Streptococcus pneumoniae is the leading bacterial cause of pneumococcal diseases, of which pneumonia is the main cause of death amongst the immunocompromised, elderly over the age of 50 and children under the age of 5. Although vaccines such as pneumococcal polysaccharide vaccine 23, pneumococcal conjugate vaccine 7, 10 and 13 are available, they are expensive to produce and distribute. Moreover, the variation in serotype distribution across geographical locations and rise in dominance of disease due to non-vaccine serotype coverage has led to significant attention towards the development of alternate vaccine candidates such as pneumococcal surface protein A (PspA). A potential dry powder vaccine formulation containing polymeric nanoparticles (NPs) adsorbed with PspA4Pro and formulated into nanocomposite microparticles (NCMPs) using L-leucine (L-leu) to be delivered via inhalation was developed. Poly(glycerol adipate-co-ω-pentadecalactone), PGA-co-PDL, NPs with either anionic or cationic surface charge of optimum size (~200-250 nm) to be effectively taken up by the lung dendritic cells (DCs) were successfully produced. The NPs were then surface adsorbed with PspA4Pro (~20 µg of PspA4Pro per mg of NPs) and spray-dried using L-leu as a microcarrier to produce NCMPs with a product yield of 55.55±6.64% for the PspA4Pro adsorbed anionic NPs/NCMPs and 53.98±2.23% for the PspA4Pro adsorbed cationic NPs/NCMPs. The NCMPs produced had a corrugated and wrinkled surface morphology. The aerosol properties of anionic NPs/NCMPs determined using a Next Generation Impactor displayed a fine particle fraction (FPF) of 74.31±1.32% and mass median aerodynamic diameter (MMAD) of 1.70±0.03 μm indicating that the majority of the dose would be deposited in the respirable airways of the lungs. The anionic and cationic PGA-co-PDL NPs upon incubation with DCs for 1 h showed an effective uptake as visualised using confocal microscopy. Furthermore, the anionic NPs/NCMPs were well tolerated by the A549 cell line with a cell viability of 87.01±14.11% at 1.25 mg/ml concentration, whereas the cationic NPs/NCMPs showed a cell viability of 75.76±03.55% at 156.25 µg/ml concentration upon 24 h exposure. The PspA4Pro released from the optimised formulations largely maintained its structure as determined using SDS-PAGE and circular dichroism, and the relative antigenicity measured using ELISA was 0.97±0.20 and 0.85±0.05 for anionic and cationic formulations, respectively. Overall, the results obtained indicate the use of these NPs as novel carriers for pulmonary vaccine delivery against pneumococcal diseases.

