Lung cancer is the most common cause of cancer-associated mortality in males and females globally. Widespread research is currently focused on the development of novel approaches for targeting non small cell lung cancer with different therapeutic nanotechnologies. In this study, a sensitive and selective HPLC method was developed for the quantification of afatinib (AFT) in formulations. Novel drug delivery systems based on cationic (CL) and pH-sensitive liposomes (PSL) for AFT were developed, with different ratios of lipid to AFT, using a film hydration method. The obtained liposomes had a small particle size of less than 50 nm with a low polydispersibilty index and acceptable zeta potential. The highest Encapsulation Efficiency (EE%) of AFT reached 43.20%, 50.20%, and 52.01% for NL (Non targeting liposomes), PSL, CL, respectively at the 1:0.5 ratio of AFT: lipids. The in vitro release study confirmed that all formulations had sustained release profiles in pH 7.4. However, in acidic pH values, PSL exhibited fast release. The stability study, conducted at 4°C and 25°C for 1 month, showed that the characteristics of liposomes in liquid form did not change significantly over this period. In vitro cytotoxicity studies revealed high antitumor activity of PSL on all cell lines at 0.75 μM concentration after 24 h exposure, based on using the Annexin V assay. A proteomics study identified 12 proteins which can be used as biomarkers capable of prediction of treatment response and choice of therapy for two different types of human NSCLC cells (H-1975 and H-1650). Spray drying was used to produce nanocomposite microparticles (NCMPs) using L-leucine and coated using different ratios of chitosan for the optimized PSL NPs. The particles had a corrugated surface except at high CH ratios, where more homogenous and smooth particles with some small indentations were obtained. The powder properties showed good flow properties and reproducible size. Coated NCMPs showed a delayed drug release profile compared to PSL NPs and the best correlation with the Higuchi model. A stability study at 40°C/ 75% ± 5% relative humidity (RH) showed large changes in the drug content for all coated NCMPs powders. Analysis of the in vitro aerosolization performance demonstrated a mass median aerodynamic diameter (MMAD) of 3.24 – 5.85 μm and fine particle fraction (FPF%) of 54.20-33.66%. The particle size of the reconstituted powders was ˂ 100 nm, which is within the size range to be effectively taken up by tumor cells. Assessment of the stability of spray dried liposomes after 3 months of storage at 40 °C/75% RH, showed that fusion and aggregation of the liposomes occurred in all samples tested. The C1NCMPs (lipid: LEU: CH ratio of 1:1.5:0.5) exhibited the highest FPF (51.2%) and fine particle dose (FPD) (40.0 μg of AFT) indicating deep lung deposition. Further cell viability studies of C1 NCMP, at a concentration of 0.75 μM on H-1975 NSCLC cell line showed a good toxicity profile comparable to PSL nanoparticles (NPs). The obtained data indicates that pulmonary delivery of PSL NCMPs is a potential new clinical strategy for better targetability and delivery of AFT for the treatment of lung cancer.

