Extracellular signal-regulated kinase 5 (ERK5) is the newest member of the mitogen- activated protein kinase (MAPK) family following the discovery of ERK1/2, JNK and p38 MAPK. ERK5 consists of an N-terminal kinase domain, similar to the other MAPK members, and a large C-terminal domain, which is unique in structure and function from the other MAPK members. ERK5 is activated in response to a multitude of extracellular stimuli, including pro-angiogenic factors in endothelial cells. The physiological importance of ERK5 was highlighted following Erk5 gene ablation in mice, where a severe disruption to cardiovascular development and loss of vascular integrity was observed, resulting in embryonic lethality. This study investigated the differences in ERK5 activation between vascular endothelial growth factor (VEGF) stimulation of primary human dermal microvascular endothelial cells (HDMECs) compared to epidermal growth factor (EGF) stimulation of HeLa (immortalised epithelial cervical cancer cell line) cells. It was discovered that in contrast to other growth factors, VEGF appeared unique in its ability to induce ERK5 phosphorylation in HDMECs, stimulating ERK5 activity via a VEGFR-2/PLCγ-dependent pathway. Utilisation of the innovative Phos-tagTM reagent in SDS-PAGE facilitated the novel discovery that VEGF was only able to induce phosphorylation of the threonine (Thr)218/tyrosine (Tyr)220 residues present within the activation loop of the kinase domain. In contrast, EGF stimulation of HeLa cells resulted in phosphorylation of ERK5 on Thr218/Tyr220 as well as additional C- terminal residues such as Thr732. It was further demonstrated that contrary to EGF, VEGF stimulation of HDMECs did not evoke a nuclear translocation of ERK5, instead, ERK5 appeared to localise to the cytoplasm and plasma membrane. The analysis of intracellular signalling pathways following treatment with small-molecule inhibitors against MAPK/ERK kinase 5 (MEK5) and ERK5 kinase activity, revealed that VEGF-mediated ERK5 activation regulated phosphorylation of AKT in HDMECs. Furthermore, with an apparent co- localisation of ERK5 and AKT in the cytoplasm and at the plasma membrane of HDMECs, it was hypothesised that the two proteins were interacting partners. Adenoviral-mediated expression of FLAG-tagged ERK5 revealed that VEGFR-2, ERK5 and AKT co- immunoprecipitated in HDMECs, suggesting the possibility of a complex between these proteins at the cell periphery following VEGF stimulation and VEGFR-2 internalisation. Taken together, this study shows that VEGF appears to induce a unique activation of ERK5 in endothelial cells, which facilitates its interaction with AKT and subsequent regulation of AKT phosphorylation, ultimately regulating endothelial cell survival.

Epilepsy effects up to 1% of the population, and up to 30% of people with epilepsy are pharmacoresistant—they continue to experience seizures despite treatment with maximal doses of multiple antiepileptic drugs. The causes of drug resistance in epilepsy remain poorly understood. In this work, I have used genetic and genomic analysis techniques to explore the causes of epilepsy pharmacoresistance. It has been reported that epilepsy pharmacoresistance results from impaired drug penetration into the epileptic focus secondary to a localized dysregulation of drug transporters. Solute carrier (SLC) transporters form the largest superfamily of multidrug transporters. I used novel in silico and stringent ex vivo strategies for identifying the SLCs that are dysregulated in the pharmacoresistant epileptic human hippocampus. I discovered that the SLCs dysregulated in the pharmacoresistant epileptic human hippocampus are either small metal ion exchangers or transporters of neurotransmitters, not antiepileptic drug transporters, and most likely contribute to pharmacoresistance by enhancing the intrinsic severity of epilepsy. This finding supports the newly-proposed and intuitive ‘intrinsic severity hypothesis’ of epilepsy pharmacoresistance. According to the intrinsic severity hypothesis, pharmacoresistance in epilepsy results from the increased dysfunction of the biological pathways which underlie epilepsy. Hence, I proceeded to perform genome-wide genetic and genomic analyses in order to find the most important pathways underlying epilepsy and pharmacoresistance in epilepsy. I performed an integrative analysis of previously published large-scale gene expression profiling studies on brain tissue from epilepsy surgery; the largest and most robust microarray analysis of brain tissue from surgery for pharmacoresistant mesial temporal lobe epilepsy; and the first-ever genome-wide association study (GWAS) of pharmacoresistant focal epilepsy. By integrating the results of the genetic and genomic studies, I was able to show that pharmacoresistance is the result of accumulation of deleterious genetic variants of increasing severity and/or numbers within the genes that constitute the core pathways underlying epilepsy. I also found that the pathways disrupted in pharmacoresistant epilepsy, at both the genetic and genomic levels, belong to many different diverse and disparate