Capillary electrophoresis with boron doped diamond electrode for amperometric detection of low molecular weight biological substrate

The research work in this thesis included the sensitive and selective separation of biological substance by capillary electrophoresis with a boron doped diamond electrode for amperometric detection. Chapter 1 introduced the capillary electrophoresis and electrochemical detection. It included the different modes of capillary electrophoresis, polyelectrolyte multilayers coating for open tubular capillary electrochromatography, different modes of electrochemical detection and carbon based electrodes. Chapter 2 showed the synthesized and electropolymerized N-acetyltyramine with a negatively charged sulfobutylether-β-cyclodextrin on a boron doped diamond (BDD) electrode followed by the electropolymerzation of pyrrole to form a stable and permselective film for selective dopamine detection. For comparison, a glassy carbon (GC) electrode with a combined electropolymerized permselective film of polytyramine and polypyrrole-1-propionic acid was used for selective detection of dopamine. The detection limit of dopamine was improved from 100 nM at a GC electrode to 5 nM at a BDD electrode. Chapter 3 showed field-amplified sample stacking using a fused silica capillary coated with gold nanoparticles embedded in poly(diallyldimethylammonium) chloride, which has been investigated for the electrophoretic separation of indoxyl sulphate, homovanillic acid and vanillylmandelic acid. The detection limit of the three analytes obtained by using a boron doped diamond electrode was around 75 nM, which was significantly below their normal physiological levels in biological fluids. This combined separation and detection scheme was applied to the direct analysis of these analytes and other interfereing chemicals including uric and ascorbic acids in urine samples without off-line sample treatment or preconcentration. Chapter 4 showed the selective detection of Pseudomonas Quinolone Signal, PQS for quorum sensing from its precursor HHQ, using a simply boron doped diamond electrode. Furthermore, by combining poly(diallyldimethylammonium) chloride modified fused silica capillary with a BDD electrode for amperometric detection, PQS was separated from HHQ and other analogues. The detection limit of PQS was as low as 65 nM. Different P. aeruginosa mutant strains were studied. Chapter 5 showed the separation of aminothiols by layer-by-layer coating of silica capillary with a boron doped diamond electrode. The capillary was layer-by-layer coated with the polycation poly(diallyldimethylammonium) chloride and negatively charged silica nanoparticles. All the aminothiols was separated and detected using a BDD electrode in an acidic electrolyte. It was a novel scheme for the separation and detection of glutathione reduced and oxidized forms, which is important for estimated overstressed level in the human system.

The purification and characterization of the high and low molecular weight forms of rabbit intestinal adenosine deaminase

Accepted Version

The gut microbiota as a contributing factor to antipsychotic-induced weight gain and metabolic dysfunction

Schizophrenia represents one of the world’s most devastating illnesses due to its often lifelong course and debilitating nature. The treatment of schizophrenia has vastly improved over recent decades with the discovery of several antipsychotic compounds; however these drugs are not without adverse effects that must be addressed to maximize their therapeutic value. Newer, atypical, antipsychotics are associated with a compilation of serious metabolic side effects including weight gain, insulin resistance, fat deposition, glucose dysregulation and ensuing co-morbidities such as type II diabetes mellitus. The mechanisms underlying these side effects remain to be fully elucidated and adequate interventions are lacking. Further understanding of the factors that contribute these side effects is therefore required in order to develop effective adjunctive therapies and to potentially design antipsychotic drugs in the future with reduced impact on the metabolic health of patients. We investigated if the gut microbiota represented a novel mechanism contributing to the metabolic dysfunction associated with atypical antipsychotics. The gut microbiota comprises the bacteria that exist symbiotically within the gastrointestinal tract, and has been shown in recent years to be involved in several aspects of energy balance and metabolism. We have demonstrated that administration of certain antipsychotics in the rat results in an altered microbiota profile and, moreover, that the microbiota is required for the full scale of metabolic dysfunction to occur. We have further shown that specific antibiotics can attenuate certain aspects of olanzapine and risperidone–induced metabolic dysfunction, in particular fat deposition and adipose tissue inflammation. Mechanisms underlying this novel link appear to involve energy utilization via expression of lipogenic genes as well as reduced inflammatory tone. Taken together, these data indicate that the gut microbiota is an important factor involved in the myriad of metabolic complications associated with antipsychotic therapy. Furthermore, these data support the future investigation of microbial-based therapeutics for not only antipsychotic-induced weight gain but also for tackling the global obesity epidemic.

