Drug-induced liver injury (DILI) is a serious adverse drug reaction (ADR) that is frequently encountered during drug development, representing a major cause of drug attrition. Furthermore, DILI is also a serious concern in the clinic, accounting for approximately half of all acute liver failure cases. Paracetamol overdose (acetaminophen; APAP) accounts for the majority of DILI-associated cases of ALF encountered in patients. The identification and development of novel biomarkers of DILI that are sensitive, specific, and rise early during hepatotoxicity are urgently required in the clinic and in the laboratory. Two liver-specific microRNAs (miRNAs) have recently been described that serve as sensitive and early markers of APAP-induced acute liver injury (APAP-induced ALI) in a mouse model. Together with the superior liver-enrichment of some liver-enriched miRNAs, these potential markers need to be assessed in patients for the clinical promise. Moreover, further work is warranted to test these potential markers in alternative pre-clinical models with other compounds to gain a better understanding regarding sensitivity of release, mechanism of release and circulatory kinetics. Furthermore, in separate work discussed in this thesis, the mammalian biological clock has been found to exert a powerful influence on the physiology of mammalian systems. This regulation hinges on the complex interplay between the clock genes and their products that oscillate over a twenty-four hour period and promote a diurnal variation in numerous output pathways. Emerging evidence suggests that the efficacy and toxicity of many drugs follow a diurnal rhythm and that this may be at least partly attributable to the clock-mediated regulation of drug targets and pathways of drug metabolism. APAP and FS represent two compounds that elicit hepatotoxicity in the mouse through two distinct mechanisms. Both APAP and FS are known to exhibit circadian variation in their toxicology and/or pharmacology. However, little is known about the molecular mechanisms that govern these differences in circadian variation. Two liver-enriched miRNAs (miR-122 and miR-192) were tested alongside serum ALT activity, the gold-standard marker of ALI, for sensitivity and time of release in a mouse model of APAP-induced ALI. At 2 hours after APAP administration, miR-122 (ΔΔCt 75.0, P=0.02) was significantly higher compared to controls (ΔΔCt 4.1) while ALT levels were in the normal range (21 U/L) indicating earlier release of miR-122. In a sensitivity study, miR-122 was not more sensitive than ALT at a 300 mg/kg dose of APAP compared to controls (mean values 300 mg/kg vs 0 mg/kg: ALT = 491 U/L vs 38.1 U/L; miR-122 = ΔΔCt 572.9 vs ΔΔCt 209.4). In patients, serum miR-122 and miR-192 were substantially higher in APAP-induced ALI patients, compared to healthy controls (median ΔΔCt miR-122: 1,265 versus 12.1, P < 0.0001; miR-192: 6.9 versus 0.44 P < 0.0001). A heart-enriched miR-1 showed no difference between APAP-ALI patients and controls, whereas miR-218 (brain-enriched) was slightly higher in the APAP-ALI cohort (ΔΔCt 0.17 versus ΔΔCt 0.07 P = 0.01). In a cohort comprised of patients who presented early (median time of presentation since APAP overdose = 8 hours), miR-122 was significantly raised in patients who develop ALI (> 3 x ULN serum ALT activity) compared to those that did not (median ΔΔCt 3.48 vs ΔΔCt 0.16, P<0.0001). In contrast, presentation ALT levels were not different between patients who developed ALI compared to those that did not (median ALT = 21 U/L vs 19 U/L). Moreover, miR-122 was significantly raised in patients who develop coagulopathy (INR > 1.3) compared to those that did not (ΔΔCt vs 3.48 vs ΔΔCt 0.17, P=0.0004). In contrast, presentation ALT levels were not different between patients who developed ALI iv compared to those that did not (median ALT = 21 U/L vs 19 U/L). In chronotoxicity studies, both APAP exhibited greater toxicity in the evening (mean ALT = 12785, 66% survival) compared to morning (mean ALT = 380, 100% survival) whereas FS showed greater toxicity after morning administration (mean ALT=561, 100% survival) compared to evening administration (mean ALT = 69.2, 100% survival). Circadian variation in APAP-induced ALI was associated with 38% lower (P=0.003) GSH levels and 20% higher (P=0.024) Cyp2e1 levels at 21:00h compared to 09:00. This work confirms that miR-122 is released earlier than ALT in a young mouse model of APAP-induced ALI. Furthermore, it is shown for the first time that circulating liver enriched miRNAs are higher in patients following an APAP overdose. Also, plasma miR-122 is raised at emergency room presentation when serum ALT activity is in the normal range. Further clinical development of blood-based miR-122 is warranted, this work suggests that miR-122 analysis at the point of hospital admission can predict risk of subsequent liver injury in patients. Finally, APAP and FS exhibit circadian variation in their toxicity in a mouse model associated with circadian variation with genes involved in drug metabolism and drug detoxification. Profiling of the hepatic proteome over the circadian phase is now warranted.