functional domains, for example ‘axon guidance’, ‘transmembrane transport of small molecules’ and ‘cell death signalling via NRAGE, NRIF and NADE’. However, using network analysis techniques, I showed that these seemingly unrelated pathways form a coherent highly interconnected network, and it can be expected that changes in one pathway in this network will have a cascading effect on the rest of the network. The most important pathways in these networks are the central ‘hub’ pathways, which I identified using betweenness centrality network analysis. I then performed the first-ever genetical genomics study in epilepsy using hippocampal samples from resective surgery for refractory mesial temporal lobe epilepsy. By integrating genome-wide genetic, genetical genomic and genomic studies, and then performing pathway, network and centrality analysis, I identified the most important putative central causal pathways underlying epilepsy pharmacoresistance: 'transmembrane transport of small molecules' and 'Deleted in colorectal cancer (DCC) mediated attractive signalling'. In conclusion, by performing genome-wide genetic, genetical genomic and genomic studies, followed by integrative analysis, pathway construction and network mapping, I have identified most important putative central causal pathways underlying epilepsy pharmacoresistance.

Protein therapeutics or biologics represent 30 % of current licensed pharmaceutical products. In general, biologics offer superior safety profiles compared to small molecules. However, significant clinical concerns have emerged in terms of development of anti-drug antibodies (ADAs), a phenomenon that is covered under the term, immunogenicity. Anti-drug antibodies can alter pharmacokinetics, reduce efficacy of the therapeutic and also can in some cases induce allergic reactions. Human recombinant interferon beta (IFN-β) is a biologic used for the treatment of multiple sclerosis (MS) – a chronic, inflammatory and demyelinating disease of the central nervous system. Long-term treatment with IFN-β has been shown to lead to the development of anti-IFN-β antibodies that can cause total loss or reduced efficacy. Anti-drug antibodies can be non-neutralising (N-NAb) and neutralising (NAb) depending on the site to which they bind. This study aimed to conduct a systematic review to determine factors affecting the formation of neutralising antibodies against three different formulations of IFN-β Avonex™, Rebif™ and Betaseron/Betaferon™. Findings from the systematic review highlight the relative differences in immunogenicity risk of different IFN-β formulations Avonex™, Rebif ™ and Betaseron/Betaferon™ with Avonex™ having the lowest risk, Rebif™ has moderate risk and Betaseron/Betaferon™ has high risk. Characterising the immunoglobulin profile of the IFN-β ADAs from the plasma of ADA positive MS patients revealed that IFN-β ADAs are predominantly of the IgG1 and IgG4 subclass. We also characterised the neutralising potential of the major ADA IgG4 subclass using a IFN-β bioactivity assay and show that IgG4 antibodies may likely contribute to the neutralisation activity. The potential of the neutralising ADAs to cross-react with endogenous IFN-β was investigated using an in vitro bioactivity assay. Findings from this set of experiments revealed varying degrees of neutralisation of endogenous IFN-β. We next explored the potential immunological consequence of ADA with regards to formation of immune complexes and activation of complement. The interaction of ADAs with the biologic can result in formation of immune complex. Immune complexes can activate the complement system. The data revealed IFN-β ADAs can form immune complexes with IFN-β and therefore activate complement. We also attempted to identify IFN-β linear epitopes that the ADAs had the ability to bind. However, a combination of multipin peptide technology and in vitro peptide competitive binding assay failed to reveal a definitive linear epitope although there was some evidence for the existence of potential linear epitopes. We also examined the involvement of T helper cells and T regulatory cells (Tregs) in ADA development. The data revealed no significant differences in the frequency of Tregs among IFN-β ADAs positive, negative and healthy donors. Attempts were also made to identify T helper epitopes within IFN-β that could potentially drive immunogenicity. Using T-cell epitope prediction tools (IEDB-AR and ProPred) and T-cell functional assays we identified an immunogenic sequence of 36 amino acids within IFN-β (position 130-166). Our data revealed that one IFN-β peptide within this sequence is a potential T-cell immunogenic epitope. In addition we identified a possible association of one IFN-β derived peptide with the DRB1*1501HLA haplotype. In summary, the results presented in this thesis have provided essential information on subclass profile of IFN-β ADAs, the possible involvement of T helper cells and potential antibody epitopes within IFN-β. Future studies should be aimed at providing greater detail on the evolution of the ADA response and test strategies to remove immunogenic determinants from IFN-β.

