Tuberculosis (TB) remains one of the most devastating diseases in humans. Nowadays, tuberculosis therapy is not sufficient to control the TB epidemic and only lasts for 6 months to cure patients and prevent relapse; therefore, the treatment of Mycobacterium tuberculosis (Mtb) is particularly challenging (1). New antibiotics, mainly those that are derived from new chemical classes, are more likely to be more effective against resistant strains. Moreover, expanding the knowledge of the mode of action of drugs has important implications in tackling TB. Only empirical approaches can be adopted in the journey of discovering new anti-tubercular drugs until a clear picture of latency and persister cells’ physiology is achieved. Mtb has the extraordinary ability to survive under hypoxia, suggesting a high degree of metabolic plasticity. The flexibility conferred by a modular respiratory system is critical to the survival of Mtb, thereby also making it a promising area of research for new drug targets. This thesis aimed towards the characterisation of cytochrome bd-I quinol oxidase (bd-I), a respiratory component that is believed to operate during both the replicative and “dormant” Mtb phenotypes. The essential nature of Mtb bd-I, which has no human homologue, has been confirmed in a recent deep sequencing study of genes required for Mtb growth by Griffin et al. (2), further confirming its potential as a novel target. Recombinant Mtb bd-I was successfully expressed under the control of the pUC19 lac promoter in the Escherichia coli ML16 bo3/bd-I and MB44 bo3/bd-I/bd-II knockout strains, allowing “noise-free” measurement of the enzyme. Initial steady-state kinetics of the enzyme was presented using a range of quinol substrates, revealing a substrate preference for dQH2 over Q1H2 and Q2H2. A number of bd-I inhibitors were identified and their pharmacodynamic profiles against Mtb H37Rv were determined. In addition, a pharmaco-metabolomics platform was initiated to explore the cellular response of Mtb to current first-line TB drugs as well as in house bd-I and type II NADH inhibitors. The initial findings are discussed in the context of the known mode of action of the drugs and future research needs in drug discovery of this devastating disease.

Introduction: Glucose-regulated protein 78-kDa (GRP78) is an endoplasmic reticulum (ER)-resident molecular chaperone that is essential for correct protein folding and assembly in the ER lumen. Micro-environmental stress and a requirement for increased protein synthesis, typical of solid tumours, leads to a disruption of ER homeostasis, and accumulation of misfolded proteins. The ability of GRP78 to dissociate from several important ER-resident transmembrane proteins under conditions of ER stress leads to a cascade of signal transduction pathways, known as the unfolded protein response (UPR), that modulate cell survival or, if the stress is significantly severe, apoptosis. GRP78 has been found to be overexpressed in a variety of cancers compared with benign tissue and has been associated with poor outcome. In-vitro data indicate that GRP78 expression is often associated with aggressive phenotype and drug resistance. Thus, GRP78 has potential as a biomarker for tumour behaviour and treatment response. For stage III colorectal cancer, there is overwhelming evidence to recommend the use of fluoropyrimidine-based adjuvant chemotherapy. Unfortunately, a large proportion of patients do not benefit from adjuvant chemotherapy, and biomarkers that can determine the likelihood of response to chemotherapy remain elusive. The benefit of chemotherapy in stage II disease is less certain and markers that could reliably predict benefit would be particularly useful in this population. This study explores a potential mechanistic relationship between GRP78 and 5-FU sensitivity using both siRNA transfection and treatment with an engineered fusion protein, epidermal growth factor (EGF)-SubA, which has been demonstrated to cause highly selective cleavage of GRP78 at a single amino acid point. It was then examined whether GRP78 may have prognostic or predictive value in the context of colorectal cancer patients treated with fluoropyrimidine-based chemotherapy. The potential therapeutic value of targeting GRP78 in vitro using EGF-SubA is also examined. Methods: Colon cancer cell lines were used to examine response to 5-FU upon modulation of endogenous GRP78 using siRNA technology and EGF-SubA. Apoptosis and cell cycle progression were assessed using flow cytometry. Immunohistochemistry was used to characterise GRP78 expression in a large cohort of colorectal cancers on tissue microarrays and the results were correlated with clinicopathological parameters and with 5-year survival for the whole cohort and those treated with fluoropyrimidine-based (5-FU) adjuvant chemotherapy. The action of EGF-SubA upon colon cancer cells was examined using western blotting, MTT assay and flow cytometry. Results: GRP78 promotes apoptosis in response to 5-FU. Better overall 5-year survival was associated with high GRP78 expression (P=0.036). Stage III patients with high GRP78 showed significant benefit from adjuvant chemotherapy (P=0.026), whereas patients with low GRP78 failed to benefit (P=0.805). Low GRP78 was an independent poor prognostic indicator of overall 5-year survival (P=0.005; HR=1.536; 95%CI 1.139-2.122). Colon cancer cells expressing EGFR were highly sensitive to EGF-SubA, demonstrating reduced proliferation and cell cycle arrest. However, EGF-SubA did not induce significant apoptosis and reduced the effectiveness of 5-FU in vitro. Conclusion: This study demonstrates a mechanistic relationship between GRP78 expression and response to 5-FU. GRP78 expression may provide a useful additional risk stratification to inform the adjuvant treatment of colorectal cancer. EGF-SubA does not have therapeutic value in colorectal cancer but is a useful tool for studying GRP78 and the UPR.