Drug hypersensitivity reactions are a significant cause of patient morbidity and mortality. They are difficult to predict in the clinic and during the drug development process. This is because mechanisms have not been fully elucidated. In fact, recent data using T-cells cloned from hypersensitive human patients have questioned the long-standing hapten concept, which states that a drug must bind irreversibly to protein to initiate a T-cell response. The work described herein was performed to investigate the chemical and cellular mechanisms of β-lactam-induced skin and liver injury and in particular to understand the relationship between drug-protein adduct formation and the activation of antigen-specific T-cells. Β-lactam antibiotics, such as piperacillin, provide the cornerstone of treatment and reduce the rate of decline in lung function in patients with cystic fibrosis, but use is limited by a high frequency of hypersensitivity reactions. Using the lymphocyte transformation test, drug-responsive lymphocytes were found in approximately 70% of clinically diagnosed piperacillin hypersensitive patients. By cloning over 400 antigen-specific CD4+, CD8+ and CD4+CD8+ T-cells, the T-cell proliferative response and cytokine secretion to piperacillin was shown to be concentration-dependent and highly drug-specific. Mass spectrometry revealed irreversible binding of piperacillin to selective lysine residues on albumin and a synthetic albumin conjugate stimulated piperacilin-specific clones via a processing-dependent pathway. These results describe the cellular processes that underlie piperacillin hypersensitivity. Approximately 20% of hypersensitive patients with cystic fibrosis develop multiple reactions that restrict therapeutic options. To explore the mechanistic basis of multiple β-lactam hypersensitivity, albumin binding profiles and T-cell responses against three commonly prescribed drugs; piperacillin, meropenem and aztreonam, were studied. PBMC responses were characterized using the lymphocyte transformation test and IFN-γ ELIspot. Clones were generated and found to proliferate and release cytokines following stimulation with all three drugs. However, crossreactivity with the different drugs was not observed. Each compound formed distinct haptens with lysine residues on albumin, which may explain the highly drug-specific T-cell response. These data indicate that multiple β-lactam reactions are instigated through priming naïve T-cells against the different drugs. The role of the adaptive immune system in reactions that target liver has not been defined. For flucloxacillin, a delay in the reaction onset and identification of HLA-B*57:01 as a susceptibility factor are indicative of an immune pathogenesis. Thus, flucloxacillin-responsive CD4+ and CD8+ T-cells were characterized from patients with liver injury. Clones expressed the gut-homing chemokine receptors CCR4 and CCR9 and secreted IFN-γ, Th2 cytokines and cytolytic molecules following drug stimulation. In contrast to the piperacillin clones, flucloxacillin clones were activated with several structurally-related β-lactam antibiotics. Furthermore, naïve CD8+ T-cells from volunteers expressing B*57:01 were activated with flucloxacillin when dendritic cells presented the drug antigen. Activation of CD8+ cells from patients and volunteers was processing-dependent and restricted by HLA-B*57:01, which effectively links the genetic association to the iatrogenic disease. In conclusion, the studies described herein provide novel insight into the way in which β-lactam antibiotics interact with protein and activate T-cells that are thought to be the ultimate mediators of drug hypersensitivity reactions in skin and liver.

