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Identification of Cathepsin B and L As Novel Uva Targets Upstream of Cutaneous Lysosomal-Autophagic Dysregulation

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Identification of Cathepsin B and L As Novel Uva Targets Upstream of Cutaneous Lysosomal-Autophagic Dysregulation Identification of Cathepsin B and L as Novel Uva Targets Upstream of Cutaneous Lysosomal-Autophagic Dysregulation Item Type text; Electronic Dissertation Authors Lamore, Sarah Diane Publisher The University of Arizona. Rights Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. Download date 27/09/2021 14:55:30 Link to Item http://hdl.handle.net/10150/228173 1 IDENTIFICATION OF CATHEPSIN B AND L AS NOVEL UVA TARGETS UPSTREAM OF CUTANEOUS LYSOSOMAL-AUTOPHAGIC DYSREGULATION by Sarah Diane Lamore _____________________ Copyright Sarah Diane Lamore 2012 A Dissertation Submitted to the Faculty of the Department of Pharmacology and Toxicology In Partial Fulfillment of the Requirements For the Degree of DOCTOR OF PHILOSOPHY In the Graduate College THE UNIVERSITY OF ARIZONA 2012 2 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE As members of the Dissertation Committee, we certify that we have read the dissertation prepared by Sarah Diane Lamore entitled "Identification of cathepsin B and L as novel UVA targets upstream of cutaneous lysosomal-autophagic dysregulation" and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy _____________________________________________________Date: April 23rd, 2012 Georg Wondrak, Ph.D. _____________________________________________________Date: April 23rd, 2012 Elaine Jacobson, Ph.D. _____________________________________________________Date: April 23rd, 2012 Terrence Monks, Ph.D. _____________________________________________________Date: April 23rd, 2012 Catharine Smith, Ph.D. _____________________________________________________Date: April 23rd, 2012 Nathan Cherrington, Ph.D. Final approval and acceptance of this dissertation is contingent upon the candidate’s submission of the final copies of the dissertation to the Graduate College. I hereby certify that I have read this dissertation prepared under my direction and recommend that it be accepted as fulfilling the dissertation requirement. ________________________________________________ Date: April 23rd, 2012 Dissertation Director: Georg Wondrak, Ph.D. 3 STATEMENT BY AUTHOR This dissertation has been submitted in partial fulfillment of requirements for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under rules of the Library. Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of source is made. Requests for permission for extended quotation from or reproduction of this manuscript in whole or in part may be granted by the copyright holder. SIGNED: Sarah Diane Lamore 4 ACKNOWLEDGEMENTS I would like to express my deepest gratitude to my advisor, Dr. George Wondrak, for constantly supporting me, challenging me, and encouraging me. He has been an exceptional mentor and is a true role model for me. Thank you to all of the lab members of the Wondrak Lab, past and present, for their contribution to this work. I would like to especially thank Angie Davis, Sara Azimian, and Chris Cabello for their friendship- my time in the Wondrak lab would not have been the same without you. I would like to thank my committee members, Dr. Nathan Cherrington, Dr. Catharine Smith, Dr. Terrence Monks, and Dr. Elaine L. Jacobson for their guidance and feedback. I feel truly fortunate to have been surrounded by such outstanding scientists during my graduate career. I would like to especially thank Dr. Elaine L. Jacobson, Dr. Mike K. Jacobson, and the Jacobson Lab manager, Donna Coyle, for their graciousness. All irradiation protocols were performed with the use of the Jacobson Lab solar simulator, and I deeply appreciate their kindness. HPV16 viral infections were performed in collaboration with the laboratory of Dr. Samuel Campos, and I would like to Dr. Samuel Campos and Dr. Christine Calton for their contribution to this work. I would also like to thank Dr. Vijay Gokhale, co-Director of the molecular modeling shared services core (AZCC), who performed the modeling of cathepsin B. Flow cytometric analyses were performed at the flow cytometry shared services laboratory (AZCC). Tissue processing and select immunohistological staining procedures (MDA-, DHP-, and Hsp70- staining of EpiDerm™ tissue) were performed at the TAC-MASS shared service core facility (AZCC). Gene expression analysis by RT- PCR was performed at the genomics shared service core facility (AZCC). Electron microscopy was performed at the Arizona Health Sciences Center Imaging Core Facility. Thank you to all of my peers and colleagues for providing such a supportive and collaborative environment during my time here at the University of Arizona. 5 DEDICATION To my family, for their constant support and encouragement. I never would have made it this far without you. To my husband Dave, who is the best husband in the world- thank you for all that you have done for me, especially over the last five years. I feel truly lucky to have you in my life. To my parents and brother, Jon, you have made me into the person I am today. Mom and Jon, thank you for always supporting me and being there for me without question. To my late father, you gave me an incredible foundation to succeed in all my endeavors and you are always with me. 6 TABLE OF CONTENTS LIST OF FIGURES .........................................................................................................12 LIST OF TABLES ...........................................................................................................16 LIST OF ABBREVIATIONS .........................................................................................17 ABSTRACT ......................................................................................................................20 CHAPTER 1: INTRODUCTION ...................................................................................22 1.1 Physiology and Function of Human Skin ..............................................................22 1.1.1 Major functions of human skin ....................................................................22 1.1.2 The Dermis ...................................................................................................22 1.1.3 The Epidermis ...............................................................................................23 1.2 Cutaneous ultraviolet exposure ..............................................................................25 1.2.1 Historical Perspective ...................................................................................25 1.2.2 Human exposure to ultraviolet radiation .......................................................25 1.2.3 Cutaneous UVB effects ................................................................................28 1.2.4 Cutaneous UVA effects ...............................................................................30 1.2.5 The role of reactive oxygen species (ROS) in UVA-photodamage .............32 1.2.6 Cutaneous Photosensitization by endogenous chromophores .....................34 1.2.7 Cellular targets of UV and activation of signaling pathways ......................36 1.3 The lysosome .........................................................................................................39 1.3.1 Lysosome structure and function .................................................................39 1.3.2 The lysosomal role in autophagy .................................................................40 1.3.3 Lysosomal lipofuscin ....................................................................................43 1.4 Cathepsins .............................................................................................................44 1.4.1 Classification and processing of cathepsins .................................................44 1.4.2 Catalytic Mechanism of Cysteine Cathepsins ..............................................47 1.4.3 Regulation of cathepsin activity ...................................................................49 1.4.4 Role of cathepsins in cutaneous homeostasis ..............................................49 1.4.5 Cathepsins in skin pathophysiology .............................................................54 1.4.6 Cathepsins as targets of solar skin damage ..................................................57 1.5 Two-dimensional difference gel electrophoresis (2D-DIGE) as a proteomic tool for the identification of novel UVA targets ..........................................................59 1.6 Hypothesis and specific aims ................................................................................61 1.6.1 Central hypothesis ........................................................................................61 1.6.2 Rationale ......................................................................................................61 1.6.3 Specific Aims ...............................................................................................61 CHAPTER 2: MATERIALS AND METHODS ...........................................................66 2.1 Chemicals ..............................................................................................................66 2.2 Cell Culture ...........................................................................................................66
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