Cardiovascular toxicity is a leading cause of drug attrition at the preclinical and clinical stages of drug development. The specific mechanisms of drug-induced cardiovascular toxicities, however, are not well understood and can occur via direct interactions of the drug with cardiomyocytes or indirectly through interactions with other components of the cardiovascular system. Indirect drug effects can target non-cardiomyocyte cells such as fibroblasts, macrophages, vascular smooth muscle cells and endothelial cells. Evidence suggests interactions between these cell types are essential to the metabolism, growth, contractile performance and rhythmicity of the myocardium (Brutsaert, 2003). Preclinical and clinical drug safety tests primarily focus on cardiomyocytes, ignoring the other cellular components of the myocardium. High content biology in combination with ATP content as a measure of cytotoxicity was used to assess the sensitivities of endothelial and fibroblast cells from different vascular beds to known structural cardiotoxins. This assay principle has previously been successful in the detection of structural cardiotoxicity in human embryonic stem cell derived cardiomyocytes (hESC-CM’s) (Pointon et al., 2013). The endothelial cells displayed no significant difference in their compound responses, while the fibroblasts showed variation; imatinib was more potent in the cardiac fibroblasts and lapatinib more potent in the dermal fibroblasts. When the sensitivities of the cells from the myocardium (human cardiac fibroblasts (hCFs), human cardiac microvascular endothelial cells (hCMECs) and hESC-CMs) were globally compared the non-myocytes were sensitive to structural cardiotoxicity at the acute time point of 6 hour (h), whereas the hESC-CMs display toxicity only after 72 h. The conclusions from this work are that non-myocyte cells play a role in drug-induced cardiovascular toxicity since early sensitivity is displayed, however current in vitro models lack the complexity required to investigate the translation of this in vivo. Multiple types of cardiac microtissue models were developed and their responses to reference structural cardiotoxins and inotropes evaluated using ATP content and video-based edge monitoring of contractility, respectively. Compound responses highlighted the promotion of maturity in the cardiac tri-cultured microtissue. Further investigations into gene expression and calcium (Ca2+) handling suggested SR function and Ca2+ handling maturity had been promoted and that the ca2+ handling protein S100A1 plays a critical role in this maturity. These findings show adult cardiac non-myocyte cells can be used to promote contractile maturity of hESC-CM’s in vitro when co-cultured as a three dimensional (3D) microtissue. The induction of maturity was very much multi-parameter dependent requiring a) both non-myocyte cell types, b) cells of cardiac origin and c) a 3D culture environment. More advanced human relevant in vitro models that better reconstitute the in vivo cellular physiology of the heart could allow improved in vitro to in vivo correlation in future drug safety screens.

