The emergence of multidrug resistant Gram-negative bacteria such as Pseudomonas aeruginosa and Acinetobacter baumannii has been recognized worldwide and some clinical isolates of these bacteria are now resistant to most of the antibiotics currently available. Unfortunately, there are at present not enough new antimicrobial drugs being produced by the pharmaceutical industry to keep pace with the continuing development of antibiotic resistance. Colistin is an old antibiotic and in the form of colistin methanesulphonate sodium (CMS) has re-emerged as a major treatment for burn patients colonised with Acinetobacter baumannii. However, this antibiotic can lead to profound toxicity and for this reason the blood levels of CMS should be carefully monitored. Unfortunately the methods available for measuring it in serum are not robust and are not able to differentiate between the two forms of colistin i.e. colistin sulphate (CLS) and CMS. In this study the chemical (Thin Layer Chromatography and High Performance Liquid Chromatography) and microbiological methods for CLS and CMS analysis were investigated to develop a methodology for reproducible quantification of CLS and CMS in water or serum. Since CMS in aqueous solution has the potential to hydrolyze to produce a complex mixture of colistin sulphomethylated derivatives as well as colistin base, the optimized chemical and microbiological methods were used to determine the degradation of CLS or CMS in aqueous solution and serum. The bacteriostatic and bactericidal activity of CLS and CMS were probed by calculations of minimum inhibitory concentrations and time survivor studies. Depending on their concentrations, both antibiotics were found to exhibit bacteriostatic and bactericidal properties against a range of Gram-negative bacteria. Membrane damage caused by both forms of colistin was investigated using Acinetobacter lwoffii R46383 anddetennining intracellular potassium leakage and 260nm absorbing materials leakage. CLS was shown to cause substantial membrane damage, indicated by rapid, gross potassium leakage, while the effect of CMS on the membrane appeared to be more subtle, with cells exhibiting a more concentration dependent loss of potassium. Adsorption isotherms ofCLS gave results that were indicative of high affmity isotherm (H- shape), while adsorption isotherms of CMS were indicative of co-operative sorption (S- shape). Investigations into CLS and CMS cytotoxicity were performed using normal rat kidney (NRK-52E) cell line; the investigations revealed that neither forms of colistin has a major adverse effect on the rat renal cells even at concentrations higher than the therapeutic doses.

Ageing is one of the leading health problems faced by society today. An understanding of the fundamental mechanisms of ageing has the potential to provide preventative therapies for multiple degenerative diseases. Drosophila melanogaster is a powerful model organism in which to study the ageing process because the rate of ageing in fly populations is easy to manipulate by either genetic or environmental means. Drosophila are also inexpensive, short lived, easy to collect and can be raised in large numbers (allowing sex and age- specific effects to be determined). Although a large body of data exists m mammals linking ageing and age-related degeneration to the accumulation of molecular cross-links such as advanced glycation end- products (AGEs) prior to the work presented in this thesis only one paper had studied such cross links in Drosophila. Accordingly I hypothesised: [1] That acid stable chemical species existed within Drosophila melanogaster (of which AGE-like entities represent a subset) • .• 1 I,. [2] That such chemical species !represented the stable products of damage to macromolecules within Drosophila' melanogaster and as such had the potential to play either a causal or a correlative role in the ageing process within this organism. To pursue this line of investigation it was first necessary to develop novel analytical methods to investigate the accumulation of damaged compounds in flies. Simple preparative techniques were developed to produce digests of whole Drosophila melanogaster for use in three dimensional (3D) fluorimetry, H NMR spectrometry and mass spectrometry. My initial data clearly indicated the presence of species which either increased or were lost with advancing population age. If they played a role in ageing then interventions that altered the rate of this process would be expected to alter the rates of accumulation or disappearance of these species. Accordingly, environmental (DR and low temperature) and genetic interventions (mutated IIS activity) were used to alter the rates of ageing of multiple cohorts of Drosophila. Populations subject to interventions which slowed ageing rates also showed a reduced rate of accumulation of signals consistent with damage (putative advanced glycation end products) as compared to cohorts under normal conditions. IH NMR spectrometry and mass spectrometry also revealed distinct age associated spectral changes. 3 My recognition that AGE-like molecules had the potential to activate Phase II detoxification mechanisms, together with the body of data on chemical changes within ageing flies that I generated using my novel techniques allowed the first critical test of the broad spectrum detoxification hypothesis of ageing (sometimes known as the "Green Theory" of ageing). The results I obtained were entirely consistent with the predictions of this theory. In conclusion, my work represents the first application of a range of analytical techniques to identify and quantify compounds associated with, and possibly causing, different rates of ageing in Drosophila melanogaster. These techniques will facilitate the identification of novel compounds that either increase or decrease during ageing in this organism and will improve our understanding of the ageing process in this key model system.