Transposable elements constitute 45% of the human genome contributing to our evolution, creating new exons, structural variation and influencing the regulation of transcription. SINE-VNTR-Alus (SVAs) are a hominid specific retrotransposon that are still actively retrotransposing in the human genome today. The structure and sequence of SVAs, in particular their variable number tandem repeat (VNTR) domain, suggest their potential for influencing the regulation of gene expression through binding of transcription factors, differential methylation patterns and formation of secondary structures along with potential for genetic variation between individuals. This project has identified novel regulatory domains and genetic variation within elements belonging to a hominid specific group of retrotransposons. A global analysis undertaken of their distribution identified their preference for genic regions over gene deserts and their insertion into functional regions of the genome such as promoters and introns. An in depth analysis of two SVA insertions, one upstream of the FUS gene and another upstream of the PARK7 gene, demonstrated the ability of SVAs to affect reporter expression in vitro and in vivo. Both of these SVAs were identified as polymorphic in their central VNTR regions and the PARK7 SVA also demonstrated different copy numbers of repeats it its 5’ CCCTCT domain. Analysis of the PARK7 SVA insertion and gene in cell lines indicated the SVA is not epigenetically silenced, as dogma might suggest to suppress retrotransposition, but present in a transcriptionally active region of the genome. There is increasing evidence for loss of silencing of retrotransposons including within the human brain which would allow for greater influence of potential transcriptional properties embedded within SVAs impacting on genomic function.

Dendritic cells (DCs) are antigen-presenting cells crucial for the initiation and coordination of primary adaptive immune responses. Immature DCs (iDCs) express low levels of MHC class II and co-stimulatory molecules such as CD80, CD86, and CD40, with high phagocytic capacity and limited ability to induce antigen-specific T cell activation. DC maturation is associated with up-regulation of co-stimulatory molecules and cytokine production, rendering the DCs competent in T cell activation and the elicitation of an immune response. DC function and co-stimulatory receptor gene expression are known to be regulated by intracellular redox status, NF-kB and MAPKs signalling pathways. Intracellular reactive oxygen species (ROS) levels influence DC maturation and function. The transcription factor, Nrf2, is essential for maintaining intracellular redox homeostasis. In response to oxidative stress, Nrf2 induces the transcription of a set of cytoprotective and antioxidant genes, including heme oxygenase-1 (HO-1), that are required for detoxification of xenobiotics and their reactive metabolites and the nullification of oxidative insult. It is now emerging that Nrf2, and its gene product, HO-1, play pivotal roles in regulation of immune responses. However, the key signalling mechanisms involved in Nrf2 and HO-1-mediated altered DC function has not been fully elucidated and requires further investigation. In addition, the role of ROS in the absence of Nrf2 or HO-1 activity, in DC activation and function has not been investigated. Using immature bone marrow-derived DCs (iDCs) from Nrf2 +/+ and Nrf2 -/- mice, we demonstrate in the first part of the work presented in this thesis, that Nrf2 deficiency in iDCs resulted in increased ROS levels, enhanced iDCs co-stimulatory receptor expression, and increased iDC-mediated antigen-specific CD8 T cell stimulatory capacity in response to an antigenic peptide. Using antioxidant vitamins to reset ROS levels in Nrf2 -/- iDCs, we show that elevated ROS was not responsible for the altered phenotype and function of these DCs. Additionally, using appropriate pharmacological inhibitors, we demonstrate that the altered Nrf2 -/- iDC phenotype and function did not require NF-kB, ERK or JNK activity but was dependent on p38MAPK-CREB/ATF1 activity. Based on these experimental results, we conclude that Nrf2 regulates DC maturation and function by modulating intracellular signalling pathways independent of intracellular ROS levels. In the second part of the study, we demonstrate that inhibition of HO-1 activity in iDCs resulted in