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Summary Parasitic Nematodes Cause Summary Parasitic nematodes cause enormous public health, agricultural and economic problems worldwide, as pathogens of humans, livestock and crops. Their impact is increasing due to lack of full efficacy of current anthelmintics and development of resistance by the nematodes, therefore there is an urgent need for an alternative. Plant cysteine proteinases (CP) from papaya (“papain”), fig (ficin), and pineapple (bromelain) have been shown to be effective on gastrointestinal (GI) nematodes. The enzymes attack by digesting the cuticle leading to rupture and death of the nematode. The nematode cuticle is composed of collagens and cuticlins but the specific molecular target(s) of the proteinases have yet to be identified. There are about 160 collagen genes and 8 identified functional cuticlins genes in the C. elegans genome. This study identified some of the molecular targets and thereby began to define the mechanism of action of this new class of anthelmintics, through imaging, proteomic, immunohistochemical, and automated motility assay techniques using a free-living nematode, Caenorhabditis elegans and murine GI nematode Heligmosomoides bakeri as target organisms. Through proteomic approach, Col-87 and Cut-19 were identified as CP targets on C. elegans and H. bakeri cuticles respectively, cuticle globin and chemosensory protein that localise in the cuticle were also target for CPs. Immunohistochemical staining indicated that DPY-7 collagen is also a target for CPs on the cuticle of C. elegans. Imaging showed that there was a marked difference in degree of damage done to the two model worms used. H. bakeri was most affected by CPs as its entire cuticle was digested more rapidly when compared to the two strains of C. elegans. Motility of the three strains of C. elegans was affected by exposure to CPs, in a concentration- , time- and CP type-dependent manner. In papaya latex supernatant (PLS), there was no detectable statistically significant difference in susceptibility between wild-type and cystatin- null mutants. Papain affected the motility of worm types and was more effective than PLS. 1 CP's mechanism of attack on the nematode cuticle is by degrading the structural proteins, leading to loss of integrity, motility and finally death of the nematode. Keywords: C. elegans, H. bakeri, cysteine proteinases, papaya latex supernatant, proteomics, cuticle, motility, 2 Acknowledgements I wish to thank Tertiary Education Trust Fund and Enugu state University of Science and Technology Enugu, Nigeria, for their sponsorship. I also wish to thank my wife and kids for their love, support, patience and understanding throughout the programme, especially during the period of writing this thesis. I also wish to thank Mr Chukwuma John Martin Nwatu for his love and support. I wish also to thank immensely my supervisor, Dr. David Buttle for his support, encouragement and guidance from the beginning of my admission to the University of Sheffield to the completion of this thesis. I thank specially Prof. Mark Dickman for his advice and for allowing me to use their LC/MS/MS facility free of charge, I thank also Alison for assisting me during the MS work. I also wish to acknowledge the following people for their candid support and help: Dr. Ian Duce who helped me by providing advice on cuticle preparation and helping me use the scanning electron microscopy facility at Nottingham and, together with Dr. Andrew Phiri provided me with the C. elegans, Prof Jerzy Benhke for donating the H. bakeri, Ann Lowe for allowing me the use of the stereo microscope and other uncountable assistance in the lab. I also wish to acknowledge Dr. Iain Johnstone (School of Life Sciences University of Glasgow) for donating the DPY-7 mAB and the C. elegans strain MQ375. I also wish to thank Prof. David Sattelle and Dr. Freddie Partridge of University College London for advice and for allowing me use the worm watcher with charge. I thank also Tim, Oumo and Said who worked with me in the lab and helped to maintain the C. elegans culture. You guys are wonderful, thank you all. Also I wish to thank Fiona Wright and Kevin Oxley for their assistance, and the ever lovely Paula Blackwell for her care and support. I acknowledge the support from my fellow PhD students: Fawaz, Debo, Shola, and many others who encouraged me with good tension-killing jokes in one way or the other. 3 Contents Summary …………………………………………………………………………….……....1 Acknowledgement..……………………………………………………………………....….3 Table of contents………………………………………………………………………….….4 List of Figures……………………………………………………………………………….11 List of Tables………………………………………………………………………………...15 Abbreviations………………………………………………………………………………..17 Chapter 1 ....................................................................................................................... 19 1.1 General introduction ................................................................................................... 19 1.1.1 Overview ....................................................................................................................... 19 1.1.2 Concept of Parasitism ................................................................................................... 20 1.1.3 Helminths ...................................................................................................................... 20 1.1.4 Platyhelminths .............................................................................................................. 20 1.1.5 Trematoda ..................................................................................................................... 21 1.1.6 Cestodes ........................................................................................................................ 22 1.2 Nematoda ................................................................................................................... 23 1.2.1 Overview ....................................................................................................................... 23 1.2.2 Distribution and Life cycle of parasitic nematodes ....................................................... 24 1.2.3 Burden of nematode parasite infections ...................................................................... 26 1.2.4 Treatment of STHs and nematode resistance to anthelmintic ..................................... 30 1.3 Nematode body and functions ..................................................................................... 31 1.3.1 Structure and composition of nematode cuticle .......................................................... 32 1.3.2 Cuticle collagen ............................................................................................................. 34 1.3.3 Cuticlins ......................................................................................................................... 36 1.4 Proteolytic attack on nematode cuticles ...................................................................... 37 1.4.1 Overview ....................................................................................................................... 37 1.4.2 Enzymes involved in Cuticle destruction- Peptidases (proteolytic enzymes) ............... 37 1.4.3 Phylogeny of peptidases ............................................................................................... 37 4 1.5 Papaya latex ............................................................................................................... 38 1.5.1 Papain............................................................................................................................ 40 1.5.2 Chymopapain ................................................................................................................ 41 1.5.3 Caricain .......................................................................................................................... 41 1.5.4 Glycyl endopeptidase .................................................................................................... 41 1.5.5 Ficain ............................................................................................................................. 42 1.6 Activities of CPs on nematode cuticle ........................................................................... 42 1.7 Hypothesis .................................................................................................................. 47 1.7.1 Aims ............................................................................................................................... 48 2 Chapter 2: General methods, materials, optimizations and buffers .......................... 49 2.1 Materials .................................................................................................................... 49 2.1.1 List of Chemicals and reagents...................................................................................... 49 2.1.2 List of consumables: ...................................................................................................... 50 2.1.3 List of laboratory equipment ........................................................................................ 51 2.2 Animals, culture, maintenance and buffers .................................................................. 52 2.2.1 Caenorhabditis elegans ................................................................................................. 52 2.2.2 C. elegans culture and maintenance conditions ........................................................... 53 2.2.3 Preparation
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