Epilepsy is a chronic neurological disorder which can arise following an initial insult that over time, progresses into a condition characterised by recurrent spontaneous seizures. During a latent period between the initial insult and the epilepsy condition proper, major changes occur within the brain at both a cellular and molecular level, in a process known as epileptogenesis. It is postulated that during epileptogenesis, signal transduction pathways are perturbed following the initial insult, which may bring about long term changes in gene expression profiles. For example, the expression of a host of neuropeptides is known to be modulated in response to an initial insult, including the up-regulation of the pro-convulsant tachykinins Substance P and Neurokinin B, encoded by the TAC1 and TAC3 genes, respectively. In this thesis I have explored the regulation of both of these genes by two distinct transcription factor (TF) families; the Neuron Restrictive Silencing Factor (NRSF) isoforms, and the Upstream Stimulatory Factor (USF) proteins. I demonstrate that both NRSF and USF variants regulate TAC3 promoter activity, and that NRSF isoforms can modulate endogenous NKB expression in a human neuroblastoma cell line. Furthermore, these distinct TF families are shown to work in cooperation to regulate the activity of the rat TAC1 promoter. Thus, both NRSF and USF variants are shown to be important in the regulation of pro-convulsant neuropeptides and as both NRSF and USF proteins have been shown to be induced by pro-convulsant stresses here, they are potential key TFs in epileptogenesis, responding to an initial insult, and orchestrating downstream gene expression changes. Consistent with such a model, I have also revealed that both NRSF and USF variants are modulated by anti-convulsant drug treatment. Here, three distinct anti-convulsant drugs, were found to differentially modulate the expression of both the full-length NRSF, and its truncated isoform, as well as the USF proteins USF1 and USF2. Furthermore, whilst the drugs had limited impact upon the localisation of these TFs in human neuroblastoma cells, they did affect the binding of these TFs to target DNA sequences, particularly NRSF binding to its recognition DNA sequence, the NRSE, in a number of genes. In addition, due to an increasingly appreciation of the role of cocaine and the dopaminergic pathways in seizure progression, I explored the impact of cocaine treatment on the expression of these TFs. Cocaine was found to modulate both NRSF and USF variant expression, and NRSF binding to target DNA sequences. These findings suggest that both NRSF and USF variants are important in epileptogenesis and are targets for modulation by the anti-convulsant drugs investigated here. To further explore the significance of NRSF expression in seizure progression, I explored the impact of over-expression of NRSF isoforms, modelling that which occurs in response to seizure in animal models, on global gene expression pathways. I reveal that NRSF isoform over-expression significantly modulates the expression of a host of genes with known associations with epilepsy, supporting a model that NRSF isoforms are key TFs which respond to the initial insult and coordinate long-term changes in gene expression. These findings may help our understanding of the molecular mechanisms at work during epileptogenesis, and may better our understanding of the progression of epilepsy.

Human herpes virus 6 (HHV6) infects the majority of individuals in childhood, followed by a lifelong asymptomatic latent infection. However, in immunosuppressed individuals reactivation of HHV6 can cause significant clinical pathology. Recent successes with adoptive T cell therapy against other viral infections, notably the human herpes viruses Epstein-Barr virus (EBV) and Human cytomegalovirus (HCMV), suggest that this may be a useful therapeutic approach for HHV6-driven disease in immunosuppressed individuals. However, very few studies have been carried out analysing the immune response to HHV6 in any detail. This thesis was aimed at characterising the CD8+ T cell response to HHV6 in a group of healthy individuals, with the aim of mapping and characterising novel CD8+ T cell epitopes. Initial studies included four HHV6B antigens (U11, U39, U54 and U90), predicted to be immunogenic based on their HCMV homologues. Whole antigen peptide mixes (pepmixes) were used to stimulate peripheral blood mononuclear cells (PBMC) from healthy subjects. T cell responses were analysed by intracellular cytokine staining (ICS) after overnight stimulation and/or by interferon-γ (IFN-γ) ELISpot assay after 10 days of stimulation. For responses to U11 and U90, peptides libraries were used to map minimum CD8+ restricted epitopes. Further characterisation of HHV6B-specific T cells was carried out by identifying the HLA restriction elements and determining whether these T cells were capable of killing HHV6B-infected cells. PBMC from 30 healthy donors were stimulated with pepmixes corresponding to HHV-6B antigens U11, U39, U54 and U90. A weak CD8+ response (0.02-0.2%) to U90 and U54 was observed in a number of donors. Short-term in-vitro reactivations of PBMC (in 25 healthy donors) with HHV6B pepmixes followed by analysis of antigen and peptide specific response were performed by IFN-γ ELISpot assay. T cell responses to U54, U90, U11 and U39 were observed in 88%, 84%, 76% and 72% of the donors, respectively. Subsequently, the breadth of epitope specificity within U90 and U11 was screened for 9 healthy donors; with successful identification of 10 CD8+ T cells specific (9-mer) epitopes within these antigens. Seven of them were within U90 antigens and three of them were within U11 antigens. Allelic association of the U90 epitopes were; VEESIKEIL - B40 (60), FESLLFPEL - B40 (60), NLITAAKNI - A2, ITAAKNIGI - A2, LNIDPSESI - A1, PSKSKKIKL - A29, NHCFINHFV - B39. Allelic association of the 2 U11 epitopes were LKTQRRHKF - B37 and GILDFGVKL - A2; the HLA association for FNAVYSQRV was not identified. CD8+ T cell populations specific to some of these epitopes were also able to kill HHV6B infected cells. HHV6B T cells responses are detectable in healthy donors. Peptide specific responses against U11 and U90 have been mapped and characterised. These findings are relevant to the development of T cell mediated immunotherapy of HHV6-associated diseases.

