Commit 9e297c11 authored by mk11g11's avatar mk11g11
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\chapter*{List of Abbreviations}
nAChR : nicotinic acetylcholine receptor \\
ECD : extracellular domain
ECD : extracellular domain \\
BWM: Body Wall Muscle \\
bgtx - bungarotoxin \\
RIC-3 - Resistant to Inhibitors of Cholinesterase-3 \\
GPCR - G protein coupled receptor \\
ChAt - Choline acetyltransferase \\
ACE - Acetylcholinesterase \\
VAChT - Vesicular acetylcholine transferase \\
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\addcontentsline{toc}{chapter}{Abstract}
\chapter*{Abstract}
An ability of insecticides to selectively target pests without affecting non-target species is a key determinant of success of componds used in agriculture. Neonicotinoids are a major class of insecticide which encompases seven chemical representing three distinct chemical classes, namely cyanoamidines, nitroguanidines and nitromethylenes. Neonicotinoids effectively control a wide range of insect pests and have low toxicity against mammals, however they can also negatively impact on non-target species of bees, threatening food safety. Neonicotinoids act by targeting insect nAChR, which are a major excitatory receptors in the insect central nervous system. Difficulties in heterologous expression of these proteins hinders their pharmacological characterisation and identification of molecular determinants of neonicotinoid-toxicity. This thesis describes the efforts into developing \textit{C. elegans}* as a platfom in which the mode of action and selective toxicity of neonicotinoid-insecticides can be studied.
We determined the effects of neonicotinoids on \textit{C. elegans} behaviours governed by the cholinergic neurotransmission. Cyanoamidines clothianidin, nitroguanidine thiacloprid and nitromethylene nitenpyram showed low efficacy on locomotion, pharyngeal pumping, egg-laying and egg-hatching of young adult wild-type \textit{C. elegans}. Exposure of mutant worm with enhanced cuticle permeability showed increased susceptibility of worms to all three neonicotinoids tested, suggesting an adult cuticle limits drug access. The role of the cuticle in neonicotinoids susceptibility was nvestigated in *C. elegans* cut-head preparation, in which the cuticle is removed and the effects of compounds on pharyngeal pumping are scored. Out of the three neonicotinoids applied, clothianidin showed the greatest efficacy; it stimulated pharyngeal pumping at $\geq$ 75 $\mu$M. The concentrations effective against the function of the pharynx are an order or magnitude lower than the residual, average concenntration in the soil, suggesting *C. elegans* is not impacted in the field, and at least 8-fold lower than lethal doses in insect-pests. The difference in neonicotinoid-susceptiility between adult \textit{C. elegans} and insects precludes the use of *C. elegans* pharynx as a platform for the mode of action studies, but highlights its potential as a suitable background for the heterologous expression of insect nAChRs.
Further experiments show that \textit{C. elegans eat-2} nAChR mutant is a suitable genetic background, in which the expression of heterologous nAChRs can be scored in behavioural and pharmacological assays. Expression of nAChR EAT-2 in the pharyngeal muscle rescued the blunted feeding phenotype and 5-HT insensitivity of the \textit{eat-2} mutant worm. Expression of exogenous receptor, viz. human $\alpha$7 in the pharynx of \textit{eat-2} mutant led to a cell-surface expression, as shown by staining with labelled $\alpha$-bungarotoxin. However the feeding and pharmacological phenotypes of the mutant were not rescued. \textit{C. elegans} strains in which human $\alpha$7 is expressed in the wild-type genetic background was also generated to determine whether the pharmacology of the human receptor can be inprinted on the \textit{C. elegans} pharynx. No difference in the phayngeal response to nAChR agonists cytisine, nicotine or acetylcholine were noted. The lack of apparent functionality of $\alpha$7 receptor could be due to the incorrect cellular localisation of this protein. $\alpha$-bgtx staining showed that the expression of $\alpha$7 receptor is concentrated to the specific cells of the pharyngeal muscle, however this localisation does not overlap with the localisation of native EAT-2 receptors. A transgenic strains in which exogenous proteins are expressed using EAT-2 native promoter should be made.
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\addcontentsline{toc}{chapter}{Acknowledgements}
\chapter*{Acknowledgements}
Throughout the process of PhD study, research and writing this thesis, I have received a great deal of support and help.
I would like to thank my supervisors: Dr Declan Doyle, Prof Vincent O’Connor and Prof Lindy Holden-Dye, whose expertise, knowledge, enthusiasm and motivation was invaluable. Without your guidance this PhD would have been unachievable.
I would like to acknowledge The Gerard Kerkut Charitable Trust for providing funding for this PhD project.
I am also very grateful to Prof Robert Walker, Prof Nullin Divecha and Dr James Dillon for their insightful comments and encouraging words.
My thanks go to Dr Alistair Bailey for his help with writing and formatting this thesis and for keeping me company throughout my studies.
Thank you to all members of the worm-lab and the lab of Dr Declan Doyle for all the fun we have experienced together. In particular, I would like to thank Dr Ben Yarnall, Dr Fernando Calahorro and Dr Anna Crisford for your support and technical help.
I am grateful to my all my family members who always believed in me.
Last but not least thank you to my partner, Damien Parsey for his continual emotional support, patience and understanding. I would not have made it without you.
......@@ -5,7 +5,7 @@ I, Monika Kudelska
declare that the thesis entitled:
Investigation of the selective toxicity of neonicotinoids using the nematode worm Caenorhabditis elegans % Change to dissertation title
Investigation of the selective toxicity of neonicotinoids using the nematode worm \textit {Caenorhabditis elegans} % Change to dissertation title
and the work presented in it are my own and has been generated by me as the result of my own original research.
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\pagestyle{plain}
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% Publications
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\addcontentsline{toc}{chapter}{Publications}
\chapter*{Publications}
Part of this thesis has been published in the following papers:
Kudelska, M. M., L. Holden-Dye, V. O’Connor, and D. A. Doyle. 2017. “Concentration-Dependent Effects
of Acute and Chronic Neonicotinoid Exposure on the Behaviour and Development of the Nematode
Caenorhabditis Elegans.” Journal Article. Pest Manag Sci 73 (7): 1345–51. https://doi.org/10.1002/ps.
4564.
Kudelska, M. M., A. Lewis, C. T. Ng, D. A. Doyle, L. Holden-Dye, V. M. O’Connor, and R. J. Walker.
2018. “Investigation of Feeding Behaviour in c. Elegans Reveals Distinct Pharmacological
and Antibacterial Effects of Nicotine.” Invertebrate Neuroscience : IN 18 (4): 14. https:
//doi.org/10.1007/s10158-018-0219-1.
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