Drug Induced Liver Injury (DILI) is associated with high morbidity and mortality rates. It is the leading cause of acute liver failure, accounting for 50% of all cases. Moreover, DILI is the most frequent cause of post marketing drug withdrawal and is often cited as a cause of compound attrition during the drug-development process. The High Mobility Group Box 1 (HMGB1) protein is an important inflammatory mediator which alters the immune system to tissue stress and injury. HMGB1 has been implicated in the pathogenesis of multiple inflammatory diseases including immune-mediated DILI. It has been identified as a potential biomarker of hepatic injury and a target for therapeutic intervention. Research is required to elucidate the pro-inflammatory role of HMGB1. HMGB1 has been reported to interact with a diverse range of endogenous (IL-1, DNA, nucleosomes, CXCL12) and exogenous (LPS) molecules to promote inflammation. However, the mechanisms responsible for these synergistic interactions remain poorly defined and therefore, the overall aim of this work was to characterise the interactions of HMGB1. Specifically, this work has focused on the interaction with IL-1β, which is of particular interest since both molecules often co-exist at the site of inflammation. The interaction between HMGB1 and IL-1β was investigated using combined cellular and Nuclear Magnetic Resonance (NMR) methodologies. LPS-free, isotopically labelled recombinant HMGB1 (full length protein, amino acids 1-215) and IL-1β proteins were expressed and purified from BL21 (DE3) cells. To facilitate these studies, three additional HMGB1 mutants were sub-cloned from the HMGB1 plasmid: Δ30 (1-185), A box (1-85) and B box (89-163). The recombinant proteins were characterised using Mass Spectrometry (MS) and NMR spectroscopy. Synovial fibroblasts were isolated from synovial tissue obtained from rheumatoid arthritis patients undergoing joint replacement surgery. Cells were treated with HMGB1 (full length, Δ30, A box or B box) alone or combination with IL-1β. Cell supernatants were collected after 24hr and IL-6 levels were quantified by ELISA. Untreated fibroblasts or cells treated with any HMGB1 construct, or IL-1β alone had no detectable IL-6 release (<9.375pg/mL). In contrast, full length HMGB1, the Δ30 and the B box domain (but not the A box domain) all acted in synergy with IL-1β to substantially enhance IL-6 production. In one patient, HMGB1, Δ30 and the B box in combination with IL-1β induced IL-6 levels of 28,473 ± 127pg/mL, 18,491 ± 2388pg/mL and 18,710 ± 2792pg/mL, respectively. The synergistic interaction was mediated via the Interleukin-1 receptor (IL-1R) and could be inhibited when the cells were pre-treated with 5µg/mL anakinra, a selective IL-1R antagonist but not detoxified LPS, a TLR4 receptor antagonist. To investigate if there is a direct interaction between HMGB1 and IL-1β, a comprehensive biophysical analysis was performed using NMR methodologies. However, despite performing the experiments in various ways, no evidence of a direct interaction between IL-1β and either full length HMGB1, Δ30 or the B box was detected. This suggests that the synergistic interaction between HMGB1 and IL-1β is mediated via an alternative cellular mechanism in which HMGB1 is required. In conclusion, the work presented in this thesis has identified that the B box domain of HMGB1 is critical for the synergistic effect observed with IL-1β. However, this is not due to the formation of a binary complex between HMGB1 and IL-1β. Instead, it would appear that the synergistic effect is mediated via an alternative cellular mechanism in which HMGB1 is required and additional proteins are involved. Future work could focus on discovering what these other proteins might be. These findings help to elucidate the pro-inflammatory role of HMGB1 and provide a novel an insight into HMGB1 biology.

