Louisiana State University LSU Digital Commons LSU Doctoral Dissertations Graduate School 2016 Extraction and Biochemical Characterization of Alligator mississippiensis glycosaminoglycans and an Ex-vivo Murine Pilot Study to Test their Potential Effect on a Selected Panel of Genes Associated with Cystic Fibrosis Jose Daniel Estrada Andino Louisiana State University and Agricultural and Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_dissertations Part of the Life Sciences Commons Recommended Citation Estrada Andino, Jose Daniel, "Extraction and Biochemical Characterization of Alligator mississippiensis glycosaminoglycans and an Ex-vivo Murine Pilot Study to Test their Potential Effect on a Selected Panel of Genes Associated with Cystic Fibrosis" (2016). LSU Doctoral Dissertations. 1245. https://digitalcommons.lsu.edu/gradschool_dissertations/1245 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. EXTRACTION AND BIOCHEMICAL CHARACTERIZATION OF ALLIGATOR MISSISSIPPIENSIS GLYCOSAMINOGLYCANS AND AN EX-VIVO MURINE PILOT STUDY TO TEST THEIR POTENTIAL EFFECT ON A SELECTED PANEL OF GENES ASSOCIATED WITH CYSTIC FIBROSIS A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The School of Nutrition and Food Science by Jose Daniel Estrada Andino B.S., Escuela Agrícola Panamericana Zamorano, 2007 M.S., Louisiana State University, 2011 August 2016 This work is dedicated to my amazing wife Monika and all of my family in Honduras, Germany and Poland. ii ACKNOWLEDGEMENTS I want to express my deepest gratitude to everyone who played a role in the completion of this work. First of all I thank God for the daily assurance of purpose in my life and for all the people whom he used for my academic, professional, personal and academic growth during the past few years. To Dr. Jack Losso, thank you so much for being an admirable person, great friend, and an inspiring and always supportive mentor. Thank you for trusting in me to conduct such a relevant project for the Louisiana alligator industry and for simultaneously allowing me to have a well- rounded experience through my involvement in national new product development competitions, IFT competitions and an amazing food industry internship. Under your mentorship I have become a better person and professional. To my committee members, Dr. Joan King, Dr. Roger Laine, Dr. Grover Waldrop, Dr. Yogesh Saini, Mr. Mark Shirley and Dr. Erin Casey, thank you for your valuable time and guidance. Special thanks to Mr. Mark Shirley and Dr. Yogesh Saini for their hands-on support and training in the field and laboratory, respectively. To all my collaborators in the experiments carried out for this dissertation, Mr. Cristian Macoto, Dr. Marvin Moncada, Dr. Luis Espinoza and Ms. Jeimy Menjivar in the processing of alligator by-products, Dr. Richard Cooper for allowing me to use his molecular biology laboratory iii and equipment, Dr. George Stanley for his FT-IR training and Dr. Kenneth Smith at Thermo Fisher Scientific for his kind support with FT-IR data collection. I would also like to thank all my labmates and friends at the School of Nutrition and Food Science and around the LSU campus for all their support and encouragement expressed in many different ways. Especially, Srikanth, Namrata, Kennet, Ryan, Reynaldo, Alejandro and Damir. To my Baton Rouge parents Mark and Alicia, your friendship and care have made this town feel like home. To my SBRPC family, thank you for supporting me in prayer and also in many practical ways even before I arrived here. Finally, to my wife Monika and my family in Honduras, Poland and Germany. Thank you for supporting my goals and always believing in my potential. I would not have made it so far without all of you. Monika, your willingness to move from Germany to Baton Rouge gave me the gift of an outstanding PhD student experience next to you. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ........................................................................................................... iii LIST OF TABLES ........................................................................................................................ vii LIST OF FIGURES ..................................................................................................................... viii ABBREVIATIONS INDEX ........................................................................................................... x ABSTRACT ................................................................................................................................. xiv CHAPTER 1 INTRODUCTION .................................................................................................... 1 CHAPTER 2 LITERATURE REVIEW ......................................................................................... 5 2.1. Louisiana Alligator Farming ........................................................................................ 5 2.2. Medicinal Properties of Crocodilians ........................................................................... 6 2.3. Hyaluronic Acid and Sulfated Glycosaminoglycans .................................................... 9 2.3.1. Classification and Structures ................................................................................. 9 2.3.2. Biosynthesis .......................................................................................................... 9 2.3.3. Signaling and Biological Functions of Hyaluronan ............................................ 12 2.3.4. Degradation ......................................................................................................... 17 2.3.5. Sources ................................................................................................................ 18 2.4. Cystic Fibrosis ............................................................................................................ 21 2.4.1. Introduction ......................................................................................................... 21 2.4.2. History of Cystic Fibrosis ................................................................................... 22 2.4.3. The Cystic Fibrosis Transmembrane Conductance Regulator Protein ............... 23 2.4.4. Diagnosis ............................................................................................................. 26 2.4.5. Airway Pathophysiology in Cystic Fibrosis ........................................................ 26 2.4.6. Current Treatment ............................................................................................... 30 2.4.7. Markers of Inflammation in Cystic Fibrosis ....................................................... 35 CHAPTER 3 MATERIALS AND METHODS ........................................................................... 37 3.1. Experimental Design and Statistical Analysis ............................................................ 37 3.2. Materials ..................................................................................................................... 37 v 3.3. Extraction of GAGs from Alligator Carcasses, Feet and Backstraps ......................... 39 3.4. Extraction of GAGs from Alligator Eyeballs ............................................................. 42 3.5. Determination of Sulfated GAGs, HA and Total GAGs Content .............................. 43 3.6. GAGs Size Analysis ................................................................................................... 44 3.7. Digestion of GAGs by Type-1 Hyaluronidase and Chondroitinase ABC .................. 45 3.8. Determination of Protein and Mineral Content .......................................................... 46 3.9. Structure Characterization by FT-IR Spectroscopy .................................................... 47 3.10. Cystic Fibrosis Ex-Vivo Scnn1b-Tg Mice Pilot Study ............................................ 47 3.10.1 Transgenic Mice Generation and PCR Genotyping ............................................ 47 3.10.2 Animal Husbandry .............................................................................................. 48 3.10.3 Trachea Harvesting ............................................................................................. 48 3.10.4 MTEC Isolation ................................................................................................... 49 3.10.5 MTEC Submerged Culture ................................................................................. 50 3.10.6 MTEC Differentiation in Air-Liquid Interface Culture ...................................... 50 3.10.7 MTEC Treatment with AEB GAGs .................................................................... 51 3.10.8 Cell Lysis and RNA Purification ........................................................................ 52 3.10.9 cDNA Generation and PCR Microarray ............................................................. 53 3.10.10 Transglutaminase Activity Assay .................................................................... 56 CHAPTER 4 RESULTS AND DISCUSSION ............................................................................