Acute respiratory distress syndrome (ARDS) is an inflammatory disease in humans where no satisfactory therapeutic options exist. Regulation of the immune system by neurotransmitters has been identified as a possible exploitable strategy to treat inflammatory conditions. Antidepressant drugs have been found to have secondary actions including anti-inflammatory activity in a wide range of disease states in vivo. The cellular effects of these drugs that contribute to this effect have yet to be elucidated. The aim was to examine whether anti-depressants have a direct effect on the immune system or an effect on the target tissue such as lung cells. This thesis utilised both primary lung epithelium cells along with the cell lines A549, 16HBE14o- and BETIY02. The macrophage cell line RAW 264.7 was used to determine effects of anti-depressants on immune cells. Mitochondrial function was used as a measure of cell viability while morphological techniques were used to determine levels of apoptosis and necrosis. Cellular cytokine release was used as a determination of inflammation. Nitric oxide production by activated immune cells was measured using the nitrite assay and the expression of inducible nitric oxide synthase measured by Western blot. The BETIY02 cell line was found to have the most similar characteristics to primary lung epithelium cells and could be used as an effective substitute for large-scale screening and mechanistic studies especially in inflammation. Fluoxetine and desipramine reduced the inflammatory signals coming from lung cells exposed to lipopolysaccharide (LPS). In addition to this they proved effective in reducing immune cell activation as evidenced by reduced NO release following LPS exposure via a mechanism of inhibiting iN OS protein expression. Fluoxetine and desipramine exhibited anti-inflammatory effects on both the lung cells and the immune cells, identifying possible therapeutic potential for ARDS.

Nanomaterials are generally defined as chemical substances or materials that contain particles with one or more dimensions less tl1an 100 nanometres in size. They may be either engineered or naturally occurring, but have unique properties due to a vastly increased surface area to volume ratio when compared to non-nano (bulk) materials. This provides the potential for the development of a wide range of enhanced formulations with superior efficacy including applications in oral health care .. As the properties of a material change at the nano-scale, there are concerns that the toxicological profile of these materials may also change. Size is only one factor; changes in shape, surface chemistry, chemical composition, porosity and solubility all contribute to the overall biological toxicity profile of a nano-scale ingredient. Established links between the specific properties of a nanomaterial and toxicity are not well understood, leaving an important data gap in the literature. The purpose of this work was to utilise in vitro oral epithelial models for the assessment of safety profiles of nanomaterials for applications in next generation oral care products. Four commercially sourced nanomaterials were analysed, alongside respective bulk counterparts already found within oral care product formulations. These nanomaterials comprised of two nano-zinc oxides (ZnO), silicon dioxide (SiOz), titanium dioxide (TiOz) and hydroxyapatite (Cas(OH)(P04)'). Comprehensive characterisation of each material was carried out using a range of analytical techniques to identify any structure-function relationships in vitro. Initial toxicity screening experiments were conducted using a non-keratinised oral epithelial cell monolayer (H376 cell line) with both cell viability and lysis analysed using MTT and LDH assays respectively. Materials were investigated further using two 3-dimensional tissue models representative of the main tissue types constituting the human oral mucosa: non-keratinised buccal (RHO) and keratinised gingival (GIN-lOO) models. Nanomaterial uptake in the models was investigated using confocal microscopy with a styrl dye (FM 1-43). This led to the development of a novel, high throughput fluorescent assay as a potential method for screening nanoparticle-uptake. Results highlighted the complexities involved with nano-characterisation in biological media using current techniques. A wide variety of particle shapes and sizes were recorded between different nanomaterials, with results being dependent upon the sample preparation steps and specific methods of analyes used. These disparities represent the current challenges experienced by both researchers and regulators of nanotechnology at the present time. ZnO \vas observed to be the most cytotoxic material during monolayer screening, at concentrations exceeding 0.3125% w/v when delivered in protein-free media. Differences between bulk and nanomaterial properties were recorded for all the materials, except for Ti02, but these did not necessarily transfer to effects seen in the more representative 3-D models. Cytotoxicity results from both R1--10 and GIN-lOO models exemplified the disparity between sensitivity of monolayer and the natural stratified tissue structure of human oral mucosa. Keratinised gingival tissue models showed slgnificantly greater durability over the less robust buccal model, in both cytotoxicity assays and IL- lcx cytokine response. Of all materials examined, cellular uptake was only observed for nano-Si02. This was the only material detected trafficking inside the cell using the FM 1-43 styryl dye assay, with confocal data serving to verify the analysis of nanoparticle Internalisation using fluorescence. In conclusion, nanomaterials pose considerable difficulties during formulation and analysis in health care products. The risk of potential uptake and bioaccumulation or translocation to particularly sensitive areas of the body also requires further investigation. Nanomaterials have to be assessed on a case by case basis, and robust/consistent regulatory strategies developed to enable industry to produce and market novel but safe nanoparticle containing formulations. Risks to human health may be less of a hazard when applied to fully functioning healthy human tissue, especially in comparison to existing bulk material effects and current, accepted irritant ingredients (e.g. Sodium lauryl sulphate).