Suppressor of cytokine signalling 3 (SOCS3) is a potent inhibitor of the mitogenic, migratory and pro-inflammatory pathways responsible for the development of neointimal hyperplasia (NIH), a key contributor to the failure of vascular reconstructive procedures. However, the protein levels of SOCS3, and therefore its potential to reduce NIH, is limited by its ubiquitylation and high turnover by the proteasome. I hypothesised that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consequently, the aim of this PhD was to identify the mechanisms promoting the rapid turnover of SOCS3. Initial experiments involved the identification of residues involved in regulating the turnover of SOCS3 at the proteasome. I assessed the ubiquitylation status of a panel of FLAG tagged SOCS3 truncation mutants and identified a C-terminal 44 amino acid region required for SOCS3 ubiquitylation. This region localised to the SOCS box which is involved in binding Elongin B/C and the formation of a functional E3 ubiquitin ligase complex. However, the single lysine residue at position 173, located within this 44 amino acid region, was not required for ubiquitylation. Moreover, Emetine chase assays revealed that loss of either Lys173 or Lys6 (as documented in the literature) had no significant effect on SOCS3 stability 8 hrs post emetine treatment. As mutagenesis studies failed to identify key sites of ubiquitylation responsible for targeting SOCS3 to the proteasome, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate was employed. These data were searched for the presence of a Gly-Gly doublet (+114 Da mass shift) and revealed 8 distinct sites of ubiquitylation (Lys23, Lys28, Lys40, Lys85, Lys91, Lys173, Lys195, Lys206) on SOCS3 however Lys6 ubiquitylation was not detected. As multiple Lys residues were ubiquitylated, I hypothesised that only a Lys-less SOCS3, in which all 8 Lys residues were mutated to Arg, would be resistant to ubiquitylation. Compared to WT SOCS3, Lys-less SOCS3 was indeed found to be completely resistant to ubiquitylation, and significantly more stable than WT SOCS3. These changes occurred in the absence of any detrimental effect on the ability of Lys-less SOCS3 to interact with the Elongin B/C components required to generate a functional E3 ligase complex. In addition, both WT and Lys-less SOCS3 were equally capable of inhibiting cytokine-stimulated STAT3 phosphorylation upon co-expression with a chimeric EpoR-gp130 receptor. To assess whether SOCS3 auto-ubiquitylates I generated an L189A SOCS3 mutant that could no longer bind the Elongins and therefore form the E3 ligase complex required for ubiquitylation. A denaturing IP to assess the ubiquitylation status of this mutant was performed and revealed that, despite an inability to bind the Elongins, the L189A mutant was poly-ubiquitylated similar to WT SOCS3. Together these data suggested that SOCS3 does not auto-ubiquitylate and that a separate E3 ligase must regulate SOCS3 ubiquitylation. This study sought to identify the E3 ligase and deubiquitylating (DUB) enzymes controlling the ubiquitylation of SOCS3. Our initial strategy was to develop a tool to screen an E3 ligase/DUB library, using an siARRAY, to sequentially knockdown all known E3 ligases in the presence of a SOCS3-luciferase fusion protein or endogenous SOCS3 in a high content imaging screening platform. However, due to a poor assay window (<2) and non-specific immunoreactivity of SOCS3 antibodies available, these methods were deemed unsuitable for screening purposes. In the absence of a suitable tool to screen the si-ARRAY, LC-MS-MS analysis of a SOCS3 co-immunoprecipitate (co-IP) was investigated. I performed a SOCS3 under conditions which preserved protein-protein interactions, with the aim of identifying novel E3 ligase and/or DUBs that could potentially interact with SOCS3. These data were searched for E3 ligase or DUB enzymes that may interact with SOCS3 in HEK293 cells and identified two promising candidates i) an E3 ligase known as HectD1 and ii) a DUB known as USP15. This thesis has demonstrated that in the presence of HectD1 overexpression, a slight increase in K63-linked polyubiquitylation of SOCS3 was observed. Mutagenesis also revealed that an N-terminal region of SOCS3 may act as a repressor of this interaction with HectD1. Additionally, USP15 was shown to reduce SOCS3 polyubiquitylation in a HEK293 overexpression system suggesting this may act as a DUB for SOCS3. The C-terminal region of SOCS3 was also shown to play a major role in the interaction with USP15. The original hypothesis of this thesis was that stabilisation of endogenous SOCS3 by inhibiting its ubiquitylation has the potential to limit vascular inflammation and NIH. Consistent with this hypothesis, immunohistochemistry visualisation of SOCS3, in human saphenous vein tissue derived from CABG patients, revealed that while SOCS3 was present throughout the media of these vessels the levels of SOCS3 within the neointima was reduced. Finally, preliminary data supporting the hypothesis that SOCS3 overexpression may limit the proliferation, but not migration, of human saphenous vein smooth muscle cells (HSVSMCs) is presented. It is expected that multiple E3 ligases and DUBs will contribute to the regulation of SOCS3 turnover. However, the identification of candidate E3 ligases or DUBs that play a significant role in SOCS3 turnover may facilitate the development of peptide disruptors or gene therapy targets to attenuate pathological SMC proliferation. A targeted approach, inhibiting the interaction between SOCS3 and identified E3 ligase, that controls the levels of SOCS3, would be expected to reduce the undesirable effects associated with global inhibition of the E3 ligase involved.