DCs with raised intracellular ROS levels, a mature phenotype, impaired phagocytic and endocytic function, and increased capacity to stimulate antigen-specific CD8 T cells. Interestingly, our results reveal that the increased ROS levels following HO-1 inhibition did not underlie the changes in phenotype and functions observed in these iDCs. Importantly, we show that the p38MAPK-CREB/ATF1 pathway was involved in the mediation of the phenotypic and functional changes arising from HO-1 inhibition. Furthermore, up-regulation of HO-1 activity rendered iDCs refractory to lipopolysaccharide-induced activation of p38MAPK-CREB/ATF1 pathway and DC maturation. Finally, we demonstrate that treatment of iDC with the HO-1 substrate, heme, recapitulated the effects that result from HO-1 inhibition. Based on these experimental results, we conclude that HO-1 regulates DC maturation and function by modulating the p38MAPK-CREB/ATF1 signalling axis. Collectively, the work described in this thesis highlights the importance of the redox regulators, Nrf2 and HO-1, in controlling DC immune functions. This work supports the basis for utilisation of Nrf2 and HO-1 as potential molecular targets for pharmacological intervention in disease states that arise from dysregulated redox function and the design of new pharmacological strategies aimed at modulating DC function in the therapy of immune diseases.

Idiosyncratic drug hypersensitivity remains a major challenge as it causes high morbidity and mortality. This is complicated by the multiple risk factors implicated and the inability to predict these reactions during the early stages of drug development. Thus, this study attempted to delineate the molecular pathomechanism(s) involved in sulfamethoxazole (SMX) hypersensitivity. The reactive metabolite, nitroso-SMX (SMX.NO) generated through the hepatic bioactivation of SMX has long been hypothesised as a major trigger of these reactions. SMX hypersensitivity has been used as a paradigm to study the role of drug metabolism in the activation of T-cells as the synthetic nitroso metabolite is available for functional studies. Metabolism of SMX in hepatic tissue has been extensively studied. CYP2C9 and Myeloperoxidase (MPO) are implicated in the formation of SMX.NO. However, it is unclear whether the SMX.NO generated in the liver migrates to the skin; the primary target in SMX hypersensitivity. It is possible that localised SMX metabolism by immune cells resident in the skin are implicated in the observed reactions. ELISA data revealed SMX metabolism in EBV-transformed B-cells used as antigen presenting cells (APCs). SMX-metabolism was significantly inhibited by methimazole. Furthermore, Western blotting and RT-PCR analyses suggested the presence of low concentrations of MPO in EBV-transformed B-cells. Interestingly, RT-PCR revealed mRNA expression of flavine containing monooxygenases (FMO1-5), TPO and LPO but the protein levels of these enzymes were not detected in immune cells. Subsequent experiments involved the generation and LC-MS/MS characterization of SMX.NO-modified MPO adducts. Although SMX.NO formed both the sulphinamide and N-hydroxysulfinamide adducts, drug specific T-cell clones failed to proliferate in response to drug-modified peptides. Since SMX.NO binds to multiple cellular proteins, it is assumed that peptides derived from the modified protein interact with a number of diverse HLA molecules to activate T-cells. However, the HLA molecules that interact with SMX.NO-modified peptides have not been defined. This study therefore examined the HLA molecules that present SMX.NO (derived peptides) to T-cells. T-cell clones (TCCs) were generated from 5 hypersensitive patients with cystic fibrosis. Fast growing TCCs from 2 SMX hypersensitive patients were used for HLA restriction studies. Drug-specific proliferative response, cytokine secretion and cytolytic markers were measured using [3H]-thymidine incorporation and ELIspot assays. Anti-human class I and class II (DR, DP, and DQ) antibodies were used to determine HLA restriction of drug-specific T-cell activation. APCs expressing similar or different HLAs were used to define the alleles involved in the presentation of SMX.NO-derived antigens to T-cells. A total of 1578 clones were tested for SMX.NO reactivity. Seventy-seven CD4+ clones were activated to proliferate and secrete IFN-ϒ, IL-5, IL-13 and granzyme-B by SMX.NO. Only one TCC