Cell instructive biointerfaces are versatile tools to mimic a natural cellular environment and control cell fate in vitro. The particular interest lies in combining information gained from surface and interface analysis tools with biological analysis to explore and understand fundamental processes such as neuronal stem cell differentiation at the biointerface. A major challenge in biointerface design is to mimic and study the complex interactions of the natural processes in the extracellular matrix (ECM) with artificially designed surfaces and interfaces. In the past, peptide surfaces have been used as ECM mimics, however, more research is required in this field to tune the properties of peptide surface to modulate the outcome of stem cell fate. The present work aims to address this challenge by designing new synthetic peptide surfaces with well controlled composition and functionality able to impart have control over the differentiation of neuronal stem cells with the ultimate goal to relate surface properties and stem cell response to understand and control how neuronal networks function. Compositionally well-defined surfaces of two short laminin peptide sequences, Arg-Gly-Asp (RGD) and Ile-Lys-Val-Ala-Val (IKVAV) were prepared of various ratios via the “grafting from” stepwise approach. The surface modification was confirmed with surface analysis techniques to indicate successful peptide functionalization. The neural progenitor stem cells (NPSCs) were set up from embryonic rat hippocampi (E18). Immunocytochemistry (ICC) observed cell viability and differentiation to specific NPSCs lineages for βIII-Tubulin and GFAP. The surface characterizing techniques of WCA, AFM, ToF-SIMS, and XPS validated the successful orthogonal functionalization of controllable peptides composition surfaces with the increase of RGD composition a relative decrease in the IKVAV composition was observed. The increase in the normalized total ion fragments of RGD in the ToF-SIMS measurements can be related linearly to the % area coverage of neurons versus astrocytes observed on the controllable peptide composition surfaces. Well-defined peptide surfaces were designed and successfully demonstrated that the amount of RGD peptide composition present on the surface influences cell adhesion, proliferation and differentiation to a desirable cell fate or controllable cell population (i.e. neurons and astrocytes). Recent technological developments demonstrate a shift from two-dimensional (2D) surfaces to three-dimensional (3D) surfaces, so we used the designed versatile 2D surfaces as the template to design comparable 3D surfaces to examine the biological response of NPSCs to both microenvironments. This study proposes the design of novel 3D nanoparticles (NPs) made of gold and surface-conjugated with differentiation-inducing peptides. The NPs-peptides will guide stem cell differentiation to neurons that self-aggregate around the NPs by cell-cell contacts to form neurospheres. The neural stem cells will establish 3D structures biomimicking the cytoarchitecture of the brain. Successively, they could be used as an alternative 3D in vitro model for neurotoxicity testing of drugs and chemicals. Size-controllable NPs will be surface-conjugated with RGDC and IKVAVC(19) peptides via the SPPS “grafting to” approach. Two different sizes of NPs were characterized as AuNPs and SiO2@AuNPs characterization and validated by TEM, DLS, -potential, EDX; and the surface functionalization on NPs was successfully confirmed by UV/vis spectroscopy and -potential. The NPSCs were set up from E18 rat hippocampi and cell viability and differentiation to specific NSPCs lineages was stained for βIII-Tubulin and GFAP. AuNPs and SiO2@AuNPs peptide immobilized surfaces supported cell adhesion, proliferation, and survival. The confocal light scanning microscope (CLSM) images indicate that the RGDC functionalized AuNPs and SiO2@AuNPs surfaces induced a preferential differentiation towards a neurons cell fate and the IKVAVC(19) functionalized surfaces of AuNPs and SiO2@AuNPs favored an astrocytes cell. The cellular uptake of functionalized AuNPs by the cells in the neurospheres was observed via TEM micrographs, whilst the micrographs of functionalized SiO2@AuNPs surfaces suggest that they were not taken-up into the cells. Hence, indicating that there is a difference in the cellular uptake mechanism for the functionalized AuNPs and SiO2@AuNPs, which might be due to the agglomeration of the nanoparticles than the individual size of the nanoparticles. In conclusion, the relation between the 2D and 3D surfaces may provide new insight in understanding how surface properties affect the regulation of physiological relevance in directing neural cell differentiation, which will be essential to understand how neural networks function.

In this thesis, thermodynamic-based mathematical modelling is combined with experimental in vitro extracellular flux analysis in order to assess drug redox cycling and cellular bioenergetics. It is widely accepted that pharmacological activity of certain classes of drugs (e.g. anticancer, antimalarial) is related to their ability to accept one or two electrons. However, pharmacological activity via redox cycling is an understated mechanism of toxicity associated with many classes of drugs. In particular, oxidative stress as a result of redox cycling plays a pivotal role in the cause of cardiac toxicity. For example, doxorubicin is an anti-neoplastic drug used to treat cancer. It has strong links to redox cycling-induced cardiac toxicity associated directly with elevated levels of reactive oxygen species (ROS) and oxidative stress within the mitochondria. The underlying mechanisms of redox cycling is very difficult to elucidate, due to the fleeting existence of the radical species. However, assessment of such cellular bioenergetics can be ameliorated with the aid of computational assistance. In chapter 2 the development of a novel thermodynamic-based in silico model of doxorubicin redox cycling is described, which is parameterized using data from in vitro extracellular flux analysis. The model is used to simulate mitochondrial-specific ROS, with its outputs confirmed against in vitro data. Chapter 3 describes construction of a pH-dependent thermodynamic model of hepatic glycolytic flux, used to determine the role of the monocarboxylate transporter 1 flux during extracellular acidification. Finally, chapter 4 describes a thermodynamic-based in silico model of mitochondrial bioenergetics, capable of simulating oxygen consumption rates of a cohort of in vitro human primary hepatocyte data. The model is finally used to simulate perturbations in key bioenergetic variables and reaction fluxes, illustrating the resulting changes on mitochondrial pH, membrane potential and subsequent oxygen consumption rates.

