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University of Florida Thesis Or Dissertation Formatting A FUNCTIONAL AZASUGAR BIOSYNTHETIC CLUSTER FROM CHITINOPHAGA PINENSIS By CLARIBEL NUÑEZ A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2019 © 2019 Claribel Nuñez To me ACKNOWLEDGMENTS I am grateful to many people that have supported me throughout my graduate school journey. Firstly, I would like to thank my mentor, Professor Nicole A. Horenstein for giving me the opportunity to work in her lab. She has been one of my biggest advocates and one of the major forces that kept me striding through both personal issues and graduate school work over the past five years. Much of what I have accomplished and how I have evolved as a scientist is thanks to her guidance and encouragement. I would also like to thank my committee members Prof. Jon Stewart, Prof. Rebecca Butcher, Prof. Ron Castellano, and Prof. Valerie De Crecy for their advice throughout my research, as well as access to their laboratory equipment and lab space. In addition, I would like to thank Dr. Ion Ghiviriga for his help in analyzing my NMR samples, as well as the Mass Spectrometry facility at UF for their assistance in analyzing my samples, especially Dr. Kari Basso and Dr. Manasi Kamat. Secondly, thank you to my friends and family for providing their encouragement and understanding. I am very fortunate to have met so many wonderful friends that have supported me as a graduate student. I’m grateful for “El Corillo”—Carla, Rebeca, Johnny, Jose, Rene, Kathy, Lorena, Paul, Glenda, Angie, Camilo, Christian, Lorraine, Andreina, Gabriel—for our Latin dance nights, karaoke nights, and cookouts that helped pick me up when I was feeling homesick. Especially my dear friends, Dr. Veronica Negron and Dr. Melissa Cruz-Acuña for feeding me when I was sick and for reassuring me whenever I lost confidence and motivation. Grateful for my roommate Lindsey for her words of encouragement and for helping me take care of my pup, Oaklee, whenever I had to work late nights in the lab. I’m grateful for my best friend, Trisha Ramdihal, for our late nights of de-stressing and motivational conversations, that helped me push forward on bad days. I would also like to extend my gratitude to my godfather, Dr. 4 Thomas Jordan, for being someone that I could always count on by providing emotional and financial support throughout my years as a graduate student. Thirdly, I’d like to acknowledge my friends and colleagues in the Chemistry department that have helped me along the way. Thankful for the past and present members of the Horenstein group, including Dr. Jeffrey Arciola, Dr. Alican Gulsevin, Dr. Timothy Gould, Maria Chiara Pismataro, Hailey Beal, Haoxi Li, and Dr. Marta Quadri. I would like to extend my appreciation to Marta for not only being a wonderful colleague, discussing research results and ideas even though my work was not her expertise, but also for being a great friend; her wonderful meals that helped keep me alive, her ability to help me save money by always finding deals and coupons when grocery shopping, and for sometimes being the only person willing to go out with me to help release some stress even though we were always so busy. I’m grateful for my wonderful friends Dr. Michelle Nolan, Dr. Danielle Fagnani, and Kathryn Olsen for always making mental health checks, for providing scientific advice and always being available to help edit any of my documents. I would not have survived graduate school without the help and support from each and every one of these individuals, and for that I am eternally grateful. Finally, I thank the University of Florida and the Florida Education Fund’s McKnight Doctoral Fellowship for their financial support over my doctoral study period. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ........................................................................................................... 4 LIST OF TABLES ...................................................................................................................... 8 LIST OF FIGURES .................................................................................................................... 9 LIST OF ABBREVIATIONS.................................................................................................... 13 ABSTRACT ............................................................................................................................. 16 CHAPTER 1 INTRODUCTION ............................................................................................................. 19 1.1 Introduction to Secondary Metabolites ......................................................................... 19 1.2 Glycosidase Inhibitors .................................................................................................. 20 1.3 Azasugars ..................................................................................................................... 21 1.3.1 Overview ............................................................................................................ 21 1.3.2 Therapeutic Potential and Applications of Azasugars .......................................... 23 1.3.2.1 Azasugars as antidiabetic agents ............................................................... 23 1.3.2.2 Azasugars in lysosomal storage diseases ................................................... 23 1.3.2.3 Additional potential therapeutic applications of azasugars ......................... 25 1.3.3 Biosynthetic Pathways of Azasugars ................................................................... 26 1.3.3.1 Biosynthesis of nojirimycins ..................................................................... 26 1.3.3.2 Biosynthesis of DMPD ............................................................................. 29 1.3.3.3 Biosynthesis of nectrisine ......................................................................... 29 2 IDENTIFICATION AND CHARACTERIZATION OF DAB-1 IN C. PINENSIS .............. 31 2.1 Overview ...................................................................................................................... 31 2.2 Results and Discussion ................................................................................................. 32 2.3 Experimental ................................................................................................................ 43 2.3.1 General Methods ................................................................................................ 43 2.3.2 Growth Conditions for Chitinophaga pinensis and Culture Extract Purification .............................................................................................................. 43 2.3.2.1 Rich media growth conditions ................................................................... 43 2.3.2.2 Minimal media growth conditions ............................................................. 44 2.3.2.3 Purification methods ................................................................................. 44 2.3.3 Glycosidase Inhibition Assays ............................................................................ 47 2.3.3.1 and -glucosidase assays ....................................................................... 47 2.3.3.2 Assays on multi-well plate reader ............................................................. 47 2.3.3.3 Jack Bean -mannosidase assay ................................................................ 47 6 3 HETEROLOGOUS EXPRESSION OF AZASUGAR BIOSYNTHETIC SIGNATURE IN E. COLI ........................................................................................................................ 49 3.1 Overview ...................................................................................................................... 49 3.2 Results and Discussion ................................................................................................. 49 3.3 Experimental ................................................................................................................ 62 3.3.1 General Methods ................................................................................................ 62 3.3.2 Cloning of the pETBlue2-Cpin2154-Cpin2153-Cpin2152 Expression Construct ................................................................................................................. 62 3.3.3 Expression of Cpin2154, Cpin2153, Cpin2152 in E. coli and purification of nectrisine ................................................................................................................. 63 3.3.3.1 Rich media growth .................................................................................... 63 3.3.3.2 Minimal media growth .............................................................................. 64 3.3.3.3 Purification methods for compounds produced in E. coli ........................... 64 3.3.4 Glycosidase Inhibition Assays ............................................................................ 65 4 BIOINFORMATIC ANALYSIS OF THE AZASUGAR BIOSYNTHETIC SIGNATURE ..................................................................................................................... 66 4.1 Overview ...................................................................................................................... 66 4.2 Results and Discussion ................................................................................................. 68 4.3 Experimental ...............................................................................................................
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