was CD8+No cross reactivity with SMX was observed. The SMX.NO-specific response of clones was blocked with antibodies against MHC class II and HLA-DQ. Clones from 2 patients (Patient 1: HLA-DQB1*05:01:01G/ DQB1*06:03:01G; Patient 2: HLA-DQB1*02:01:01G/DQB1*02:01:01G) were used to define the DQ alleles involved in the presentation of SMX.NO derived antigens. SMX.NO-specific responses were detected with heterologous APCs expressing HLA-DQB1*05:01 (patient 1) and HLA-DQB1*02:01 (patient 2), but not other HLA-DQB1 alleles. Activation of PD-1 on T-cells is thought to inhibit antigen-specific T-cell priming and regulate T-cell differentiation. Thus, this study sought to measure the drug-specific activation of naïve T-cells after perturbation of PD-L1/PD-1 binding and investigate whether PD-1 signalling influences the differentiation of T-cells. Naive T-cells were co-cultured with monocyte-derived dendritic cells in the presence of SMX.NO for a period of 8 days (±PD-1/2 block) and T-cell priming investigated using readouts for proliferation and cytokine secretion. Priming of naïve T-cells against SMX.NO was found to be more effective when PD-L1 signalling was blocked. Drug-specific TCCs generated through priming and from hypersensitive patients were found to secrete IFN-γ, IL-5 and IL-13. More detailed analysis revealed two different cytokine signatures. Clones secreted either FasL/IL-22 or granzyme B. The FasL/IL22 secreting clones expressed the skin homing receptors CCR4, CCR10 and CLA and migrated in response to CCL17/CCL27. PD-1 was stably expressed at different levels on clones; however, PD-1 expression did not correlate with the strength of the antigen-specific proliferative response or the secretion of cytokines/cytolytic molecules. In conclusion, this study used a variety of in vitro assays to investigate the multiple factors involved in the pathomechanism of SMX hypersensitivity. A clear understanding of mechanisms of drug hypersensitivity will provide insights that aid drug design and reduce the frequency of such reactions.

Warfarin is a challenging drug to dose accurately, especially during the initiation phase because of its narrow therapeutic range and large inter-individual variability. Therefore, the aim of this thesis was to investigate the use of pharmacogenetics and clinical data to improve warfarin therapy. Genetic variants in cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase (VKORC1) are known to influence warfarin dose. Therefore we developed a pharmacogenetic dosing algorithm to predict warfarin stable dose prospectively in a British population based on 456 patients who started warfarin in a hospital setting and validated it in 262 retrospectively recruited patients from a primary care setting. The pharmacogenetic algorithm which included CYP2C9*2, CYP2C9*3 and VKORC1-1693 together with body surface area, age and concomitant amiodarone use, explained 43% of warfarin dose variability. The mean absolute error of the dose predicted by the algorithm was 1.08 mg/day (95% CI 0.95-1.20). 49.6% of patients were predicted accurately (predicted dose fell within 20% of the actual dose). The HAS-BLED score, a bleeding risk score has recently been suggested for use in the management of patients with atrial fibrillation. We validated HAS-BLED performance in predicting major bleeding using a prospective cohort with 6 months follow-up (n=482) (c-statistic 0.80 95% CI (0.71-0.90). Factors significantly associated with major bleeding in our cohort (p≤0.1) were concurrent amiodarone use, labile INR, concurrent clopidogrel use, bleeding predisposition, concurrent aspirin use and CYP2C9*3. Adding a genetic covariate (CYP2C9*3) to the HAS-BLED score did not significantly improve its performance in predicting major bleeding. Considering CYP2C9*3 is a rare allele, our study was underpowered and requires further investigation in a larger cohort. A retrospective study of 97 Caucasian children was conducted to gain greater understanding of the factors that affect warfarin anticoagulant control and response in children. Results from multiple regression analysis of genetic and non-genetic factors showed that indication for treatment (Fontan or non-Fontan group), VKORC1 -1693, and INR group explained 20.8% of variability in proportion time in which INR measurements fell within the target range (PTTR); CYP2C9*2 explained 6.8% of the variability in INR exceeding target