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Phenotypic and Molecular Analysis of Desensitization to Levamisole in Male and Female Adult Brugia Malayi

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Phenotypic and Molecular Analysis of Desensitization to Levamisole in Male and Female Adult Brugia Malayi Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Phenotypic and molecular analysis of desensitization to levamisole in male and female adult Brugia malayi Mengisteab T. Wolday Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Toxicology Commons Recommended Citation Wolday, Mengisteab T., "Phenotypic and molecular analysis of desensitization to levamisole in male and female adult Brugia malayi" (2019). Graduate Theses and Dissertations. 17809. https://lib.dr.iastate.edu/etd/17809 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Phenotypic and molecular analysis of desensitization to levamisole in male and female adult Brugia malayi by Mengisteab Wolday A thesis submitted to the graduate faculty in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Major: Toxicology Program of Study Committee: Richard J. Martin, Major Professor Alan P. Robertson Aileen F. Keating The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this thesis. The Graduate College will ensure this thesis is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Mengisteab Wolday, 2019. All rights reserved. ii DEDICATION This Thesis research is dedicated to my loving and caring parents Tesfaldet Wolday and Mizan Teckleab, and my siblings Mussie, Selam, Daniel, Yonas, Alek and Abraham, and my son Yosias. iii TABLE OF CONTENTS Page LIST OF FIGURES .........................................................................................................................v LIST OF TABLES ........................................................................................................................ vii NOMENCLATURE .................................................................................................................... viii ACKNOWLEDGMENTS ............................................................................................................. ix ABSTRACT ................................................................................................................................... xi CHAPTER 1. GENERAL INTRODUCTION ................................................................................1 1.1 Introduction ......................................................................................................................... 1 1.2 Thesis Organization ............................................................................................................. 2 1.3 References ........................................................................................................................... 3 CHAPTER 2. LITERATURE REVIEW .........................................................................................5 2.1 Parasitic Nematodes ............................................................................................................ 5 2.2 Lymphatic filariasis (Elephantiasis) .................................................................................... 6 2.3 Nicotinic acetylcholine receptors (nAChRs) ..................................................................... 12 2.3.1 Parasitic nematode nAChRs ...................................................................................... 13 2.4 Anthelmintics and Anthelmintic Resistance ...................................................................... 15 2.4.1 Anthelmintics ............................................................................................................ 15 2.4.2 Imidazothiazoles ........................................................................................................ 15 2.4.3 Anthelmintic resistance ............................................................................................. 16 2.5 References ......................................................................................................................... 18 CHAPTER 3. PHENOTYPIC AND MOLECULAR ANALYSIS OF DESENSITIZATION TO LEVAMISOLE IN ADULT BRUGIA MALAYI...................................................................26 3.1 Abstract .............................................................................................................................. 26 3.2 Significance ....................................................................................................................... 27 3.3 Introduction ....................................................................................................................... 28 3.4 Materials and Methods ...................................................................................................... 30 3.4.1 Parasites ..................................................................................................................... 30 3.4.2 nAChR Primer Design............................................................................................... 30 3.4.3 Worminator assay ...................................................................................................... 31 3.4.4 RNA extraction and reverse transcriptions by PCR .................................................. 32 3.4.5 SYBR Green quantitative RT-PCR ........................................................................... 32 3.4.6 Data analysis.............................................................................................................. 33 3.5 Results ............................................................................................................................... 34 3.5.1 Expression of nAChR subunit genes at the whole worm level ................................. 34 3.5.2 Levamisole transiently inhibits B. malayi motility ................................................... 35 3.5.3 Significant up regulation of Bma-unc-38 increases recovery and motility ............... 38 3.5.4 Worms under flaccid paralysis show significant upregulation of Bma-unc-29 ........ 41 3.5.5 Desensitization and relative expresssion in male B.malayi .......................................... 42 iv 3.6 Discussion .......................................................................................................................... 46 3.6.1 Interpretation of the Worminator assay ..................................................................... 47 3.6.2 Gender based variation in desensitization ................................................................. 48 3.7 References ......................................................................................................................... 51 CHAPTER 4. CONCLUSION AND FUTURE DIRECTIONS ...................................................62 4.1 Conclusion ......................................................................................................................... 62 4.2 Future Directions ............................................................................................................... 65 v LIST OF FIGURES Page Figure 2-1 Global distribution of neglected tropical diseases. ....................................................... 6 Figure 2-2 Global distribution of lymphatic filariasis indicated in red. ......................................... 7 Figure 2-3 Adult female Brugia malayi in well plate. .................................................................... 9 Figure 2-4 Life cycle of Brugia malayi. ....................................................................................... 12 Figure 2-5 Structure of the nicotinic acetylcholine receptor. ...................................................... 13 Figure 3-1 A representative gel picture of whole worm PCR reveals the presence of different nAChR amplicon expression. .................................................................... 35 Figure 3-2 In vitro effects of levamisole on the motility of adult female B. malayi a dose response curve observed at the 3 minute time point following. ............................... 36 Figure 3-3 In vitro effects of levamisole on the motility of B. malayi following exposure to various concentrations of levamisole. ...................................................................... 38 Figure 3-4 Time-course of the motility of adult female B. malayi following exposure to 100 µM of levamisole. ................................................................................................... 39 Figure 3-5 Quantitative gene expression of nAChRs in female B. malayi after incubation for 4 h with 100 µM levamisole and when they were completely desensitized. Bma-unc-38 is significantly up-regulated. ............................................................... 40 Figure 3-6 Motility of adult female B. malayi versus time in the presence and absence of 100 µM levamisole. .................................................................................................. 41 Figure 3-7 B. malayi incubated in 100 µM levamisole for 1 h, showed significant upregulation of Bma-unc-29. .................................................................................... 42 Figure 3-8 Motility of adult B. malayi
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