The non-nucleoside reverse transcriptase inhibitor nevirapine (NVP) is widely used in the treatment of human immunodeficiency virus infection in many sub- Saharan countries. However, NVP-treated individuals have a 5% risk of developing hypersensitivity reactions (HSRs), ranging from maculopapular exanthema to life-threatening severe cutaneous eruptions and hepatotoxicity. A number of clinical (gender and CD4+ T cell count) as well as immunogenetic factors (MHC class I and class II alleles) have been associated with NVP hypersensitivity, but the predictive value is poor. Gene expression analyses provide an unbiased method for determining which immune-related mechanisms are involved in the pathogenesis of NVP hypersensitivity. mRNA microarray analysis demonstrated that expression of CD177, a neutrophil specific antigen, was significantly increased in our cohort of NVP-treated patients from Malawi (p ≤ 0.001), but also after in vitro treatment of NVP-hypersensitive patients from Liverpool (p < 0.05). Interestingly, CD177 protein expression did not increase in patient samples treated with NVP in vitro. A case-control study (n = 288) of NVP-hypersensitive and tolerant patients showed that none of the investigated CD177 polymorphisms were associated with NVP-induced HSRs. Of the clinical factors analysed, only CD4+ T cell count was significantly associated with NVP hypersensitivity (p < 0.001). The polymorphisms rs45441892 and rs10425385 in CD177 have previously been associated with an increased proportion of CD177-positive granulocytes. We confirmed these results in our NVP-naïve, healthy volunteer cohort (n = 35) for rs10425385, but not rs45441892. Previous reports have identified HLA-C*04:01 as a susceptibility marker for NVP-induced HSRs in patients from Malawi. Expression levels of miR-148a, a microRNA known to downregulate HLA-C expression, were analysed in serum samples from NVP-hypersensitive and tolerant individuals (n = 96). However, paradoxically, a statistically significant increase in miR-148a expression was found in NVP-hypersensitive patients at the time of reaction (p = 0.008). Additionally, the serum expression levels of 84 miRNAs were analysed in hypersensitive and tolerant patients treated with NVP (n = 24). Twenty-one miRNAs were differentially expressed in tolerant and hypersensitive samples. Of these miRNAs, miR-205 showed the highest increase in NVP-hypersensitive patients (p < 0.01). Besides HLA-C*04, several other HLA-alleles have been reported as risk factors for NVP hypersensitivity. A summary meta-analysis of published data indicated that four HLA-allelotypes (HLA-B*35, -B*58:01, -C*04, -DRB1*01) might be common risk factors of NVP-induced HSRs for different ethnicities. The results presented in this thesis highlight that various genetic, immunological and clinical factors may contribute to the pathogenesis of NVP hypersensitivity. Further understanding of the complex interactions may ultimately lead to the characterisation of true causative associations facilitating the precise prediction of hypersensitivity reactions to NVP.

Nrf2 is a transcription factor that, under conditions of chemical stress, is able to evade its cytosolic repression and translocate to the nucleus to initiate the transcription of a battery of cytoprotective genes, such as those involved in the detoxication of xenobiotics. Nrf2 has previously been shown to afford protection against chronic and acute renal injury, yet, relatively little is known about the mechanism by which Nrf2 affords this protection, and the extent of its transcriptional roles in the kidney. This thesis seeks to further our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney. Using an iTRAQ-based proteomic approach to quantify protein expression levels in the kidneys of Nrf2+/+ and Nrf2-/- mice, acutely treated with vehicle or the potent Nrf2 inducer CDDO-Me (3 mg/kg), we demonstrated that 189 proteins were differentially expressed in the Nrf2-/- mouse kidney, compared to Nrf2+/+, and 42 proteins were differentially expressed in the CDDO-Me treated Nrf2+/+ mouse kidney, compared to vehicle. The key finding was that the kidneys of Nrf2-/- mice are deficient in proteins that mediate cellular redox balance, the metabolism of a range of xenobiotics, and the regulation of core metabolic processes, including energy metabolism and the synthesis and recycling of amino acids. Functional demonstration of a reduction in energy metabolism was