range within the first week of treatment; CYP2C9*2, VKORC1 -1693, age and INR group explained 41.4% of warfarin dose variability and VKORC1 -1693 explained 8.7% of haemorrhagic events. The contributions of CYP2C9 and VKORC1 polymorphism were small in the above outcomes. We therefore went onto explore other genetic markers using genome-wide scanning. Two SNPs on chromosome 5, rs13167496 and rs6882472 were found to be significantly associated at a genome-wide significance level with PTIR. However, none of SNPs were significantly associated with warfarin stable dose, INR values exceeding the target range within the first week of treatment and bleeding complications. Because of our small sample size, these findings will need to be validated in a replication cohort. Finally, we have validated and evaluated the performance of Genie HyBeacon®, a point of care therapy (POCT) instrument to genotype 135 samples for CYP2C9*2, CYP2C9*3 and VKORC1 -1693. We showed that the instrument accuracy was >98% (agreement with ABI Taqman® genotyping), it was relatively simple to use and had a good turn-around time (1.6 hours) making it suitable for clinical use. In conclusion, the results presented in this thesis demonstrate how knowledge of pharmacogenetics may help in assessing improvement in the quality of care of patients on warfarin. However, for personalized medicine to be widely adopted in clinical practice, payers need evidence of clinical- and cost-effectiveness. How such evidence is produced and evaluated varies in different healthcare settings, which further increases the challenge of implementing personalised medicine into the clinic.

The failure of highly active antiretroviral therapy may be due to pharmacological factors such as drug transporters and metabolism enzymes. Drug transporters and metabolism enzymes played complementary roles in drug absorption, distribution, metabolism and excretion by biotransformation and counter-transport, particularly in the intestine while nuclear receptors as transcription factors regulate the expression of drug transporters and metabolism enzymes. In this thesis, a positive correlation between nuclear receptors expression and the expression of ABC transporters and OATP transporters in intestine were observed while a negative correlation was found between the gene expression of nuclear receptors and cytochrome P450 enzymes in intestine. Single nucleotide polymorphisms in genes could potentially impact on gene expression of drug transporters and metabolism enzymes. The polymorphisms of nuclear receptors were associated with the expression of ABC transporters. Drug concentrations have a high inter-individual variability in patients receiving the same dose of antiretroviral drugs, which could affect outcome of antiretroviral therapy. There are many factors that may affect plasma concentrations such as age, gender, body weight, ethnicity, genetic factors and so on. In a Ghanaian cohort, a negative correlation was found between the body weight and the EFV plasma concentration. Genetic factors such as the polymorphisms of cytochrome P450 enzymes also influenced efavirenz plasma concentrations. Meanwhile, efavirenz plasma concentrations were associated with the viral load in plasma within a UK cohort. Nanomedicine involves new and promising technologies that may enable and improve the targeted delivery of antiretroviral drugs. The permeability of lopinavir in the Caco-2 cell line was improved by formulation of nanodispersions. However, the permeability of efavirenz was decreased for all nanodispersions in MDCKII and MDCKII-ABCB5 cell lines. Comparing efficiency of efavirenz nanodispersions transcellular permeability in MDCKII and MDCKII-ABCB5 cell lines indicated that ABCB5 is able to transport efavirenz when incubated as dissolved molecule or nanodispersion. It is support by the copy number variation of ABCB5 had no relationship with EFV plasma concentrations. In summary, this thesis has attempted to determine the pharmacological factors influencing pharmacokinetics of HIV drugs, including drug transporters, metabolism enzymes and nuclear receptors. Data illustrating the factors that influence efavirenz plasma concentrations which are important for viral suppression were also generated. Furthermore, the nanodispersion technology is worthy of further study in order to improve drug delivery and drug distribution of antiretroviral drugs.

