this research. I would also like to thank my committee members, Dr. Raoul Kopelman, Dr. Mark Meyerhoff, and Dr. Leslie Satin for their feedback and suggestions for my project and for taking the time to serve on my committee. I am grateful for the camaraderie and mentorship of all former and present Kennedy lab members, especially Dr. Ting Zhang for training me in islet isolation and microfabrication, Mahmoud El Azzouny for sharing his metabolomics expertise, Shusheng Lu for his assistance in insulin secretion measurements and for helpful discussions about islets, Erik Guetschow for his droplet microfluidics expertise and advice, and Thitaphat (Non) Ngernsutivorakul for taking over the fiber optic detector project and helping to push it to completion. I would also like to thank my collaborators at the University of Illinois at Chicago, Dr. David Eddington, Dr. José Oberholzer, and Elizabeth Ferraz-Samar for their work on the droplet microfluidic project, and Dr. Francis Esmonde-White for his work and guidance on the fiber optic detector project. Finally, I would like to thank my family and friends for their support and encouragement throughout this endeavor. iii TABLE OF CONTENTS DEDICATION ...................................................................................................................ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF FIGURES ..........................................................................................................vi LIST OF APPENDICES ................................................................................................. viii LIST OF ABBREVIATIONS .............................................................................................ix ABSTRACT .................................................................................................................... xii CHAPTER 1. Introduction ............................................................................................... 1 Diabetes Background ................................................................................................... 1 Microfluidic Monitoring of Cells .................................................................................. 10 Metabolomics ............................................................................................................. 15 Dissertation Overview ................................................................................................ 20 References ................................................................................................................. 21 2+ CHAPTER 2. Sequential Detection of [Ca ]i and Insulin Secretion On-Chip ................ 28 Introduction ................................................................................................................ 28 Experimental Procedures ........................................................................................... 31 Results ....................................................................................................................... 38 Discussion.................................................................................................................. 44 Conclusion ................................................................................................................. 49 References ................................................................................................................. 50 CHAPTER 3. Development and Optimization of a Sample Preparation Method for Islet Metabolomics ................................................................................................................ 53 Introduction ................................................................................................................ 53 Experimental Procedures ........................................................................................... 55 Results and Discussion .............................................................................................. 60 Conclusion ................................................................................................................. 78 References ................................................................................................................. 80 iv CHAPTER 4. Application of Metabolomic Method to the Study of Oxidative Stress in Islets .............................................................................................................................. 84 Introduction ................................................................................................................ 84 Experimental Procedures ........................................................................................... 86 Results ....................................................................................................................... 91 Discussion................................................................................................................ 101 Conclusion ............................................................................................................... 108 References ............................................................................................................... 110 CHAPTER 5. Summary and Future Directions ............................................................ 114 Summary.................................................................................................................. 114 Future Directions ...................................................................................................... 118 References ............................................................................................................... 126 APPENDICES ............................................................................................................. 128 v LIST OF FIGURES Figure 1.1. KATP-dependent pathway of GSIS. ................................................................ 3 Figure 1.2. Anaplerotic and cataplerotic pathways .......................................................... 4 Figure 1.3. Interaction of glucose and fatty acid metabolism. .......................................... 5 Figure 1.4. Schematic of islet transplantation. ................................................................. 7 Figure 1.5. Optical configurations for detection on microfluidic chips. ........................... 13 Figure 2.1. Image of PDMS chip for fraction collection.................................................. 33 Figure 2.2. Schematic of capillary collection chips. ....................................................... 35 2+ Figure 2.3. [Ca ]i measurements on-chip. .................................................................... 36 Figure 2.4. Schematic of microfluidic devices for aqueous droplet extraction and analysis. ........................................................................................................................ 37 Figure 2.5. Insulin immunoassay reproducibility. ........................................................... 39 Figure 2.6. Representative calibration of standard insulin using continuous flow collection method. ......................................................................................................... 40 Figure 2.7. Electropherogram with fluorescein tracer. ................................................... 41 2+ Figure 2.8. Measurement of [Ca ]i and insulin secretion from a representative group of 8 islets using continuous flow collection method. .......................................................... 42 Figure 2.9. Representative calibration of standard insulin using droplets generated from a 96-well plate. .............................................................................................................. 43 Figure 2.10. Electropherograms with and without rhodamine tracer. ............................ 44 2+ Figure 2.11. [Ca ]i and insulin secretion measured from 20 islets with segmented flow collection method. ......................................................................................................... 44 Figure 2.12. Design and performance of single channel electrophoresis chip. ............. 46 Figure 3.1. Typical metabolite chromatograms from islet extracts ................................ 59 Figure 3.2. Metabolomic sample preparation method. .................................................. 60 Figure 3.3. Metabolism quenching method comparison ................................................ 62 Figure 3.4. Extraction solvent effect on peak areas. ..................................................... 63 Figure 3.5. Glutamate chromatographic peak shapes by extraction solvent. ................ 64 Figure 3.6. RSE dependence on sample size. .............................................................
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