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Reticulitermes Flavipes RETICULITERMES FLAVIPES (ISOPTERA: RHINOTERMITIDAE) PROTEOMICS By CHARLES JERRY BOWEN, JR. Bachelor of Science Midwestern State University Wichita Falls, Texas 1994 Master of Science Midwestern State University Wichita Falls, Texas 2002 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY May 2007 C O P Y R I G H T By C. Jerry Bowen May 2007 RETICULITERMES FLAVIPES (ISOPTERA: RHINOTERMITIDAE) PROTEOMICS Dissertation Approved: Bradford M. Kard Dissertation Advisor Jack W. Dillwith Kristopher L. Giles Mark E. Payton A. Gordon Emslie Dean of the Graduate College iii ACKNOWLEDGEMENTS I would like to express my deepest gratitude to Dr. Jack W. Dillwith for all he has done to help me with this project. Without his guidance, this project would have been impossible to complete. Dr. Brad Kard was invaluable as my committee chair and major advisor. Also, I would like to extend my sincerest gratitude to Dr. Kris Giles and Dr. Mark Payton for their participation on my committee. I would like to thank Robin Madden and Dr. Bryna Donnelly for both their technical assistance ‘at the bench’ and their friendship. I would like to thank the people of the Department of Entomology and Plant Pathology for allowing me to be a part of the whole and their patience in waiting to apply a termiticide until my research was complete. My thanks to the department head, initially, Dr. Russell Wright during the early days, and later, Dr. Jonathan Edelson for their support. I would like to thank Oklahoma State University and The Nature Conservancy for allowing me to collect termites on their facilities. Thank you to Michael P. Doss and Greg H. Broussard for assistance collecting ‘wild’ termites from the field. Also, thank you to Doug Keuhl for assistance sorting termite instars in the laboratory. I would like to thank Don Arnold for his patience when the termites I was collecting damaged several of the slide boxes. I would like to thank Dr. Steve Hartson, Janet Rogers, and Lisa Whitworth of the Oklahoma State University Biochemistry and Molecular Biology Core Facility. I would also like to thank Dr. Patricia Ayoubi and Dr. Peter Hoyt for their assistance assimilating the molecular functions. I would also like to thank Hua Weng for maintaining and updating local protein databases. I would like to thank my grandmother, Clara Riordan, my parents, Don and Joan Riddles, and Charles and Elizabeth Bowen. I would also like to thank Pam Hathorn and my friends for their support over the past several years. Without you, I could never have made it this far. I would like to thank Matrix Science, Inc. (Mascot) and Syngene Ltd.(Dymension) for their consent to reproduce images generated by their software. To those whom I have failed to name, I thank you as well. iv TABLE OF CONTENTS Chapter Page I. INTRODUCTION .................................................................................................. 1 II. OBJECTIVES ......................................................................................................... 6 III. LITERATURE REVIEW ........................................................................................ 7 IV. MATERIALS AND METHODS .......................................................................... 18 Objective I – Develop a system for analyzing the termite proteome. .............. 19 Part 1: Optimize solubilization and staining ........................................ 19 Part 2: Optimize isoelectric focusing ................................................... 26 Part 3: Optimize SDS-PAGE ................................................................ 28 Objective II – Develop standard protein reference map for Reticulitermes flavipes ............................................................. 32 Objective III – Initial characterization of the putative R. flavipes proteome ... 34 Objective IV – Test for differential protein expression among R. flavipes colonies .................................................................. 40 Objective V – Test for differential protein expression between worker and solder castes .......................................................... 42 V. RESULTS AND DISCUSSION ............................................................................ 43 Objective I – Develop a system for analyzing the termite proteome ............... 43 Objective II – Develop standard protein reference map for Reticulitermes flavipes ............................................................. 52 Objective III – Initial characterization of the putative R. flavipes proteome ... 78 Objective IV – Test for differential protein expression among R. flavipes colonies ................................................................ 