Impact of the smoke-free workplace legislation on smoking behaviour, risk perception & stigmatisation

The Republic of Ireland became the first European country to implement nationwide smoke-free workplace legislation. Aims: To determine prevalence of smoking among bar workers and estimate the impact of the smoke-free workplace legislation on their smoking behaviour to that of a comparable general population sample. To approximate the influence of tobacco control measures on risk perception of second-hand smoke (SHS) among the general population. To explore the de-normalisation of smoking behaviour and the potential increased stigmatisation of smokers and their smoking. Methods: Prevalence estimates and behavioural changes were examined among a random sample of bar workers before and 1 year after the smoke-free legislation; comparisons made with a general population sub-sample. Changes in risk knowledge related to SHS exposure were based on general population data. Qualitative interviews were conducted among a purposive sample of smokers and non-smokers four years after the implementation of the legislation. Results: Smoking prevalence was extremely high among bar workers. Smoking prevalence dropped in bar workers and significantly among the general population 1 year post ban while cigarette consumption dropped significantly among bar workers. Disparity in knowledge between smokers and non-smoker of risk associated with SHS exposure reduced. Lack of understanding of the risk of ear infections in children posed by SHS exposure was notable. Evidence for advanced de-normalisation of smoking behaviour and intensification of stigma because of the introduction of the legislation was dependent on many factors, quality of smoking facilities played a key role. Conclusions: Ireland’s smoke-free legislation was associated with a drop in prevalence and cigarette consumption. Disparity in knowledge between smokers and non-smokers of the risk posed by SHS exposure reduced however the risk of ear infections in children needs to be effectively disseminated. The proliferation of ‘good’ smoking areas may diminish the potential to reduce smoking behaviour and de-normalise smoking.

Rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultra-fast performance liquid chromatography

The research work in this thesis reports rapid separation of biologically important low molecular weight compounds by microchip electrophoresis and ultrahigh liquid chromatography. Chapter 1 introduces the theory and principles behind capillary electrophoresis separation. An overview of the history, different modes and detection techniques coupled to CE is provided. The advantages of microchip electrophoresis are highlighted. Some aspects of metal complex analysis by capillary electrophoresis are described. Finally, the theory and different modes of the liquid chromatography technology are presented. Chapter 2 outlines the development of a method for the capillary electrophoresis of (R, S) Naproxen. Variable parameters of the separation were optimized (i.e. buffer concentration and pH, concentration of chiral selector additives, applied voltage and injection condition).The method was validated in terms of linearity, precision, and LOD. The optimized method was then transferred to a microchip electrophoresis system. Two different types of injection i.e. gated and pinched, were investigated. This microchip method represents the fastest reported chiral separation of Naproxen to date. Chapter 3 reports ultra-fast separation of aromatic amino acid by capillary electrophoresis using the short-end technique. Variable parameters of the separation were optimized and validated. The optimized method was then transferred to a microchip electrophoresis system where the separation time was further reduced. Chapter 4 outlines the use of microchip electrophoresis as an efficient tool for analysis of aluminium complexes. A 2.5 cm channel with linear imaging UV detection was used to separate and detect aluminium-dopamine complex and free dopamine. For the first time, a baseline, separation of aluminium dopamine was achieved on a 15 seconds timescale. Chapter 5 investigates a rapid, ultra-sensitive and highly efficient method for quantification of histamine in human psoriatic plaques using microdialysis and ultrahigh performance liquid chromatography with fluorescence detection. The method utilized a sub-two-micron packed C18 stationary phase. A fluorescent reagent, 4-(1-pyrene) butyric acid N-hydroxysuccinimide ester was conjugated to the primary and secondary amino moieties of histamine. The dipyrene-labeled histamine in human urine was also investigated by ultrahigh pressure liquid chromatography using a C18 column with 1.8 μm particle diameter. These methods represent one of the fastest reported separations to date of histamine using fluorescence detection.

The Ford administration, Angola and the perception of Post-Vietnam US credibility, 1974-1976

This thesis is a study of how the Gerald Ford administration struggled to address a perceived loss of US credibility after the collapse of Vietnam, with a focus on the role of Secretary of State Henry Kissinger in the formulation, implementation and subsequent defence of US Angolan policy. By examining the immediate post-Vietnam period, this thesis shows that Vietnam had a significant impact on Kissinger’s actions on Angola, which resulted in an ill conceived covert operation in another third world conflict. In 1974, Africa was a neglected region in Cold War US foreign policy, yet the effects of the Portuguese revolution led to a rapid decolonization of its African territories, of which Angola was to become the focus of superpower competition. After South Vietnam collapsed in April 1975, Kissinger became fixated on restoring the perceived loss of US prestige, Angola provided the first opportunity to address this. Despite objections from his advisors, Kissinger methodically engineered a covert program to assist two anti-Marxist guerrilla groups in Angola. As the crisis escalated, the media discovered the operation and the Congress decided to cease all funding. A period of heated tensions ensued, resulting in Kissinger creating a new African policy to outmanoeuvre his critics publicly, while privately castigating them to foreign leaders. This thesis argues that Kissinger’s dismissal of internal dissent and opposition from the Congress was influenced by what he perceived as bureaucrats being affected by the Vietnam syndrome, and his obsession with restoring US credibility. By looking at the private and public records – as expressed in government meetings and official reports, US newspaper and television coverage and diplomatic cables – this thesis addresses the question of how the lessons of Vietnam failed to influence Kissinger’s actions in Angola, but the lessons of Angola were heavily influential in the construction of a new US-African policy.