This thesis identies, inter alia, the socio-economic factors that affect malaria incidence at both the household and district levels and investigates how these differ across rural and urban settlement-types. In addition, state level data for India are used to examine the effect of aggregate income relative to that of public health expenditure on malaria incidence. The household and district-level analysis focuses on the state of Uttar Pradesh and exploits the National Family Health Survey, which is the Demographic Health Survey (DHS) for India, for two time periods - 1992-93 and 1998-99 - and combines these data with the district-level census data for 1991 and 2001. A key theme of the micro-level analyses is whether household wealth exerts a negative impact on malaria incidence. Wealth is measured using the DHS data by constructing a consumer durable asset-index by Principal Components Analysis and malaria incidence was modelled using a probability model. The household-level analysis reveals that the relationship between socio-economic status and malaria incidence is not always negative. For example, owning a water pump, indicative of a higher socio-economic status, has a positive impact on malaria incidence and being of a lower caste has a negative impact. Variables that support the negative socio-economic status and health relationship include having an electricity connection in the house, having access to a protected public drinking water supply rather than an open source, and living farther away from open water sources. The aggregate (or panel data) analysis was undertaken using data for 15 states in India covering the time period 1978 to 2000. The aggregate analysis reveals that income has a negative impact on malaria incidence but direct expenditure on health is more effective in bringing about a decline in malaria incidence - an increase of a rupee in aggregate income per person reduces malaria incidence by 0.1 percent whereas an equivalent increase in real health expenditure per capita results in a 0.4 percent decline in malaria incidence. The research undertaken for this thesis is unique in using the DHS to identify the factors aecting malaria incidence and shows that these data are very useful in exploring the relationship between malaria incidence and a host of socio-economic factors in order to identify areas for effective policy intervention. Such a holistic approach is critical in controlling and, eventually, eradicating malaria rather than relying primarily on more direct treatment strategies based on insecticide-treated bed nets and drug therapy. The areas where public spending could be directed to attack malaria identied by the empirical analysis include education, particularly raising awareness on prophylactic measures through adult literacy centres, controlling the breeding of mosquitoes in open water collection sites such as public taps and around water pumps and improving water flow in agricultural fields to prevent stagnant water collection.

Background: Cardiovascular disease (CVD) is one of the major causes of morbidity and mortality worldwide. Clinical guidelines, based on the results of randomised controlled trials, state that effective secondary prevention therapies should be prescribed following a diagnosis of particular CVD unless there are contraindications. Although evidence shows that use of evidence based pharmacotherapies after diagnosis of CVD reduces mortality and disease progression, many inequalities exist in prescribing practice. Many studies have documented that women and the elderly are less likely to receive evidence based therapies than men and the young, respectively. Greater socioeconomic deprivation has also been shown to be associated with lower rates of prescribing of therapies. However, prior studies have all focussed on one particular CVD or failed to adjust for confounders. Also, few studies have examined trends in the prescribing of evidence based pharmacotherapies over time and documented whether prescribing inequalities are static, narrowing