demonstrated by assessing total NADPH and GSH, of which Nrf2-/- mouse kidneys had 35% and 30% less than their Nrf2+/+ counterparts, respectively. A single acute dose of CDDO-Me failed to augment the expression of proteins, other than Nqo1, that were shown to be regulated by Nrf2 at the basal level in the mouse kidney, however qPCR analysis of these kidneys revealed that CDDO-Me has an effect at the transcriptional level which has not fully translated within the timeframe of this study. In summary, we have provided evidence that Nrf2 regulates the expression of an array of proteins that contribute to cell defence and the maintenance of homeostasis in the kidney, supporting current interest in Nrf2 as a novel therapeutic target in a number of renal diseases. MicroRNAs are a recently discovered RNA-regulatory element that show promise in their use as biomarkers of physiological and pathological events. In order to provide insight into the microRNAs under Nrf2 control in the kidney, we performed an unbiased microRNA array analysis on kidney homogenates from Nrf2+/+ and Nrf2-/- mice, treated with vehicle or CDDO-Me, and then validated several promising microRNA candidates using targeted qPCR analysis. Of particular note are miR-466h-3p, the expression of which was significantly increased in the CDDO-Me treated Nrf2+/+ mouse kidney and decreased in the Nrf2-/- mouse kidney, compared to their respective controls, and miR-28c and 144, which were both significantly decreased in the CDDO-Me treated Nrf2+/+ mouse kidney, and increased in the Nrf2-/- mouse kidney. This novel analysis represents the first step in characterising the renal Nrf2 microRNA-ome, which could reveal novel mechanisms of Nrf2 function and markers of its activity that could translate to the clinic. Recent interest in the use of CDDO-Me as a therapeutic intervention for late-stage chronic kidney disease has culminated in a phase III clinical trial (BEACON), which was subsequently terminated due to unforeseen adverse cardiac events, of which the cause has yet to be identified. In order to determine whether the drive to produce more potent Nrf2 inducers has inadvertently led to the generation of inherently more toxic compounds, the relationship between potency towards Nrf2 and toxicity was evaluated for CDDO-Me and related triterpenoids, and other classes of Nrf2 inducer. Using a rat H4IIE-ARE8L luciferase reporter cell line to determine in vitro therapeutic indices, it was discovered that within the compounds tested an increase in potency toward Nrf2 of four magnitudes results was associated with an increase in toxicity of only two magnitudes, resulting in a relative increase in in vitro safety. This data indicates that it is possible to generate potent Nrf2-inducers that are not inherently toxic, and suggests that therapeutic targeting of Nrf2 continues to hold promise as a novel treatment for a range of diseases. In summary, by using a proteomic approach we have identified an array of renal Nrf2-regulated proteins that contribute to various cytoprotective and metabolic processes in the kidney, supporting current interest in the therapeutic targeting of Nrf2 as treatment for renal disease. Additionally, the microRNAs under Nrf2 regulation in the kidney have also been identified, and represent the first step in fully characterising the Nrf2 microRNA-ome. Finally, it was shown that the drive to produce more potent Nrf2 inducers has not led to the generation of inherently more toxic compounds; indeed an increase in potency is associated with a relative increase in in vitro safety, suggesting that the targeting of Nrf2 is still a promising therapeutic route. Overall, the work presented in this thesis has furthered our understanding of the physiological, pharmacological and toxicological roles of Nrf2 in the kidney.