Ovarian cancer is the fifth most common cancer which affects women in the United Kingdom. The 5-year survival rate is less than 45% despite improvements in chemotherapeutic regimens. Platinum-based therapy in combination with other chemotherapy including paclitaxel is currently the best standard of care for ovarian cancer. However, many women relapse with drug-resistant disease and this has led to the development of alternative drug therapies. This research evaluated two statins, simvastatin and pitavastatin, in several ovarian cancer models. Statins competitively inhibit 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) in the mevalonate pathway, resulting in the cellular depletion of the isoprenoid, geranylgeranyl diphosphate, and a reduction in the prenylation and localisation of many proteins including Ras, Rho and Rab involved in cell signalling. This is likely to have contributed to the decrease in cell proliferation, induction of apoptosis, and simultaneous induction and inhibition of autophagy observed when ovarian cancer cells were exposed to statins, although the mechanisms remain poorly defined. The concentration of statins required to cause cell death in ovarian cancer cells was significantly higher than that achieved in patients receiving a standard 40 mg dose for hypercholesterolaemia. Continual inhibition of HMGCR for several days was necessary to induce cell death. Lipids consumed in the diet may reverse the cytotoxic effects of the statins, suggesting that patients receiving statins for cancer therapy may require dietary modification. Studies evaluating statins in combination with carboplatin or targeted therapeutics demonstrated limited synergy, and in some cases, profound antagonism, and therefore, statins may be best evaluated as single agents. Statins retained cytotoxic activity in ovarian cancer cells resistant to chemotherapy, supporting the use of statins in chemoresistant disease. These observations will help to inform the design of future clinical trials evaluating statins in ovarian cancer.

Background: It is now widely accepted that physical training therapy is more reliable and effective than medication in improving fatigue in patients with Multiple Sclerosis (MS). However, efforts are still being made to maximise the benefits of training and hence increase independence and optimise the levels of daily functioning in those patients. The present research is a step in that direction. Towards this end, a 12-week training programme with new design guidelines has been developed and tested on a group of fatigued Kuwaiti MS patients with no previous training experience. Method: This was a mainly quasi controlled study (ABA design) with an independent assessor studying the effects of 12-week period of exercise on fatigue measure using the Modified Fatigue Impact Scale (MFIS). In addition to fatigue, a range of both quantitative and qualitative data were collected and these were: Expanded Disability Status Scale (EDSS), , Hospital Anxiety and Depression Scale (HADS), Barthel index (BI), Berg Balance Scale (BBS), Timed Up and Go (TUG), the 10-metre walk, the 6-minute walk, and the 9-Peg-hole test. Handgrip strength, knee, elbow and hip flexion and extension and shoulder adduction were also assessed. Two short and semi-structured interviews were administered to obtain additional information on the impact of fatigue on the participants and their attitude towards training. The training programme consisted of 3x30-minute weekly sessions of resistance or combined training. Initially, combined training consisted of 15 minutes of treadmill walking, followed by 2 sets of 9 stretching and strengthening exercises, 15-20 repetitions. For resistance training, participants performed 3 sets of 15-20 reps of stretching and strengthening exercise. In week 7 onward, treadmill walking time was increased by 5 minutes, and resistance training sets were increased by one. Results: We recruited 26 patients (mean age 37.54, 8 males and 18 females). Following the 12-week training programme, tests showed significant improvements in fatigue, fine mobility and static and dynamic balance (TUG); short duration walking speed (10m test); exercise tolerance (6-minute walk); balance and fear of fall (BBS); left and right shoulder flexion and adduction, and left and right elbow, knee and hip flexion and extension (MMT). All these gains were still maintained four weeks after the training intervention had been terminated. However, there was no significant effect of training on anxiety and depression (HADS); mobility disability (EDSS); personal basic functional activities (B1); fine motor coordination and finger dexterity (9-HolePeg) and hand grip (JAMAR). Results of the study also show that support for Kuwaiti MS patients is lacking and that the rigid cultural norms place those patients under a great deal of unnecessary stress and pressure Conclusion: MS patients with mild to moderate disabilities can gain immensely from individualised and customised resistance and combined training programmes. There is good reason to suggest that their gains can be increased even further with improved attitude towards training. Societal stressors are a significant confounder and these need to be addressed before an randomised controlled trial is carried out.