Dendritic cells (DCs) are potent innate antigen presenting cells which are able to sense and engulf pathogens from sites of infection, which are then processed and presented to adaptive T lymphocytes in secondary lymphoid organs. Therefore, they are critical for the initiation and modulation of primary antigen-specific adaptive immune responses. They also play a critical role in the maintenance of T cell tolerance. T cells play vital roles in mediating both cellular and humoral-specific adaptive immune responses. There are various types of T cells present within the immune system including the cytotoxic CD8 T cells and T helper CD4 cells. CD4 T cells can be further subdivided into various subtypes examples of which include T helper 1 cells (Th1), Th2, Th17 and T regulatory cells. Each subset has its own distinctive function, transcriptional regulation and effector cytokine profile. It is established that appropriate DC and T cell immune function is highly dependent on their intracellular redox status. Cellular redox homeostasis is maintained through a balance between oxidising agents e.g. reactive oxygen species (ROS) and anti-oxidant or reducing agents e.g. glutathione (GSH). Excessive ROS production resulting in oxidative stress is extremely deleterious to the cell and if left unimpeded can result in cell necrosis, tissue damage and the onset of disease. As a result, mammalian cells have evolved an inducible adaptive defence system which provides protection against such oxidative or chemical insult. The functionality of this cellular defence system is principally governed by the activity of the redox-sensitive transcription factor Nrf2. In response to oxidative stress, Nrf2 induces the transcription of a battery of cytoprotective and antioxidant genes involved in GSH synthesis, detoxification of xenobiotics and their reactive metabolites and the maintenance of cellular redox homeostasis. It is now emerging that Nrf2 plays a pivotal role in immunity. However, its precise role in DC and T cell function is unclear. Using immature bone marrow-derived DCs (iDCs) from Nrf2+/+ and Nrf2-/- mice, the work presented in this thesis demonstrates that Nrf2 deficiency in iDCs resulted in lowered GSH levels, enhanced iDCs co-stimulatory receptor expression, impaired endocytic and phagocytic capacity, and increased iDC-mediated antigen-specific CD8 T cell stimulatory capacity in response to both an antigenic and self-peptide. Furthermore, artificially lowering GSH levels in the iDCs did not recapitulate the Nrf2 deficient iDC phenotype. Moreover, Nrf2-/- DCs exhibited an enhanced capacity to present cell-associated peptide antigens to antigen-specific CD8 T cells, resulting in increased CD8 T cell effector function. Loss of Nrf2 in LPS-stimulated DCs results in a lowered Th1 cytokine profile. These results have implications for Nrf2 in DC-mediated CD8 T-cell immunity, peripheral CD8 T cell tolerance and CD4 effector differentiation. The role of Nrf2 in T cell function is poorly understood. Using thymocytes, splenocytes and lymph-node derived T cells from Nrf2+/+ and Nrf2-/- mice, we demonstrate that loss of Nrf2 did not affect the development of CD4+CD25+ natural occurring Treg, mature CD4 and CD8 T cell populations within the thymus. Furthermore, Nrf2 deficiency did not alter the composition of CD4 and CD8 T cell populations within secondary lymphoid organs. It was observed that splenic Nrf2-/- naïve T cells exhibited enhanced ROS generation, accompanied by low level increases in T cell activation markers. However, the marginal augmentation of Nrf2-/- naïve T cell activation status did not result in increased T cell receptor (TCR)-triggered T cell proliferation. In contrast, Nrf2 deficient effector T cells exhibited enhanced TCR/CD3-triggered proliferation, associated with increased Th1 and decreased Th2 effector function. Importantly, we demonstrated that Nrf2-/- effector T cells secreted increased levels of IL-17A and IL-22, a signature cytokine profile indicative of the more recently identified Th17 cell lineage. This was also observed under Th17 polarising conditions, further suggesting that loss of Nrf2 predisposes effector Th17 development. The implications of the latter findings are significant given the pivotal role that Th17 cells play in the pathogenesis of a variety of autoimmune diseases including multiple sclerosis (MS) and Systemic lupus erythematosus (SLE). Nrf2 plays a critical role in the detoxification of xenobiotics in the liver, which as the primary drug-metabolising organ, is subjected to an array of xenobiotics and their respective metabolites. Individuals vary in their responses to xenobiotic exposure from adaptation to severe adverse drug reactions. However, it is unknown whether this human disparity in drug response is a consequence of inter-individual variation in the Nrf2 adaptive defence system to xenobiotic stress. In light of this, we aimed to firstly investigate whether variation in the Nrf2 adaptive system was present within individuals in response to a chemical inducer of Nrf2, CDDO-Me. To address this issue, basal and induced Nrf2 protein levels and downstream NQO1 expression were measured in activated human T cell blasts, in response to increasing concentrations of CDDO-Me. Examination of various donor-derived T cells, demonstrated that humans vary in their Nrf2 response to CDDO-Me, with respect to nuclear Nrf2 and NQO1 mRNA expression. Therefore we concluded that inter-individual variation does exist in the human’s Nrf2 adaptive system in response to a known Nrf2 probe. Overall, the experimental results obtained in this PhD programme have provided detail on the role of Nrf2 in immune cells and highlights the potential for therapeutically targeting Nrf2 in immune-mediated disease.