Drug-induced liver injury (DILI) is a major cause of attrition during drug development, and also a leading cause of hospital admissions. In order to identify novel biomarkers that may improve the diagnosis and treatment of DILI, well characterised hepatotoxins such as acetaminophen (APAP) are used. High mobility group box 1 (HMGB1) protein is a ubiquitously expressed protein involved in the facilitation of gene transcription. However, extracellular HMGB1 acts as a key mediator of inflammation. During APAP-induced liver injury (AILI), HMGB1 is passively released from necrotic hepatocytes in a fully reduced, hypoacetylated isoform, or actively released from immune cells following acetylation of HMGB1 at key lysine residues within nuclear localisation sequences. Extracellular HMGB1 can bind to a range of receptors including TLR2, TLR4, TLR9, CXCR4 and RAGE to mediate cytokine-inducing or chemotactic effects and has been shown to be a more sensitive and predictive serum biomarker of AILI outcome than ALT. Additionally, anti-HMGB1 antibodies have been used to successfully attenuate injury in animal models of inflammatory conditions, including DILI. Mechanistic understanding of HMGB1 regulation and function is essential for this emerging biomarker of AILI. A murine monoclonal anti-HMGB1 antibody (2G7) has previously been used to attenuate AILI. A chimeric anti-HMGB1 monoclonal antibody (h2G7) was investigated, alongside 2G7 and a human isotype control antibody (E2) in an experimental model of AILI. C57BL/6 mice dosed with 530mg/kg APAP by intraperitoneal (i.p.) injection, followed by antibody or PBS treatment i.p. at 2hr post-APAP and euthanisation at 10hr post-APAP showed that 300µg h2G7 treatment afforded protection from AILI by significantly attenuating serum ALT, miR-122 and chemo/cytokine increases seen in the isotype control group. Liver histology and MPO revealed reduced neutrophil infiltration and activity in anti-HMGB1 treated animals. No significant differences were seen between 2G7 and h2G7 animals, and there was no effect on hepatic GSH in antibody-treated animals. Mechanism of action studies using Fc region modified forms of h2G7 ruled out complement-mediated and FcγR-mediated effects of h2G7 in the AILI model, as these did not significantly affect the h2G7 effect on AILI. Redox modulation of HMGB1 function had been proposed as a mechanism of regulation of proinflammatory activity of HMGB1. Investigations into the cytokine-inducing capability of different redox isoforms of HMGB1 in THP-1 and RAW264.7 cells demonstrated that HMGB1 needs to be in a Cys23-Cys45 disulphide conformation with a reduced thiol at Cys106 in order to induce cytokine release via NF-κB activation. During apoptosis, HMGB1 can be oxidised by activated caspases. Oxidation has previously been shown to regulate the structure of proteins and facilitate other post-translational modifications (PTMs). LC-MS/MS characterisation of HMGB1 from APAP overdose patients with high levels of apoptosis revealed ubiquitination at Lys30 (HMGB1-Ub). Ubiquitination was shown in vitro not to direct HMGB1 for proteasomal degradation, but HMGB1-Ub was detectable in apoptotic microparticles (MP), membrane vesicles that are shed from a cell during late apoptosis. In summary, HMGB1 was shown to be critical for the coordination of AILI, as antibody-mediated inhibition lead to a reduction in hepatic injury. Chimeric anti-HMGB1 antibody was effective at attenuating liver injury and advances anti-HMGB1 antibody treatment towards clinical utility. HMGB1 redox regulation of inflammatory signalling and the identification of HMGB1-Ub demonstrate the importance of PTMs in governing HMGB1 function. HMGB1 shows promise as an acute, sensitive and predictive biomarker of AILI in man. Through the use of in vitro and in vivo systems, the work presented in this thesis expands our current understanding of the mechanistic and functional roles of HMGB1 in DILI and has further defined mechanisms that can be used to inform the clinical situation.

Epilepsy is one of the most common chronic neurological disorders, and the symptomatic form is characterised by the occurrence of spontaneous recurrent seizures following a neurological insult. One third of epilepsy patients are resistant to antiepileptic drugs (AEDs), therefore the development of novel treatments is required. Glutamate is implicated in epilepsy however, only a few glutamate receptor antagonists have been successful in epilepsy trials. An indirect means of modifying glutamate-mediated excitation, such as targeting nitric oxide (NO), might be a reasonable alternative approach. Selectively targeting NO signalling pathway by two drug interventions was investigated in this thesis. Post-synaptic density 95 blocking peptide (PSD95BP) is a protein that uncouples GluN2 subunits of the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor from neuronal NO synthase to prevent downstream neurotoxic signalling of NO. In contrast, 1400W is a highly specific inducible NO synthase inhibitor that binds to the guanidine recognition site of the enzyme, competing with