Chronic cough is defined as a cough that lasts for greater than 8 weeks in duration and has been estimated to have a prevalence of 11-13% of the population. Limited research has been conducted exploring its impact on the wider community. In up to 42% of chronic cough cases, the cough persists despite medical management, these cases tend to be labelled as refractory chronic cough. Pharmacological treatments are limited often with undesirable side effects. Research into non-pharmacological treatments for refractory chronic cough has been limited. An internet based European survey explored the impact of chronic cough (January 2012 - April 2013). A systematic review investigated the effectiveness of non-pharmacological interventions for refractory chronic cough. A single blinded multi-centre randomised controlled trial (RCT) investigated the efficacy of a non-pharmacological intervention (Physiotherapy, Speech and Language Therapy Interventions, - PSALTI) on cough related quality of life (QoL), cough frequency, severity, sensitivity, vocal performance, anxiety and depression alongside a control intervention. In total 1120 responses were collected and analysed from the European survey. Findings identified that cough impacted upon QoL, mood and ability to undertake activities and limited/ no effectiveness of medication; also a wish for more patient information to be available. PSALTI trial showed statistically significant differences between groups for the outcomes; QoL, cough frequency and urge to cough, improvements were significantly greater in the PSALTI group compared with control. There were no significant changes in outcomes from 4 weeks to 3 months suggesting that observed improvements were maintained. This thesis has identified the impact of chronic cough in Europe. It identified the need to improve the management of chronic cough and the information available for patients. This thesis also provides the first evidence within a single blinded multi-centre RCT that PSALTI is an effective treatment option for people with refractory chronic cough.

Lung cancer is a worldwide health problem associated with poor prognosis. The survival at 5 years remains between 5% and 15% in spite of the development of new drugs. One of the main reasons for this is the disease being diagnosed in late stages when curative treatments might not be available. Therefore, some of the most important factors within improving prognosis are both refining diagnostic techniques for early detection, and better assessing tumour response to treatment. Here lies a need for novel diagnostic tools for lung cancer. Spectroscopic and spectrometric analysis of the molecular underpinnings of the disease may provide biochemical signatures for use in diagnostics. Selected Ion Flow Tube – Mass Spectrometry (SIFT-MS) and Fourier Transform Infrared (FTIR) Spectroscopy may provide the gold standard of diagnostic assessment that is needed. Given both techniques previous contributions and technological advancements, their clinical requirements are being increasingly met. This is leading towards the opportunity for the study of lung cancer to benefit from the rapid, non-destructive and sensitive qualities they have to offer. In this thesis, both techniques have been used with the aim of improving the diagnosis and management of lung cancer.

Ovarian cancer is treated in most cases with a combination of surgery and chemotherapy. However, despite the overall improvement in survival rates, over the last 30 years there has not been a break-through therapeutic development. Following first-line treatment, the majority of patients experience periods of relapse characterised by resistance to anticancer drugs. Factors which contribute to ovarian cancer chemoresistance have been identified as potential targets for drug discovery, such as the Bcl-2 family of proteins, which regulates apoptosis. The strategy of targeting the anti-apoptotic members of the Bcl-2 family that are overexpressed in ovarian cancer has led to the discovery of BH3-mimetics. ABT-737, a selective BH3-mimetic, has been found to be synergistic with chemotherapy in ovarian cancer. In this study the activity of navitoclax, an orally available analogue of ABT-737 and obatoclax, a pan-Bcl-2 inhibitor, were evaluated in a series of ovarian cancer models. Navitoclax demonstrated synergy with both carboplatin and paclitaxel. Obatoclax potently inhibited the growth of ovarian cancer cell cultures. In part this was due to the anticipated induction of apoptosis, but in other cell lines an additional mechanism of cell death was implicated. Surprisingly, obatoclax was neither synergistic with carboplatin nor paclitaxel. These observations may be used to inform the design of clinical trials of these agents in ovarian cancer.