or widening. This project aims to describe the pharmacotherapy received by patients with CVD in Scotland, and to describe the factors associated with prescribing of evidence based pharmacotherapy. Methods: In this retrospective cohort study I examined a linked database of primary care records (Continuous Morbidity Records) and secondary care records (Scottish Morbidity Records) covering 238064 individuals in Scotland (approximately 6% of the total population) from 1997 to 2005. Patients with a first diagnosis (defined as a first hospitalisation or first recording of the diagnosis in primary or secondary care) of myocardial infarction (MI), angina, and peripheral arterial disease (PAD) were identified. Patients who died within the first 30 days of diagnosis/first hospitalisation were excluded from further analysis. Data on prescribing of evidence based therapies (angiotensin converting enzyme inhibitors (ACEI)/angiotensin receptor blockers (ARBs), β-blockers, statins and antiplatelet agents [aspirin or clopidogrel]) within 30 days of diagnosis was obtained from primary care database records. Multivariable logistic regression was conducted to examine the association between prescribing of evidence based pharmacotherapies and age, sex, socioeconomic status, comorbidities and year of diagnosis. Results: Between 1997 and 2005, 4305 (83.4%) patients with a first diagnosis of MI, 7210 (98.6%) with angina, and 3385 (95.8%) with PAD had survived to 30 days after their first diagnosis. Increasing age was associated with lower odds of being prescribed evidence based therapies. This association persisted after adjustment for sex, socioeconomic status, year of diagnosis, and comorbidities. In general, older patients ≥ 85 were significantly less commonly prescribed evidence based therapy (EBTs), however they were significantly prescribed nitrates (OR 1.29; 95% CI 1.05-1.59, P < 0.01) for angina. Generally men were more likely to be prescribed evidence based therapies than women. After adjustment, prescribing of evidence based therapies was significantly higher in men with a MI for β-blockers (OR 1.18; 95% CI1.04-1.33, P < 0.01), ACEI/ARBs (OR1.26; 95% CI1.05-1.47, P < 0.01) in angina, and statins in men (OR 1.39; 95% CI1.01-1.93, P < 0.04) with PAD and coronary heart disease (CHD). In contrast, men diagnosed with isolated PAD were significantly less commonly prescribed statins than women (OR 0.73; 95% CI0.59-0.91, P < 0.004). Prescribing of evidence based therapies varied negligibly between the most deprived and least deprived patients. These minor differences disappeared after adjustment except for β-blockers which were significantly less likely to be prescribed for patients who had been diagnosed with angina and were residing in quintile 9 compared to the least deprived area (OR 0.76, 95% CI 0.58-1.00, p= 0.05). Prescribing of evidence based therapies increased between 1997 and 2005, particularly for ACEIs/ARBs, β-blockers, statins and antiplatelet agents. Generally the presence of comorbidities was associated with lower odds of being prescribed evidence based therapies. When comparing prescribing rates between the different diagnoses, patients with a first MI were more likely to be prescribed ACEI/ARBs, β-blockers, statins, aspirin and clopidogrel compared to angina. All evidence based therapies were less likely to be prescribed for those with PAD compared to patients with a MI or angina. Conclusion: In conclusion, I have shown that prescribing of evidence based therapies has improved over time, though rates remain low. Prescribing evidence based therapies is inequitable, though not always significant, for age, sex, and socioeconomic status. Concomitant disease decreased the odds of being prescribed evidence based therapies. More studies are needed to identify the reasons for the prescribing inequalities and low rates observed. Further studies are needed to examine the existence of other inequalities in using evidence based therapies such as dosing and to find strategies to improve prescribing rates.