L-arginine and preventing neurotoxic production of NO. These compounds are effective in animal models of stroke and were tested here in a C57BL/6J mouse model of epileptogenesis. Kainic acid (KA) is widely used to induce status epilepticus (SE) in animals and the resulting neuropathology mimics that seen in humans with temporal lobe epilepsy. Repeated low dose administration of KA via intraperitoneal injection every 30 minutes consistently induced generalised seizures but was associated with inter-animal variability in KA sensitivity, acute seizure severity and mortality rate. Extra-dural telemetry electrodes were implanted in mice for electroencephalography (EEG) recordings. Two algorithms, measuring spike frequency and EEG coastline respectively, were used to quantify epileptiform activity. Mice that received drug interventions following KA-induced SE had significantly lower mean spike frequency and fewer extended coastline epochs per day than the control group at both 7 and 14 days after the initial insult. Label-free proteomics quantification showed significant changes to the hippocampal protein profile as a result of both PSD95BP and 1400W administration following KA-induced seizures. Drug treatment, singly or in combination, also reversed the effects of KA on the expression of both transforming growth factor β1 and inwardly rectifying potassium channel 4.1 in the hippocampus. Surgical implantation of extra-dural electrodes significantly lowered the seizure threshold to KA and was associated with an increase in brain expression of pro-inflammatory cytokines, suggesting that careful consideration is required in studies involving intracranial surgery to assess epileptogenesis or AED effects. Current research investigating novel therapeutic agents is focusing on non-NMDA glutamate receptors, such as α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and kainate receptors, as potential AED targets. There are no compounds targeting the NO signalling pathway currently being investigated for the treatment of epilepsy. The data reported in this thesis give cause for optimism but further studies are needed to fully investigate the antiepileptic and possible antiepileptogenic properties of PSD95BP and 1400W and true efficacy will ultimately require clinical evaluation.

A substantial proportion of older adults with non-inflammatory joint pain do not consult their general practitioner (GP) despite apparent clinical need. This thesis describes the development, execution, and interpretation of an original study using conjoint analysis – a fairly novel approach with some advantages over conventional observational and qualitative studies - to understand the relative importance of need-related and service-related factors on the decision to consult the GP. Background reading, a systematic review of previously published conjoint analysis studies, and a series of developmental studies involving patients and members of the public informed the design of the main study. A partial-profile choice-based conjoint (PPCBC) questionnaire was chosen, comprising 10 choice tasks using a combination of selected attributes (pain characteristics, pain disruption to everyday life, comorbidity, assessment and investigations available, available treatment options, and perceived GP attitude). The PPCBC questionnaire was postally-administered to 1170 adults aged 50 years and over with hip, knee, or hand pain identified from an existing population cohort study in North Staffordshire. 863 questionnaires were returned (adjusted response rate 74%; mean age: 70 years; 55% female) and well-completed ( < 5% missing data). The extent to which pain disrupted everyday life (1.10 logits) and perceived GP attitude (0.86 logits) were the most important determinants of the decision to consult the GP. Service factors were highly influential with a ‘negative’ GP attitude potentially outweighing the perceived value of optimal assessment and management. Latent class analysis identified possible subgroups with differing strengths of preference. Conjoint analysis is feasible and offers unique insights into the relative importance of actual and hypothetical services. While it presents many challenges - extensive developmental testing, complex design and analysis procedures, ability to integrate findings from a range of different methods – it can provide important information on patients’ preferences for existing and emerging treatments and models of care.