117 Objective V – Test for differential protein expression between worker and soldier castes .................................................................. 131 VI. SUMMARY AND CONCLUSIONS .................................................................. 179 VII. LITERATURE CITED ........................................................................................ 183 VIII. APPENDICES .................................................................................................... 203 Appendix A – Flowcharts ............................................................................. 204 Flowchart Illustrating Overall Project ................................................ 205 v Flowchart Illustrating Objective I – Develop a system for analyzing the termite proteome ........................................... 206 Flowchart Illustrating Objective II – Develop standard protein reference map for Reticulitermes flavipes ............................... 209 Flowchart Illustrating Objective III – Initial characterization of the putative R. flavipes proteome ......................................... 210 Flowchart Illustrating Objective IV – Comparing Multiple Termite Colonies....................................................................... 211 Flowchart Illustrating Objective V – Comparing Worker/Soldier Castes .............................................................. 212 Appendix B – Suggested Reagent List......................................................... 213 Appendix C – Links ..................................................................................... 215 vi LIST OF TABLES Page Table 1. Immobiline pH strip protein loads and DDT reswelling solution volume ......... 26 Table 2. Reagent volumes for acrylamide solutions per each 10.0mL of solution .......... 29 Table 3. Reference map data ............................................................................................ 61 Table 4. Putative identifications with a ‘termite’ source ................................................ 101 Table 5. Putative worker protein identifications ............................................................ 102 Table 6. Differentially expressed proteins between worker and soldier castes from the same colony ....................................................................................... 142 Table 7. Putative soldier protein identifications ............................................................ 168 vii LIST OF FIGURES Page Fig. 1. Examples of proteins processed with and without protease inhibitor: (A) Precipitant processed with protease inhibitor; (B) Precipitant processed without protease inhibitor. ................................................................. 44 Fig. 2. Examples of gels using 8.0M treatments: (A) 8.0M urea, Triton X-100, and carrier ampholyte; (B) 8.0M urea, Triton X-100, tris, and carrier ampholyte; (C) 8.0M urea, Triton X-100, CHAPS, and carrier ampholyte. ...... 45 Fig. 3. Examples of gels using 6.0M urea and 2.0M thiourea solutions: (A) 6.0M urea, 2.0M thiourea, Triton X-100, and carrier ampholyte; (B) 6.0M urea, 2.0M thiourea, Triton X-100, tris, and carrier ampholyte; (C). 6.0M urea, 2.0M thiourea, Triton X-100, CHAPS, and carrier ampholyte; and (D) 6.0M urea, 2.0M thiourea, Triton X-100, CHAPS, tris, and carrier ampholyte. ................................................................................. 46 Fig. 4. Examples of gels generated using various pH ranges: (A) 4-7 pH; (B) 3-10 pH; and (C) 3-10NL pH. ...................................................................... 47 Fig. 5. Differences in spot patterns relative to acrylamide monomer percentage: (A) 7cm with 12.0% monomer; (B) 7cm gel with 14.0% monomer; (C) 7cm gel with 16.0% monomer; (D) 7cm gel with 18.0% monomer; (E) 24cm gel with 16.0% monomer; (F) 24cm gel with 18.0% monomer. ........ 49 Fig. 6. Comparison of gel sizes: (A) 7cm gel; (B) 11cm gel; (C) 24cm gel. .................. 50 Fig. 7. Grid for estimating pI and molecular weights. ..................................................... 55 Fig. 8. Worker caste reference Map for Reticulitermes flavipes. Inset: Overview of gel quadrants. ....................................................................... 56 Fig. 9. Worker caste reference map for Reticulitermes flavipes – Quadrant 1. Inset: Highlight of selected gel quadrant. ........................................................... 57 Fig. 10. Worker caste reference map for Reticulitermes flavipes – Quadrant 2. Inset: Highlight of selected gel quadrant. ........................................................... 58 Fig. 11. Worker caste reference map for Reticulitermes flavipes – Quadrant 3. Inset: Highlight of selected gel quadrant. ..........................................................
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