Patients living with many cancers, including ovarian cancer (OC), often suffer from a lack of adequate treatment options. In the case of OC, primary debulking surgery followed by platinum and paclitaxel chemotherapy has led to a vast improvement in patient survival over the past few decades, however, rates of drug-resistant recurrence remain high. Research into new, experimental treatment options is therefore warranted for OC and other cancers. Oncolytic viruses (OVs) are replication-competent viruses that can selectively infect and destroy cancerous cell types, while leaving healthy cells unharmed. OVs do this by exploiting differences between cancer and normal cell phenotypes. Herpes simplex virus (HSV)-1, strain 1716 is one example of this type of virus that has shown selectivity for cancer cells in previous preclinical studies, as well as high levels of safety in humans. One prominent area of current OV study seeks to investigate the ability of OVs to induce immunogenic cell death (ICD) – this term describes multiple modes of programmed death pathways that culminate in release of proimmunogenic factors, which facilitate a modification of the host immune system. Two of the most prominent of these pathways are necroptosis and immunogenic apoptosis (IA). Here, I show that while many OV cell lines express the necessary components for necroptosis, they are unable to undergo classical necroptotic death (induced by TSZ). Despite this, HSV-1716 can infect and kill a range of OC lines successfully. I showed that HSV-1716-induced cell death displays two markers of IA yet does not seem to rely solely on apoptosis to kill cells. In addition, it appears not to rely on any components of the necrosome in order to kill cells, even in cells that are competent to typical necroptosis. However, when RIPK3 is overexpressed in HeLa cells, virus-induced cell death increases, as do markers of both necroptosis and IA. To investigate the role of ICP6 in HSV-1716-induced ICD, viral and cell mutants were made possessing various forms of the protein. Full-length ICP6 protein expressed in cell lines had the effect of blocking cellular response to TSZ, but constructs lacking a region known as the RHIM did not. A functionally similar mutation was produced within the RHIM of live HSV-1716 using CRISPR/Cas9 technology, which was shown to have the effect of disrupting ICP6/RIPK3 binding – thought to be the determinant of necroptotic cell death. Despite this, no changes in cell death signalling could be determined between the viruses at all. Interestingly, when cells were infected in combination with TNF-α, or TNF-α in addition to SMAC mimetic, the RHIM-modified virus produced significantly more death than HSV-1716. This suggests that while loss of RIPK3 inhibition is not sufficient to lead to increased necrosis alone, cells infected with this virus are more sensitive to further necrosis induction. This finding may prove to have great utility for producing the next generation of oncolytic viral therapeutics which can induce greater levels of proimmunogenic cell death. From this we can conclude that HSV-1716 is capable of inducing IA in OC cells. Death is not dependent on necroptosis, however additional RIPK3 seems to sensitise cells to death by other means. Cellular binding of viral ICP6 and RIPK3 can be disrupted by modification of the RHIM, although this change has no bearing on ICD signalling alone but can sensitise cells to TNF-α-induced death.

