An analysis of ethanol-induced behavioural plasticity in Caenorhabditis elegans

Ethanol is one of the most widely used and socially acceptable drugs in the world.However its chronic use can lead to serious problems including the development ofdependence. Alcohol dependence is a chronic, relapsing disorder characterised bytolerance, withdrawal, preoccupation with obtaining alcohol, loss of control over itsconsumption and impairment in social and occupational functioning. In humans thisdevelops over years, primarily driven by adaptations in many distinct signallingpathways and neural circuits as a result of continued heavy drinking. Whilst alcoholdependence has been extensively studied our understanding of how its distinct targetsintegrate to produce various behavioural responses remains far from clear.The nematode worm Caenorhabditis elegans is a model genetic organism with asimple nervous system and well-defined behaviour. These nematodes can displayplasticity in the form of tolerance to, and withdrawal from, 5-HT or nicotine. They arethus a genetically tractable system in which to investigate the neural substrates ofadaptive responses to ethanol. In this simple system the impact of changes at themolecular level on signalling in defined neural circuits and the resultant animalbehaviour can be investigated. The aims of this thesis were to establish a C. elegansparadigm for alcohol dependence and to use this to define the genetic basis of theethanol-dependent behaviours of intoxication, tolerance and withdrawal.Evidence was provided that ethanol equilibrates rapidly across the worm cuticleindicating that the internal concentration closely approximates to the externalconcentration in which the animal is placed. Ethanol-dependent behaviours werecarefully characterised using a variety of behavioural assays. C. elegans exhibitdistinct behavioural states, corresponding to intoxication and withdrawal, whichimpair the ability to navigate towards food. Visual and automated analysis defined asub-behaviour, an increased tendency to form spontaneous deep body bends, whichwas specifically associated with withdrawal. This was ameliorated by a low dose ofalcohol supporting the contention that it arises from ethanol-induced neuroadaptation.A series of loss of function mutants, were analysed for alterations in ethanoldependentbehaviour. The absence of withdrawal in a strain of worms depleted inneuropeptides (egl-3) demonstrated that peptidergic signalling is key to the chronicadaption to, but not to the acute effects of, ethanol. However the neuropeptidereceptor NPR-1, previously shown to impact on ethanol responses in C. elegans, hadno effect on withdrawal behaviour in these assays. Alterations in intoxication andwithdrawal behaviour in strains of worms depleted in 5-HT (tph-1) and dopamine(cat-2) indicated that serotonergic and dopaminergic signalling may also be involvedin the ethanol response in C. elegans. This study has therefore provided a quantitativeanalysis of distinct ethanol-induced behavioural states and highlighted a role forneuropeptides and major classes of neuromodulatory transmitters. In particular thisdata is consistent with the emerging role of neuropeptides in ethanol withdrawal.<br/

Molecular imprinting of glycoprotein recognition sites using surface initiated radical polymerization

Since the importance of glycoproteins and their role in the development of diseases are becoming increasingly recognised there is a growing need for highly sensitive and selective glycoprotein recognition platforms. Herein, a novel molecularly imprinted glycoprotein sensor that displays a high affinity for its target glycoprotein was developed using surface-initiated radical polymerisation. The sensor was developed by first fabricating a suitable self-assembled monolayer (SAM) that was then used as the foundation from which the polymerisation procedure to later be used for the imprinting procedure was developed. Particular focus was given to establishing control over the polymerisation reaction in order to optimise the thickness of the polymer layer to the desired depth. Alongside these investigations, complexation studies aimed at elucidating the binding of a functional boronic acid monomer to the model glycoprotein, RNase B, were undertaken using mass spectrometry. Here, we aimed to optimise the binding conditions to encourage the monomer ligand to bind the glycoprotein, whilst also ensuring that the protein remained stable in these conditions. Finally, we brought together these studies to then form imprints for RNase B. Several imprints were fabricated and then extensively characterised, following which surface plasmon resonance (SPR) was used to examine their binding affinities towards the RNase B target and control glycoproteins

Precise generation of selective surface-confined glycoprotein recognition sites

Since glycoproteins have become increasingly recognized as key players in a wide variety of disease processes, there is an increasing need for advanced affinity materials for highly selective glycoprotein binding. Herein, for the first time, a surface-initiated controlled radical polymerization is integrated with supramolecular templating and molecular imprinting to yield highly reproducible synthetic recognition sites on surfaces with dissociation constants (KDs) in the low micromolar range for target glycoproteins and minimal binding to non-target glycoproteins. Importantly, it is shown that the synthetic strategy has remarkable ability to distinguish the glycosylated and non-glycosylated forms of the same glycoprotein, with >5-fold difference in binding affinity. The precise control over the polymer film thickness and positioning of multiple carbohydrate receptors plays a crucial role in achieving enhanced affinity and selectivity. The generated functional materials of unprecedented glycoprotein recognition performance open up a wealth of opportunities in the biotechnological and biomedical fields

A Randomized, Crossover Study of the Acute Cognitive and Cerebral Blood Flow Effects of Phenolic, Nitrate and Botanical Beverages in Young, Healthy Humans

Background: In whole foods, polyphenols exist alongside a wide array of other potentially bioactive phytochemicals. Yet, investigations of the effects of combinations of polyphenols with other phytochemicals are limited. Objective: The current study investigated the effects of combining extracts of beetroot, ginseng and sage with phenolic-rich apple, blueberry and coffee berry extracts. Design: This randomized, double-blind, placebo-controlled crossover design investigated three active beverages in 32 healthy adults aged 18&ndash;49 years. Each investigational beverage comprised extracts of beetroot, ginseng and sage. Each also contained a phenolic-rich extract derived from apple (containing 234 mg flavanols), blueberry (300 mg anthocyanins) or coffee berry (440 mg chlorogenic acid). Cognition, mood and CBF parameters were assessed at baseline and then again at 60, 180 and 360 min post-drink. Results: Robust effects on mood and CBF were seen for the apple and coffee berry beverages, with increased subjective energetic arousal and hemodynamic responses being observed. Fewer effects were seen with the blueberry extract beverage. Conclusions: Either the combination of beetroot, ginseng and sage was enhanced by the synergistic addition of the apple and coffee berry extract (and to a lesser extent the blueberry extract) or the former two phenolic-rich extracts were capable of evincing the robust mood and CBF effects alone