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Investigating Calcium Channel Blockers As Antimalarials

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Investigating Calcium Channel Blockers As Antimalarials Investigating Calcium Channel Blockers as Antimalarials May Rajab School of Environment and Life Sciences University of Salford Salford, UK Submitted in Partial Fulfilment of the Requirements for the Degree of Doctor of Philosophy, March 2018 Declaration I certify that this thesis, submitted to the University of Salford in partial fulfilment of the requirements for a Degree of Doctor of Philosophy, is a presentation of my own research work and has been funded by the University of Salford’s Pathway to Excellence studentship. The content of this thesis has not been submitted for any degree or other purpose at this or any other university. Although the author has carried out most experiments and analysis of this research, some parts of this thesis were carried out in collaboration with other colleagues. Wherever contributions of others are involved, every effort is made to indicate this clearly with due reference to the literature and acknowledgment of collaborative research and discussions. Dr Steve Rossington carried out the synthesis of some of the fendiline analogues which have been clearly stated within the thesis. ii Table of contents Declaration .............................................................................................................................................. ii Table of contents ................................................................................................................................... iii List of figures ........................................................................................................................................ viii List of tables ............................................................................................................................................ x Acknowledgments .................................................................................................................................. xi Abbreviations ....................................................................................................................................... xiii Abstract ................................................................................................................................................ xvii Chapter 1 ................................................................................................................................................. 1 Introduction ............................................................................................................................................ 1 1.1 Global impact of malaria ............................................................................................................... 1 1.2 Biology of the parasite .................................................................................................................. 2 1.3 Clinical symptoms of malaria ........................................................................................................ 4 1.4 Malaria control and prevention .................................................................................................... 5 1.4.1 Vector control ........................................................................................................................ 6 1.4.2 Vaccines ................................................................................................................................. 7 1.4.3 Prophylaxis ............................................................................................................................. 8 1.5 Malaria chemotherapy .................................................................................................................. 9 1.5.1 Quinoline derivatives ............................................................................................................. 9 1.5.2 Antifolates ............................................................................................................................ 12 DHFR Inhibitors ......................................................................................................................... 13 DHPS Inhibitors ......................................................................................................................... 15 1.5.3 Artemisinins ......................................................................................................................... 16 1.5.4 Antibiotics and antiparasitic drugs ...................................................................................... 18 1.6 Antimalarial drug discovery ........................................................................................................ 20 1.6.1 Traditional drug development ............................................................................................. 20 1.6.2 Drug repositioning ............................................................................................................... 22 1.7 Calcium and calmodulin .............................................................................................................. 23 1.7.1 Importance of calcium ......................................................................................................... 23 1.7.2 Importance of calmodulin .................................................................................................... 26 1.8 Calcium channels and their blockers .......................................................................................... 28 iii 1.8.1 Voltage-gated calcium channels .......................................................................................... 28 1.8.2 Calcium channel blockers ..................................................................................................... 30 1.8.3 Fendiline ............................................................................................................................... 32 1.9 Aims and Objectives .................................................................................................................... 34 Chapter 2 ............................................................................................................................................... 36 Materials and methods ......................................................................................................................... 36 2.1 In vitro cultivation of P. falciparum ............................................................................................. 36 2.1.1 Parasite strains: .................................................................................................................... 36 2.1.2 Complete media: .................................................................................................................. 36 2.1.3 Wash media: ........................................................................................................................ 36 2.1.4 Processing human RBCs for parasite culture ....................................................................... 37 2.1.5 Maintaining parasite culture ................................................................................................ 37 2.1.6 Estimating parasitaemia....................................................................................................... 37 2.1.7 Preservation of parasites in liquid nitrogen ......................................................................... 38 2.1.8 Thawing of parasites from liquid nitrogen ........................................................................... 38 2.1.9 Culture synchronisation ....................................................................................................... 38 2.1.10 Percoll purification ............................................................................................................. 39 2.2 Drug susceptibility assays ........................................................................................................... 39 2.2.1 Optimising the haematocrit levels of SG plate reader assay ............................................... 39 2.2.2 Optimisation of media levels for SG plate reader assay ...................................................... 40 2.2.3 Dose-response assay ............................................................................................................ 41 2.2.4 Adopted SG plate reader method ........................................................................................ 41 2.2.5 SG flow cytometer method .................................................................................................. 42 2.2.6 Validating the SG plate reader method ............................................................................... 42 2.3 Plate reader optimisation results ................................................................................................ 43 2.3.1 Optimising the haematocrit levels of SG plate reader assay ............................................... 43 2.3.2 Optimisation of media levels for SG plate reader assay ...................................................... 44 2.3.3 Validating the SG plate reader method ............................................................................... 44 2.4 Cytotoxicity test on mammalian cells ......................................................................................... 46 2.4.1 Cell culture media ................................................................................................................ 46 2.4.2 Cultivation of HepG2 cells ...................................................................................................
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