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Plasma Produced Reactive Oxygen and Nitrogen Species in Angiogenesis Krishna Priya Arjunan Alisa Morss Clyne, Ph.D

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Plasma Produced Reactive Oxygen and Nitrogen Species in Angiogenesis Krishna Priya Arjunan Alisa Morss Clyne, Ph.D Plasma Produced Reactive Oxygen and Nitrogen Species in Angiogenesis A Thesis Submitted to the Faculty of Drexel University by Krishna Priya Arjunan in partial fulfillment of the requirements for the degree of Doctor of Philosophy May 2011 © Copyright 2011 Krishna Priya Arjunan. All Rights Reserved. ACKNOWLEDGMENTS This dissertation would not have been possible without the guidance of many individuals who in one way or another contributed to the completion of my Ph.D. First and foremost, I would like to express my deepest gratitude to my supervisor, Dr. Alisa Morss Clyne for her encouragement and support throughout my Ph.D. It was a great experience working with her. I am grateful to Drs. Kenneth Barbee, Gary Friedman, Fred Allen and Kambiz Pourrezaei for their valuable advice on this thesis as committee members. I would like to thank A. J. Drexel Plasma Institute for providing the plasma devices for running the experiments. I would like to acknowledge Dr. Alexander Fridman whose plasma lectures helped me understand better the field of plasma physics. I wish to express my sincere thanks to Drs. Alexander Gutsol, Victor Vasilets, Jane Azizkhan- Clifford and Shivanthi Anandan for their valuable guidance, encouragement and suggestions. I will always cherish the fruitful discussions I had with these wonderful people. I would also like to acknowledge the friendship and help of past and present colleagues and friends at the Vascular Kinetics Lab and A. J. Drexel Plasma Institute, in particular, Sameer Kalghatgi, Kivilcim Buyukhatipoglu, Dannielle Solomon, Steve Kemeny, Justin Mathew, David Staack, Shailesh Gangoli, Nachiket Vaze, Halim Ayan, Tanvir Farouk, Sin Park, Ryan Robinson and Ekaterina Cerchar. Next, I extend my gratitude to my parents Arjunan Pillai and Anandavally Ammal for their love, encouragement and invaluable support throughout my life. I would also like to thank my sister, Resmi for inspiring and assuring me that there is nothing in this world that cannot be achieved if you try hard enough, and my brother, Hari for his constant support. I would like to thank my lovely niece, Avni for cheering me up on a gloomy day with her songs and laughter. Finally, I am grateful to my fiancé, Kirill for standing by me during the good times and bad. This thesis would not have been possible without his help, understanding and support. I dedicate this thesis to my loving family. i Table of Contents LIST OF TABLES ............................................................................................................ vii LIST OF FIGURES ......................................................................................................... viii ABSTRACT .......................................................................................................................xv CHAPTER 1: INTRODUCTION ........................................................................................1 1.1 Thesis overview .......................................................................................................1 1.2 Clinical motivation...................................................................................................2 1.2.1 Wound healing .........................................................................................................2 1.2.2 Ischemia ...................................................................................................................3 1.2.3 Vascularization of tissue engineering scaffolds .......................................................4 1.3 Angiogenesis ............................................................................................................5 1.3.1 Reactive species .......................................................................................................6 1.3.2 Fibroblast growth factor-2 .....................................................................................11 1.3.3 Therapeutic angiogenesis .......................................................................................13 1.3.3.1 Protein therapy .......................................................................................................13 1.3.3.2 Gene therapy ..........................................................................................................14 1.3.3.3 Cell-based therapy .................................................................................................15 1.4 Plasma Medicine ....................................................................................................16 1.4.1 Plasma ....................................................................................................................16 1.4.2 Non-thermal dielectric barrier discharge plasma ...................................................17 1.4.3 Pin-to-hole spark discharge plasma .......................................................................18 1.4.4 Clinical applications of plasma ..............................................................................18 1.4.4.1 Blood coagulation ..................................................................................................19 ii 1.4.4.2 Wound sterilization ................................................................................................19 1.4.4.3 Medical equipment sterilization .............................................................................20 1.4.4.4 Wound healing .......................................................................................................20 1.4.4.5 Dental cavity treatment ..........................................................................................21 1.4.4.6 Cancer therapy .......................................................................................................21 1.4.4.7 Skin diseases ..........................................................................................................21 1.4.4.8 Cell transfection .....................................................................................................22 1.4.4.9 Scaffold treatment ..................................................................................................22 1.5 Biologically relevant reactive oxygen and nitrogen species ..................................23 1.5.1 Reactive oxygen species (ROS) .............................................................................23 - 1.5.1.1 Superoxide (O2 ).....................................................................................................24 1.5.1.2 Hydrogen peroxide (H2O2) ....................................................................................25 1.5.1.3 Hydroxyl radical (OH∙) ..........................................................................................26 1 1.5.1.4 Singlet oxygen (O2( ∆g)) ........................................................................................27 1.5.2 Reactive nitrogen species (RNS) ...........................................................................28 - - - 1.5.2.1 Nitric oxide (NO), nitrite (NO2 ), nitrate (NO3 ), and peroxynitrite (ONOO ) ......28 1.6 Thesis organization ................................................................................................30 CHAPTER 2: REACTIVE OXYGEN AND NITROGEN SPECIES PRODUCED BY DBD AND PHD PLASMA IN LIQUID AND CELLS ....................................................52 2.1 Introduction ............................................................................................................52 2.2 Materials and methods ...........................................................................................56 2.2.1 Cell culture .............................................................................................................56 2.2.2 Plasma treatment ....................................................................................................56 2.2.2.1 Non-thermal atmospheric pressure plasma ............................................................56 iii 2.2.2.2 Pin-to-hole spark discharge plasma .......................................................................57 2.2.3 ROS/RNS measurement in liquid and cells ...........................................................58 2.2.3.1 Cumulative ROS/RNS in liquid .............................................................................59 2.2.3.2 Cumulative ROS/RNS in cells ...............................................................................60 2.2.3.3 Hydrogen peroxide (H2O2) ....................................................................................61 2.2.3.4 Hydroxyl radical and peroxynitrite (OH∙ and ONOO-) .........................................62 - 2.2.3.5 Superoxide (O2 ).....................................................................................................63 1 2.2.3.6 Singlet oxygen (O2( ∆g)) ........................................................................................64 - 2.2.3.7 Nitrite (NO2 ) .........................................................................................................65 2.2.3.8 Nitric oxide (NO) ...................................................................................................66 2.2.4 pH and temperature measurement .........................................................................68 2.2.5 Statistical analysis ..................................................................................................69 2.3 Results ....................................................................................................................69 2.3.1 DBD plasma:
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