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Sokolova Phd 2011 University of Dundee DOCTOR OF PHILOSOPHY Nitroaromatic pro-drug activation and resistance in the African trypanosome Sokolova, Antoaneta Y. Award date: 2011 Link to publication General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Download date: 28. Sep. 2021 DOCTOR OF PHILOSOPHY Nitroaromatic pro-drug activation and resistance in the African trypanosome Antoaneta Y. Sokolova 2011 University of Dundee Conditions for Use and Duplication Copyright of this work belongs to the author unless otherwise identified in the body of the thesis. It is permitted to use and duplicate this work only for personal and non-commercial research, study or criticism/review. You must obtain prior written consent from the author for any other use. Any quotation from this thesis must be acknowledged using the normal academic conventions. It is not permitted to supply the whole or part of this thesis to any other person or to post the same on any website or other online location without the prior written consent of the author. Contact the Discovery team ([email protected]) with any queries about the use or acknowledgement of this work. Nitroaromatic pro-drug activation and resistance in the African trypanosome ANTOANETA Y. SOKOLOVA PHD THESIS September 2011 Supervisor: Professor Alan H. Fairlamb Division of Biological Chemistry and Drug Discovery UNIVERSITY OF DUNDEE I Table of contents LIST OF FIGURES.....................................................................................................................................IV LIST OF TABLES ....................................................................................................................................... V LIST OF ABBREVIATIONS ........................................................................................................................VI ACKNOWLEDGEMENTS...........................................................................................................................IX DECLARATION......................................................................................................................................... X ABSTRACT..............................................................................................................................................XI CHAPTER 1 - INTRODUCTION ............................................................................................................ 1 1.1. HUMAN AFRICAN TRYPANOSOMIASIS AND THE AFRICAN TRYPANOSOME .............................- 2 - 1.1.1. Trypanosoma brucei – the aetiological agent ................................................................. - 3 - 1.1.2. Transmission and life cycle of T. brucei.......................................................................... - 4 - 1.1.3. Clinical manifestation and diagnosis of HAT................................................................ - 10 - 1.2. CHEMOTHERAPY OF SLEEPING SICKNESS .............................................................................- 14 - 1.2.1. Drugs against first-stage HAT....................................................................................... - 15 - 1.2.2. Drugs against second-stage HAT.................................................................................. - 16 - 1.2.3. Potential novel chemotherapeutics for the treatment of HAT ....................................... - 20 - 1.3. NITROAROMATIC PRO-DRUGS ..............................................................................................- 20 - 1.3.1. A historical overview..................................................................................................... - 20 - 1.3.2. Mode of action and activation of nitroaromatic drugs.................................................. - 24 - 1.3.3. Resistance to nitroaromatic drugs in trypanosomes ..................................................... - 27 - 1.4. PROJECT AIMS ......................................................................................................................- 28 - CHAPTER 2 - MATERIALS AND METHODS................................................................................... 29 2.1. MATERIALS..........................................................................................................................- 30 - 2.1.1. Analysis of nitroaromatic compounds ........................................................................... - 30 - 2.2. MOLECULAR BIOLOGY .........................................................................................................- 31 - 2.2.1. Isolation of genomic DNA from T. brucei ..................................................................... - 31 - 2.2.2. DNA amplification......................................................................................................... - 31 - 2.2.3. Agarose gel electrophoresis and extraction of DNA ..................................................... - 32 - 2.2.4. Cloning of PCR products .............................................................................................. - 32 - 2.2.5. Transformation of competent E. coli cells..................................................................... - 33 - 2.2.6. Purification of plasmid DNA......................................................................................... - 33 - 2.2.7. DNA sequencing............................................................................................................ - 34 - 2.2.8. Digestion of plasmid DNA with restriction endonucleases ........................................... - 34 - 2.2.9. Ligation ......................................................................................................................... - 35 - 2.2.10. Replacement of the thrombin recognition site in pET43.1c_TbNTRΔ83....................... - 35 - 2.2.11. Southern analysis of genomic DNA............................................................................... - 36 - 2.3. EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS .............................................- 37 - 2.3.1. Expression of recombinant TbNTR ............................................................................... - 37 - 2.3.2. Preparation of cell-free bacterial lysates...................................................................... - 38 - 2.3.3. Immobilised metal affinity chromatography (IMAC) .................................................... - 38 - II 2.3.4. Ion exchange chromatography...................................................................................... - 39 - 2.3.5. Size exclusion chromatography..................................................................................... - 39 - 2.3.6. Other chromatographic methods................................................................................... - 40 - 2.3.7. Batch affinity chromatography...................................................................................... - 40 - 2.3.8. Buffer exchange and sample concentration................................................................... - 41 - 2.3.9. Proteolytic cleavage of affinity tags .............................................................................. - 41 - 2.3.10. Protein purification under denaturing conditions......................................................... - 42 - 2.3.11. Expression and purification of recombinant EcNfsB .................................................... - 43 - 2.3.12. Cell-free expression of recombinant TbNTR ................................................................. - 43 - 2.4. PROTEIN DETECTION AND CHARACTERISATION....................................................................- 44 - 2.4.1. Protein quantification.................................................................................................... - 44 - 2.4.2. SDS-PAGE .................................................................................................................... - 44 - 2.4.3. Western blotting ............................................................................................................ - 45 - 2.4.4. Protein fingerprinting analysis...................................................................................... - 45 - 2.4.5. Nitroreductase activity in bacterial lysates................................................................... - 46 - 2.4.6. Disc diffusion test.......................................................................................................... - 47 - 2.5. CELL CULTURE OF T. BRUCEI................................................................................................- 47 - 2.5.1. Cell lines and culture conditions................................................................................... - 47 - 2.5.2. Cell density measurements ...........................................................................................
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