DIFFERENTIAL GENE EXPRESSION IN THE SKIN OF XIPHOPHORUS MACULATUS JP 163 B IN RESPONSE TO FULL SPECTRUM (10,000 K) FLUORESCENT LIGHT by Kaela Caballero, B.S. A thesis submitted to the Graduate Council of Texas State University in partial fulfillment of the requirements for the degree of Master of Science with a Major in Biochemistry August 2015 Committee Members: Ronald Walter, Chair Rachell Booth Steven Whitten COPYRIGHT by Kaela L. Caballero 2015 FAIR USE AND AUTHOR’S PERMISSION STATEMENT Fair Use This work is protected by the Copyright Laws of the United States (Public Law 94-553, section 107). Consistent with fair use as defined in the Copyright Laws, brief quotations from this material are allowed with proper acknowledgment. Use of this material for financial gain without the author’s express written permission is not allowed. Duplication Permission As the copyright holder of this work I, Kaela L. Caballero, authorize duplication of this work, in whole or in part, for educational or scholarly purposes only. DEDICATION To my parents and family, who have always supported and encouraged me, my education and my coffee addiction. And to Max, my study buddy; where you have gone, there is no need for coffee fueled late nights. Rest in peace. ACKNOWLEDGEMENTS This work could not have been accomplished without the support and mentorship that I have received throughout my life from family, teachers, mentors and friends. It is impossible to name everyone here, but I would like to use a few lines to thank those that have helped me achieve my goals. Thank you to Dr. Ron Walter for your support and mentorship this last year and half. It has definitely been a journey; you have pushed me to be better in all aspects of my life and have put up with me when I failed. My gratitude towards you cannot be adequately expressed, so I hope that through my continued success I can convey the appreciation I have for the opportunities and lessons you have allowed me to take part in. I hope that one-day I can show you that the time and support you have shown me where well spent. I would also like to thank the various current and former members of the Molecular Biosciences Research Group and Xiphophorus Stock Center Staff: Will and Mikki Boswell, Markita Savage and Natalie Taylor, and graduate students Jordan Chang and Angelica Riojas for their help, support and fellowship during my time here. Thank you to UTHSCSA coordinators of the Doctoral Bridge program, Dr. Nicquet Blake and Dr. Kay Oyajobi, for your continuous support and guidance from the time I applied to Texas State and throughout my time here. Thank you to my committee members Dr. Rachell Booth and Dr. Steve Whitten for your academic input and mentorship during my time here. It has been greatly appreciated and invaluable. v Lastly, to my parents, fiancé, family and friends: to give thanks for your continuous love, support, understanding and sacrifice is not even minimally adequate. I could not have made it this far without your love and encouragement. Thank you. vi TABLE OF CONTENTS Page ACKNOWLEDGEMENTS ................................................................................................ v LIST OF TABLES .............................................................................................................. x LIST OF FIGURES ......................................................................................................... xiii ABSTRACT ..................................................................................................................... xiv CHAPTER I. INTRODUCTION ............................................................................................... 1 Fish Models in Research ............................................................................. 1 Xiphophorus Fishes ..................................................................................... 2 Xiphophorus as a Model to Study Inherited Tumorigenesis ....................... 2 Contribution of Ultraviolet Radiation to Formation of Melanoma in Xiphophorus ................................................................................................ 5 Photoreactivation ........................................................................................ 7 Evidence for a Specific Response to Fluorescent Light ............................. 8 II. METHODS & MATERIALS .......................................................................... 12 Research Animals ..................................................................................... 12 Light Sources and Exposures .................................................................... 13 RNA Isolation ........................................................................................... 16 RNA Sequencing ...................................................................................... 17 Filtration and Read Mapping .................................................................... 18 Functional Analysis .................................................................................. 18 Quantitative Real Time PCR .................................................................... 19 NanoString ................................................................................................ 21 vii III. CO-INDUCED MODULATION OF GENE EXPRESSION IN XIPHOPHORUS SKIN ......................................................................................... 24 Introduction ............................................................................................... 24 Results ....................................................................................................... 25 A. Differential Gene Expression in Response to CO Exposure ........ 25 B. Genetic Responses Among CO Exposures ................................... 26 Discussion ................................................................................................. 30 IV. CHARACTERIZATION OF BIOLOGICAL FUNCTIONS MODULATED BY FULL SPECTRUM FLUORESCENT LIGHT EXPOSURE IN XIPHOPHORUS MACULATUS JP 163 B SKIN ................................................. 32 Introduction ............................................................................................... 32 Results ....................................................................................................... 33 A. Functional Clusters of the Shared Genetic Response ................... 33 B. Functional Clusters After 20 MIN. Exposure ............................... 41 C. Functional Clusters After 40 MIN. Exposure ............................... 45 D. Functional Clusters After 60 MIN. Exposure ............................... 49 E. Functional Clusters After 80 MIN. Exposure ............................... 52 Discussion ................................................................................................. 55 Validations ................................................................................................ 65 qRT-PCR ............................................................................................. 65 NanoString .......................................................................................... 68 V. SUMMARY & CONCLUSIONS .................................................................... 70 LITERATURE CITED ..................................................................................................... 73 viii LIST OF TABLES Table Page 2-1. Table showing read statistics for each skin sample ................................................... 17 2-2. Primer sequences for select genes used in quantitative-real time PCR validation of a select subset of significant DE genes ................................................................. 21 2-3. NanoString custom designed probe set for 19 test targets and 10 housekeeping controls ............................................................................................................... 22 3-1. Table showing the fractions of DE up and down-modulated shared genes (p-adj < 0.01, ±2 fold change) for comparisons of the 20, 40 and 60 min. gene sets ............................................................................................................. 29 3-2. Table showing the fractions of DE up and down-modulated unique genes (p-adj < 0.01, ±2 fold change) for various comparisons of the 20, 40, 60 and 80 min. gene sets ..................................................................................................... 29 3-3. Table showing the fractions of unique DE up and down-modulated genes (p-adj < 0.01, ±2 fold change) for 20, 40, 60 and 80 min. when compared to the 181 overall shared gene set ................................................................................ 29 4-1. Eight of the nineteen overall up-modulated genes clustered into 2 separate GO clusters ............................................................................................................... 34 4-2. One hundred twenty-one of one hundred-sixty one overall down-modulated genes clustered into 13 GO categories that met the cut off for functional clusters (p- value < 0.01, list not repeated) ........................................................................... 35 4-a. Table exhibiting gene name, Ensembl transcript ID, fold change, p-adj, and read counts of 6 significant DE 80 min. up-modulated genes that were found in common with the overall shared response ......................................................... 39 ix 4-b. Table exhibiting gene name, Ensembl transcript ID, fold change, p-adj,