The primary intent of this thesis is to delineate the relative roles of local membrane and systemic consequences of peritoneal dialysis therapy, with particular reference to the role of inflammation and a severe, uncommon complication, encapsulating peritoneal sclerosis (EPS). Data sources comprised observational cohort studies as well as registry data: the Stoke PD study, a single centre study with clinical data, the Global Fluid Study (GFS), a multinational study with clinical data and repeated dialysate and plasma samples, and Scottish Renal Registry (SRR) and AnzData registry data. Through a cross sectional analysis of dialysate and plasma samples from GFS for inflammatory cytokines, we demonstrated that peritoneal and systemic inflammation are mostly separate processes although there is an association for IL-6 along with a steep concentration gradient from dialysate to plasma. Peritoneal inflammation, though IL-6, is the strongest determinant of peritoneal solute transport, and systemic inflammation, though IL-6, is a strong predictor of patient survival although peritoneal may contribute to systemic inflammation. Through a nested case control study of GFS we showed that inflammatory cytokines are upregulated within the peritoneum prior to developing EPS. With a nested case control design from the Stoke PD study, we showed that a decrease in ultrafiltration, likely due to increased fibrosis causing a reduction in osmotic conductance to glucose, also predisposes to EPS. A competing risks analysis of SRR and AnzData showed that patients at a high risk of death, have a low risk of EPS. These findings provide supporting evidence for the theory that the risk of EPS develops through the accumulation of inflammation-driven fibrosis due to dialysate exposure over a long period of time. Dialysate contains high concentrations of glucose and absorption of this drives impairment of systemic glucose metabolism, demonstrated through a cross sectional analysis of GFS.

Producing therapeutic neural cell populations in vitro to treat neurodegenerative diseases is a key aim of regenerative medicine. Various protocols have been developed to produce a wide range of neural cell types in vitro, but the protocols are labour and resource intensive. Lower costs will take the cell therapy closer to clinical adoption. Cell-material interactions can be used to control cellular processes and behaviours in the place of expensive reagents. The thesis went about developing superior materials to culture neurons in vitro by using simple surface parameters. By using simple surfaces findings could be leveraged by incorporation in to other materials, and protocols to culture neurons. We have investigated the responses of primary neural tissue derived from rat ventral mesencephalon (VM), interacting with a range of surface chemical functionalities and net molecular properties by using silanes. Specific substrate functionality leads to higher ratios of neurons, longer neurites and neurosphere spreading capacity. All of these characteristics indicate a high neuro-regenerative capacity. Next it became important to optimize the amine functionalised surface with the addition of secondary amines in to the surface. The rational of adding secondary amines to the surface would produce functionalities which have a closer resemblance to biological molecules. The biomimicry in the surfaces provides extra scope for selective surface interactions to provide more control over neural cell culture which could steer protocols away from the use of expensive surfaces which are coated in extra cellular matrix molecules such as laminin. Controlling differentiation with surfaces has long been an aim in regenerative medicine to deliver productive production protocols. It has been shown that surfaces can induce differentiation of stem cells; however there is little control where stem cells and adult cells are simultaneously cultured. To achieve controlled differentiation of neural stem cells a surface gradient of amine polymer lengths, and polymer densities. That is in contrast to the surfaces used in previous chapters which had homogeneous presentations of surface chemistries.

Massive screens of chemical libraries for antimalarial activity have identified thousands of compounds that exhibit sub-micromolar potency against the blood stage of the malaria parasite Plasmodium falciparum. Triaging these compounds to establish priorities to take forward for development requires additional information regarding their activity. Key amongst their pharmacodynamics (PD) properties is the rate of kill– with a rapid cytocidal effect specifically identified as a key requirement for a Single Exposure Radical Cure and Prophylaxis (SERCaP) product. Compounds that exert an immediate cytocidal effect rapidly reduce parasite burden to ameliorate the morbidity and mortality of disease. With the overall aim to accelerate drug screening by validating a rapid rate of kill, the validation of a novel, quick (6hr) and potentially scalable bioluminescence rate of kill (BroK) assay is described here that demonstrates a good correlation with in vitro recrudescence-based rate of kill data and available in vivo clinical findings. The BRoK assay was used to screen the Medicine for Malaria Venture Malaria Box to identify compounds with rapid cytocidal activity. Seventeen compounds have an initial rate of kill greater than artemisinins, with a further 39 compounds exhibiting a rate of kill between chloroquine and artemisinins. These compounds represent potential Target Candidate Profile, compounds for a SERCaP product. This work highlights the opportunity for the BRoK assay as a hit discovery tool. In addition, the potential for this assay in lead validation through structure activity relationship studies are highlighted.