The aim of this thesis is to identify concepts of human occupation within the discourses surrounding Crisis Resolution Home Treatment Teams. The scope of the research covers specific Government policy and research related to the implementation of Crisis Resolution Home Treatment Teams in the United Kingdom, exploring theories of human occupation with the intent of proposing an additional perspective of mental health crisis. Drawing on Michel Foucault's ideas of governmentality and the influences of discourses within historical and political contexts, articles, research reports and government policy have been investigated. Documents reflecting the national context of a number of stakeholders and types of knowledge were subjected to a discourse analysis, mapping the development of mental health crisis as a discursive formation. The main findings in Part One identify how overarching discourses of neo-liberal ideas privileges economic and managerial discursive practices in shaping the remit and measurement of efficacy of this service provision. As a result, these discourses influence the focus of research onto the economic efficacy of Crisis Teams and their role in performing a gatekeeper function to hospital admission. Part Two proposes that concepts of human occupation are implicit within recent Government policy strategies, and posits an additional perspective for exploring how concepts of human occupation manifest themselves in relation to other discourses and through the mode of govern mentality. Links developed between policy rhetoric and service user discourse suggest an additional paradigm for the definition of crisis which draws upon the concepts of human occupation. Future research that can investigate mental health crisis from additional perspectives will address power relations in mental health crisis response and redress the balance of a current economic and managerial research focus in this area. Further investigation from a perspective of occupational disruption/deprivation will contribute to the development of responses to support recovery from mental health crisis, raising awareness of occupation in relation to increasing resilience and well-being. In addition, research exploring concepts of human occupation implicit within a range of social arenas will identify and look to address issues of occupational justice.

Limbal stem cell deficiency (LSCD) can be treated successfully using ex vivo limbal epithelial stem cells (LESC) derived from cadaveric donor tissue. However, shortages in such tissues and graft rejection, resulting from inflammation, are persistent issues. The purpose of this study was to optimize current culturing techniques used for LESC transplant tissue, considering expansion and cryopreservation issues surrounding the establishment of a stem cell bank. In addition, a novel anti-inflammatory biomimetic peptide was investigated to address issues surrounding amnion and steroid use in LESC transplantation, inflammation and transplant rejection. Cell cultures derived from Optisol and organ culture stored tissues were examined for optimum growth, characterized by an ability to grow to 70 % to 80 % confluence while maintaining epithelial cell morphology and the presence of positive and negative LESC markers (K3, K19, p63 and PAX-6) as identified by immunocytochemical staining and QRT-PCR. Furthermore, the effect of culture expansion and cryopreservation on stem cell characteristics was examined. A short chain IL-l receptor antagonist peptide was synthesized and characterized using mass spectroscopy (MS), high performance liquid chromatography (HPLC) and liquid chromatography-mass spectroscopy (LC-MS). Anti-inflammatory properties were investigated using ELISA detection of IL-8, IL-6 and IL- l ~ in keratocytes and LESC following stimulation with either lipopolysaccharide or recombinant human IL- l~. Feasible delivery of cells and peptide on a contact lens was investigated to assess the possibility of an "all in one" graft. Results showed that organ culture stored tissues can provide 100 % successful cell cultures using current techniques in terms of reaching confluence and maintaining LESC morphology and phenotype. Sub-culturing and cryopreservation of cultures however did not produce confluent cell sheets, as required for clinical application. The anti-inflammatory peptide was shown to effectively suppress production of key inflammatory cytokines in LESC and keratocytes by acting as an IL- l receptor antagonist and interrupting the IL- l inflammatory pathway. Binding of the peptide to the contact lens was shown to be possible. Such a scaffold also supported expansion of LESC. However, the 2.7 nmol of peptide bound to the lens did not significantly lower cytokine production. Findings suggest that it is possible to culture adequate numbers of LESC for the initiation of a stem cell bank using current techniques. However, modifications to culturing methods are needed to ensure successful sub-culturing and cryopreservation. The peptide has been shown to be effective in reducing inflammatory cytokine production, providing a possible alternative to steroids. An a11-in-one graft could provide a key development in treating LSCD. However further work is required to optimize the peptide concentration to allow effective inflammation control.

