corrections

index.Rmd

@@ -2,7 +2,7 @@
 # YAML for thesis template
 site: bookdown::bookdown_site
 documentclass: book
-bibliography: [bib/packages.bib, bib/papers.bib, bib/august.bib] # Changes to reference bib files as needed
+bibliography: [bib/packages.bib, bib/papers.bib, bib/march.bib] # Changes to reference bib files as needed
 biblio-style: apalike
 toc: False # Table of contents supplied from tex files
 lof: False # List of figures supplied from tex files

tex-files/abbreviations.tex

 \addcontentsline{toc}{chapter}{List of Abbreviations}
 \chapter*{List of Abbreviations}
 
-nAChR : nicotinic acetylcholine receptor \\
-ECD : extracellular domain \\
-BWM: Body Wall Muscle \\
+5-HT - 5-hydroxytryptamine \\
+ACE - Acetylcholinesterase \\
+AChBP - Acetylcholine Binding Protein \\
 bgtx - bungarotoxin \\
-RIC-3 - Resistant to Inhibitors of Cholinesterase-3 \\
-GPCR - G protein coupled receptor \\
+Bovine Serum Albumin (BSA) \\
+e. coli 
+polyvinylidene difluoride (PVDF)
+C. elegans
+GLIC - \textit{Gloebacter} ligand gated ion channels \\
+Green Fluorescent Protein (GFP) \\
+BWM - Body Wall Muscle \\
+Ch - choline \\
 ChAt - Choline acetyltransferase \\
-ACE - Acetylcholinesterase \\
-VAChT - Vesicular acetylcholine transferase \\
\ No newline at end of file
+dimethyl sulfoxide (DMSO) \\
+ECD - extracellular domain \\
+E/R ratio
+ELIC - \textit{Erwinia} ligand gated ion channels
+GABA - Gamma aminobutyric acid \\
+GPCR - G protein coupled receptor \\
+nAChR : nicotinic acetylcholine receptor \\
+NGM - nematode growth medium \\
+RIC-3 - Resistant to Inhibitors of Cholinesterase-3 \\
+TM - transmembrane 
+VAChT - Vesicular acetylcholine transferase \\
+MBP - Maltose Binding Protein
\ No newline at end of file

tex-files/abstract.tex

 \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. 
+An ability of insecticides to selectively target pests without affecting non-target species is key determinant of success of compounds used in agriculture. Neonicotinoids which encompass seven different types of chemical representing three distinct chemical classes, namely the cyanoamidines, nitroguanidines and nitromethylenes, are a major class of insecticides. They 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 nicotinic acetylcholine receortors (nAChRs), which are major excitatory receptors in the insect central nervous system. Difficulties in heterologous expression of these proteins hinders their pharmacological characterisation and identification of the molecular determinants of neonicotinoid-toxicity. This thesis describes efforts into developing \textit{Caenorhabditis elegans (C. elegans)} as a platform 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. 
+We determined the effects of neonicotinoids on \textit{C. elegans} behaviours governed by the cholinergic neurotransmission. The cyanoamidine represented by clothianidin, the nitroguanidine represented by thiacloprid and the nitromethylene represented by 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 cuticular 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 investigated in \textit{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 (18.75 ppm). Generally, the concentrations effective against the function of the pharynx are an order or magnitude lower than the residual, average concentration of neonicotinoids in the soil, suggesting \textit{C. elegans} is not impacted in the field, and at least several fold lower than lethal doses in insect-pests. The difference in neonicotinoid-susceptibility between adult \textit{C. elegans} and insects precludes the use of \textit{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. 
\ No newline at end of file
+Further experiments showed that \textit{C. elegans eat-2} nAChR mutant is a suitable genetic background, in which the expression of heterologous nAChRs can be scored. Expression of \textit{C. elegans} nAChR EAT-2 in the pharyngeal muscle rescued the blunted feeding phenotype and 5-HT insensitivity of the \textit{eat-2} mutant. Expression of the exogenous receptor, 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} strain 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 imposed 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 $\alpha$7 receptor is expressed in the specific cells of the pharyngeal muscle, however this localisation does not overlap with the localisation of native EAT-2 receptors. A transgenic strain in which exogenous proteins are expressed using EAT-2 native promoter should be made. 
\ No newline at end of file

tex-files/publication.tex

@@ -3,12 +3,6 @@
 
 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., 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 \textit{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.
\ No newline at end of file
+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 \textit{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.
\ No newline at end of file

tex-files/uhead.sty

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thesis-dissertaion.log

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