China is an emerging force in undertaking randomised clinical trials. The quality of trials from China may affect not only its own substantial population but also potentially contribute to health policy throughout the world. However, little is known about the quality of clinical trials conducted there. In this thesis, I will evaluate the quality of published Chinese randomised controlled trials (RCTs) by comparing them with Indian RCTs as well as a set of ‘gold standard’ trials reported in leading European and North American journals. I will also describe and contrast the quality and biases within Chinese RCTs. I then explore the reasons for these differences from the point of view of the major RCT stakeholders: Chinese clinicians and patients. The potential influences from Chinese traditional culture is also evaluated. Chinese medical journal editors need to undertake more training on the reporting of RCTs; all medical societies should take more concern about doctors’ research work; the Chinese public media should help the general population to understand more about RCT principles.

Osteoarthritis is the most widespread musculoskeletal disease worldwide among the elderly. It causes joint pain that can affect locomotion and reduce mobility. For osteoarthritis patients, maintaining walking ability is considered the most beneficial way to preserve their quality of life. Walking sticks are widely used by elderly adults and have been shown to have a supportive role on locomotion. In this thesis I carried out four experiments: The first study investigated how footwear affects the locomotion of elderly patients suffering from this disease. In the second chapter the gait of elderly walking stick users was analysed in conjunction with their responses to a questionnaire with a view to understanding the causes and context of walking stick use in their everyday environments. My findings demonstrated that the majority of participants experienced greater pain after prolonged use of their walking stick. In the last two experiments I investigated how the use of a walking stick combined with aspects of the individual’s locomotor environment (e.g. indoor and outdoor, level and sloped surfaces) to influence gait. Overall, osteoarthritis, advanced age and challenging locomotor environments can influence their quality of life and the risk of falling.

The liver has a unique connection with the immune system; harbouring vast numbers of lymphocytes, able to instigate secondary lymphoid organ-independent naive T cell activation, and promoting potent immune tolerance. We set out to determine the effect of this unique microenvironment on the biology of CD4+ T cells at three key interaction points: following migration into the parenchyma, after short-term hepatocyte contact, and at long-term tissue-residency. Modelling transmigration through hepatocytes revealed intrinsic, disease-specific cytokine responses in blood-derived CD4+ T cells, not discernible through static co-culture. However, short-term co-culture did induce activation-independent CD69 upregulation, reliant upon cell-cell contact. This phenotype mimicked the similar hepatic CD4+ CD69INT cells that we discovered in liver tissue. Unlike CD69HI cells which represented the tissue-resident memory T cells (TRM) of the liver, CD69INT cells were the most activated population, likely able to migrate to many liver and gut niches, and singularly able to produce IL-4 and IL-10. By contrast, CD69HI TRM displayed a resting phenotype, marked for more restricted movement, and produced the best multifunctional TH1 responses following stimulation. These data demonstrate the importance of studying migration, and provide detailed characterisation of CD69HI TRM and novel CD69INT cells, along with their proposed roles and generation pathways.

Heterotopic ossification (HO) is the pathological formation of ectopic bone and can be a devastating complication of military injuries. It causes multiple problems, including prosthetic limb fitting difficulties and ankylosis, ultimately causing loss of mobility, independence, and dignity. There is no effective prophylaxis and the only treatment is surgery, which causes rehabilitation delays and risks of bleeding and nerve injury. This work aimed to develop a new therapy for HO. A review of mineralised tissue analytical methods was performed and used to guide the study of human HO samples using X-ray micro computed-tomography, X-ray fluorescence, synchrotron X-ray diffraction/nano-tomography, Raman spectroscopy, and scanning electron microscopy. Screening of therapies demonstrated that hexametaphosphate (HMP) could dissolve hydroxyapatite (the mineral component of bone) under physiological conditions. Formulation engineering principles were applied to control this effect temporally and anatomically. A murine HO model was developed and used for feasibility testing of injected HMP as an HO therapy, showing that it had no adverse effect on normal bone. In summary, this project revealed the physicochemical structure of HO in unprecedented detail, discovered and developed a novel therapy, set up an animal model of HO, and showed that injected HMP is feasible in this model.