Background: Loss and bereavement are devastating life experiences for all human beings belonging to any social group, and adults with learning disabilities are not exempt from the effects of loss. Recent research has shown that adults with learning disabilities understand loss and bereavement; however, there is a lack of empirical evidence to show what adults with learning disabilities believe is useful for them to cope with loss and bereavement. Literature in this field suggests that adults with learning disabilities have been generally treated as subjects in research in the field of loss and bereavement and that most of this research has been based on case studies, observations, and other people’s perspectives. This qualitative research study aims to engage bereaved adults with learning disabilities as active members in research using Photovoice (a participatory methodology using photography and photographs for community-based research), to explore the experiences of loss in their lives and their perceptions of support available in the local area (Staffordshire, UK). Methods: A preliminary Photovoice workshop was organised for 10 bereaved adults with learning disabilities - the participants of the current research - in order to provide ethical and technical training for using a camera; engage them creatively to participate in research; and provide information on the photographs they would subsequently take as part of this research. The Photovoice workshop was followed by 10 semi-structured interviews with adults with learning disabilities to identify the support available and accessed; the impact of both loss and support; and the coping strategies used following bereavement. Interviews were facilitated through discussion of participants’ photographs taken after the Photovoice workshops. Additionally, data were gathered from focus groups with advocates (who support people to speak up by discussing, understanding their issues and finding information about their situations and options available); focus groups with professional carers; and interviews with family carers, to explore key issues and support needs and roles in supporting bereaved adults at home, in an advocacy organisation, and in a care setting. In total 27 (10 bereaved adults with learning disabilities; eight advocates; six professional carers; and three family carers) participated in the current research. All data, including the photographs taken by participants, were analysed using Thematic Analysis. Findings: The main themes emerging from the photographs taken by the bereaved adults with learning disabilities included people (the deceased family members; living family members; friends; personal assistants; support staff); a range of daily activities (such as drama; pottery; dance); and objects inside homes and outside. Thematic analysis of remaining data highlighted five main themes across the results, namely: (1) Multiple losses, (2) communications related to loss, (3) support and barriers to support, (4) impact of bereavement and (5) continuing bonds. Conclusions: The experiences of bereaved adults with learning disabilities mirrored the usual ordinariness of types of loss as people without learning disabilities but illustrate differences in the support, communications and other events after loss. Most of these differences were owing to the stigma attached to the learning disability itself. Creative methods (such as Photovoice) together with clear information are beneficial to engaging adults with learning disabilities in research and enabling them to share their viewpoints around sensitive topics. Overall, the benefits of using Photovoice extended beyond engaging participants, teaching them skills and collecting data, but also in providing evidence of its success that bereaved adults with learning disabilities are able to share their viewpoints on difficult topics such as loss and bereavement with the support of photographs for future research involving vulnerable groups. Note on terminology used: The acronym AWLD is used only in this thesis to refer to adults with learning disabilities. The author, however, does not encourage any type of labelling of this population. Note on chapter 3: Some information focusing on the use of Photovoice in this chapter is part of a published article, reproduced with kind permission from the publisher ‘© Emerald Publishing’.

Curcumin, the active constituent of the rhizome Curcuma longa has been extensively studied as an anticancer agent for various types of tumours. However, its efficacy as an anticancer agent is constrained due to poor absorption, rapid metabolism, degradation in acidic media and consequently, low oral bioavailability. In the present study, we aim to formulate a curcumin-containing mucoadhesive nanoparticulate delivery system that offers protection to curcumin from the degradative effects of the upper digestive tract system but capable of releasing the payload in the colon for the localised treatment of colorectal cancer. Such a system should have a good surface coverage locally at the tumour site and ideally, capable of traversing the tumour. Thus, nanopariculate delivery is most suited to achieve this objective. The latter was formulated using chitosan and pectin as polymers due to their biodegradability and non-toxicity. The objectives of this study were (i) to fabricate, optimize and characterize the curcumin-containing delivery system; (ii) to evaluate the mucoadhesive propensity, release of curcumin from the carrier, and stability of the formulation in various milieu; (iii) to investigate the in vitro antiproliferative efficacy of the formulation as well as the cellular uptake profiles; and (iv) to proof the concept of the formulation using animal modules. The formulated nanoparticulate system had a z-average of 206.0 nm (± 6.6 nm), zeta potential of +32.8 mV (±0.5 mV), and encapsulation efficiency of 64%. The nanoparticles were more mucoadhesive at alkaline pH compared to acidic pH. Furthermore, more than 80% release of curcumin was achieved in simulated colonic medium as opposed to negligible release in simulated gastric and intestinal fluids, respectively. The nanoparticles were taken-up by HT-29 colorectal cancer cells which ultimately resulted in a tremendous reduction in cell propagation. This anti-proliferative effect of the encapsulated curcumin was similar to that of free curcumin at equivalent doses which confirms that the encapsulation process did not affect the anticancer efficacy of curcumin. The bioavailability of curcumin from the nanoparticles was enhanced by 4-folds after oral administration after 6 hr of treatment. Moreover, the half-life, Cmax, and AUC of curcumin were significantly improved. A lower elimination rate was observed from the formulation compared to equivalent doses of free curcumin. These findings are a strong indication of the potential of the studied formulation for the possible treatment of colon cancer via oral administration.