The outcome of the deployment of cardiovascular stents in coronary arteries compromised by atherosclerosis may be affected by the deposition of a myofibroblast-driven neointimal tissue and consequent re-blockage of the vessel lumen known as instent restenosis (ISR). Its incidence is particularly high in people with diabetes unless drug-eluting stents (DES) are implanted as alternatives to the traditional bare metal stents (BMS) made of stainless steel (ST). However, the long-term outcome of the use of DES is under debate due to evidence of late thrombosis. Monocytes/macrophages (MM) play a key role in ISR participating in the different phases of the host response to the implant. This in-vitro study investigates differences in the distribution and response of MM that may be significant for a better understanding of the causes of the increased ISR occurrence among diabetic patients, particularly focusing on the trans-differentiation potential of MM into myofibroblast-like cells when in contact with ST. The overnight incubation of MM freshly isolated from blood from healthy donors (n=6) in high glucose (4S00mglL) and high free fatty acid (sodium palmitate 0.4mM) concentrations, alone or in combination, aimed at creating a diabetes-mimicking experimental model. MM response to ST seemed exacerbated by the syoergistic effect of the palmitate and glucose as shown by the occurrence of features characteristic of activation as well as by their trend to fuse into the formation of giant cells as shown by scanning electron microscopy. Similarly results from RT-PCR (n=6) suggested an increase in the expression of the mRNA for HAS3, the enzyme responsible for the synthesis of low molecular weight hyaluronic acid characteristic of the inflammatory process. Furthermore, the presence of MM positive for a-actin, as shown by immunocytochemistry, seemed to confirm the occurrence of early changes in their phenotype. This was accompanied by a significant (p<O.OS or p<O.OOS after paired Hest) increase in the release of PDGF-BB, growth factor characterizing the post-inflammatory condition, as well as in the release of 1NF-a confirming the short-term metal-induced inflammatory reaction from MM incubated on ST in comparison to control surfaces as shown by ELISA tests. Flow cytometry on buffy coats collected from three cohorts of donors (control, type 1 and type 2 diabetes) (n=7) suggested that variations among donors with diabetes in the number of circulating activated macrophages and haematopoietic progenitor cells may, once compared to clinical data, help to identify patients at high risk of developing ISR. The hypothesis of the contribution of MM in the deposition of restenotic neointimal tissue through the transformation into myofibroblasts was reinforced by results from flow cytometry that identified, both in freshly isolated buffy coats and after contact with surfaces, subpopulations co-expressing a-actin and markers for MM (CDI4 or CD68). This was also supported by the imununostaining of the cells after overnight incubation on the surfaces where a-actin-positive cells were seen. Furthermore, ELISA test on the supernatants (n=10) suggested that ST triggered a significant increase in the release of PDGF-BB and 1NF-a when compared to control surfaces. This study reinforced the hypothesis that MM can contribute to ISR through phenotypical changes and that the acute synergistic effect of glucose and lipids can affect their level of activation.

Highly active anti-retroviral therapy (HAART) has proved very successful in reducing the morbidity and mortality associated with HIV infection. This same lifespan prolongation, however, has also revealed many side effects linked to HAART, including cardiovascular disease. A once daily fixed dose drug combination pill Atripla was hoped to improve compliance and adherence. The HAART components of Atripla efavirenz, emtricitabine and tenofovir are considered to be significantly safer than the conventional reverse transcriptase inhibitors but cardiovascular effects of these drugs have yet to be investigated in vitro. The overall objective of this thesis was to examine the effects of the three components of Atripla on cardiovascular cell function.

A common treatment used by physiotherapists for patients with low back pain (LBP) is mobilisations. The aim of applying mobilisations is to increase range of movement (ROM) and reduce pain and stiffness. Therapists choose a specific dose of mobilisation for each patient, which includes a decision on the duration of applied force, commonly up to 3 minutes. Little research has been done to determine the biomechanical and analgesic effects of different durations of treatment. There is tentative evidence that increased duration beyond 3 minutes leads to an increase in range of movement and decrease in pain. This research set out to establish the biomechanical and analgesic effects of longer durations of lumbar mobilisations than commonly used in clinical practice. Only the immediate effects of a single treatment dose have been assessed to date.

Probiotics are generally live preparations of bacteria that exert beneficial effects on host health when ingested in sufficient quantities. The novel probiotic Escherichia coli strain Nissle 1917 (EcN) has been shown to have a number of beneficial effects in this context, including protection against food-borne pathogens and infectious diarrheal diseases, and maintaining remission of inflammatory bowel diseases by virtue of its anti-inflammatory properties. However, little is known regarding the mechanisms underlying the beneficial effects of this organism. The overall aim of this work was to provide a greater understanding of the mechanisms through which EcN interacts with the mammalian gastro-intestinal epithelium to benefit human health.