Adverse drug reactions (ADRs) are a major problem for drug companies and healthcare providers alike. Although ADRs can present anywhere in the body, the liver frequently effected, due to the relatively large concentrations of drugs it encounters. Drug induced liver injury (DILI) can occur through several different mechanisms. Bioactivation of drugs to reactive metabolites is believed to be a crucial step in the development of many cases of DILI. Nefazodone, an anti-depressant which was withdrawn due to hepatotoxicity, has been shown to be bioactivated to a reactive quinone-imine. The role of the mitochondria and their involvement in DILI is being increasingly recognised. The biguanides are known mitochondrial toxins, and phenformin and buformin were removed from the market due to unacceptably high incidents of lactic acidosis. The aims of this thesis were two-fold; to assess the bioactivation and irreversible binding of nefazodone and it safer analogue buspirone and to use the biguanides to assess mitochondrial toxicity in primary hepatocytes. In liver microsomes, both nefazodone and buspirone demonstrated NADPH-dependent irreversible binding, however, nefazodone irreversible binding was 9-fold that of buspirone. The metabolism of both nefazodone and buspirone was extensive and consisted mainly of hydroxylation and N-dealkylation reactions. In rat and human liver microsomes supplemented with GSH, nefazodone formed GSH conjugates with m/z 791 and m/z 807. This implied that the conjugates were formed from bioactivation of para-hydroxy nefazodone and dihydroxy-nefazodone. In rat liver microsomes, buspirone did not form any GSH conjugates. Further investigations of nefazodone and buspirone were carried out in freshly isolated rat hepatocytes. Metabolism of nefazodone and buspirone was investigated and revealed extensive metabolism of both compounds; however, GSH conjugates of neither compound were discovered. At 6 hours, both nefazodone and buspirone demonstrated significant irreversible binding (117.54±15.32 pmol equiv./mg protein and 84.43±30.93 pmol equiv./mg protein respectively) but only nefazodone demonstrated a significant decrease in cell viability (19.25±18.26 % control viability). Inhibition studies, using ABT, significantly reduced the irreversible binding of nefazodone (49.34±4.64 pmol equiv./mg protein) but did not decrease cytotoxicity. This indicated that in rat hepatocytes the parent compound may be responsible for toxicity. Mitochondrial toxicity was investigated using the model mitochondrial toxins the biguanides. Initially, studies in cultured primary rat hepatocytes demonstrated that phenformin was the most potent mitochondrial toxin and dissipated the mitochondrial membrane potential, as measured by TMRM, within 24 hours (1.028±0.39% control fluorescence). This was taken forward to investigate mitochondrial toxicity in primary rat hepatocytes. Investigations into phenformin in rat hepatocytes demonstrated a high turnover to glucuronide metabolites and the novel metabolites [O, OMe] phenformin glucuronide and [2O] phenformin glucuronide were identified. Inhibition studies of CYP450 2D were undertaken using quinine (100µM) and this demonstrated significant inhibition of phenformin up to 200µM (AUC 47.30±47.30 without quinine vs AUC 648.80±121.28 with quinine). Despite increased phenformin concentrations, inhibition of phenformin metabolism did not produce overt cytotoxicity, however, lactate concentrations correlated with increased phenformin concentration. The work presented here highlights the need for a greater understanding of the role of bioactivation and irreversible binding in hepatotoxicity. It also demonstrates that whilst irreversible binding can help inform decisions as to whether a compound progresses into clinical trials, it should be made in the context of other safety assessments. Investigations into phenformin mitochondrial toxicity, illustrates the need to assess drugs and systems fully, to establish model compounds to investigate mechanisms of ADRs. A greater understanding of in vitro systems and the tools utilised to assess them, will benefit drug discovery and development. Ultimately, understanding these in vitro tests and the model compounds used to assess them, will help bridge the gap to man.