Ovarian cancer is one of the most lethal malignancies and often presents at an advanced stage, resulting in a poor prognostic outlook. Platinum chemotherapy leads to an initial clinical response, however most patients will ultimately relapse and there remains a sub-group who are intrinsically resistant to platinum. I focussed on high grade serous ovarian cancer (HGSOC), the most common subtype of ovarian cancer. The ID8 CRISPR-generated models represented a novel and simple tool to investigate the biology of HGSOC. By using this in vivo model, I aimed to further the understanding of platinum sensitivity in HGSOC by investigating the homologous recombination pathway and the tumour microenvironment. In vitro work showed that sensitivity to PARP inhibitors was clearly correlated with defective homologous recombination but the relationship with platinum sensitivity was more complicated. Using the ID8 derivatives, in vivo cisplatin experiments identified Pten and Nf1 loss to be associated with the worst prognosis with the knockout of Brca1 or Brca2 prolonging survival. A Brca1 mutation in the PALB2 domain compared to the BRCT2 domain was found to be associated with a greater sensitivity to cisplatin. The tumour microenvironment was shown to differ between genotypes and altered with the addition of platinum chemotherapy. Specifically, the loss of Pten was associated with an immunosuppressive microenvironment with increased levels of myeloid-derived suppressor cells (MDSCs) and tumour-associated macrophages (TAMs). The chemokines, Ccl2 and Ccl7 were shown to be significantly increased in the Trp53-/-;Pten-/- genotype. I targeted both the cytokine/chemokine response directly by using a transgenic mouse model (CCR1, 2, 3, 5 receptors knockout) and the PI3K/AKT pathway by using a PI3K inhibitor (p110β) (AZD8186) to attempt to reverse the effect of Pten loss. The transgenic mouse model (GGTACKO) showed encouraging early results with a reduction in MDSCs and TAMs in the knockout mice injected with the Trp53-/-;Pten-/- genotype but a repeat experiment is required before valid conclusions can be made. The AZD8186 in vivo experiment showed a significant reduction in MDSC levels in the ascites following AZD8186 treatment in mice injected with the Trp53-/; Pten-/- genotype and a non-significant decrease in the tumour samples. There was also a reversal in the anaemia previously shown with the loss of Pten and a decrease in Ccl2 and Ccl7 expression. I have used a transplantable in vivo model for HGSOC to investigate potential mechanisms of platinum sensitivity and identified poor prognostic genotypes (Pten, Nf1). I have found Pten loss to be associated with an immunosuppressive microenvironment and highlighted potential therapeutic targets. By targeting the PI3K/AKT pathway I have shown that the effect of Pten loss can be reversed. The next step will be to determine whether this reversal results in a prolonged survival.

Mosquitoes are responsible for millions of deaths a year through the viruses and parasites they vector. Many of these vector species have successfully expanded their range into temperate climates due to a combination of climate change and the easy movement of goods and people around the world. The temperate climate of the U.K. is home to 34 native species, several of which bite humans and are capable of vectoring pathogens more commonly associated with warmer climates, therefore the threat of mosquito-borne illness in the U.K. is a very real possibility. Many vector mosquito species have evolved resistance to traditional chemical insecticides and the search for novel control strategies in endemic areas is a priority in vector control. Entomopathogenic nematodes (EPNs) are microscopic roundworms, which are obligate parasites of insects from the family Rhabditae. In particular, soil-dwelling nematodes from the genera Heterorhabditis and Steinernema. Presently EPNs are used in a range of plant-based industries as a chemical-pest control. However, previous laboratory research has shown that EPNs are capable of killing more than 250 species of insect including a selection of vector species and nuisance arthropods. This thesis is concerned with discovering whether commercially available and naturally occurring strains of EPNs from the U.K. could be used as an effective biocontrol agent for mosquito and chironomid species. This study includes a snapshot of the current EPN diversity in the U.K. which found four different Steinernema species, including the first molecular confirmation of Steinernema carpocapsae. EPNs from both field-collected and commercial sources were capable of killing and parasitizing two native and tropical mosquito species and Chironomus plumosus. Commercial strains were more effective at killing both, however, the native field-collected, mosquito species Ochlerotatus detritus was susceptible to field-caught EPNs, unlike the tropical, lab-reared Aedes aegypti. EPNs were found to be capable of tolerating the extremes of habitat that mosquito species can inhabit in laboratory tests. These studies have shown that with further research including viable field trials that EPNs could be very useful to add to a range of vector and nuisance control measures when used appropriately.

Meningococcal infection due to serogroup B disease is the leading cause of meningitis across Europe, particularly in infants. Prevention through vaccination will soon be possible utilising a new multicomponent vaccine (4CMenB or Bexsero®) principally directed against serogroup B Neisseria meningitidis. In addition to OMVs, the vaccine contains three novel recombinant proteins discovered by reverse vaccinology. Studies were performed on the biophysical characteristics of these antigens to better understand their structural properties in solution and aspects of their immunogenicity. Initially, the antibody responses to the vaccine components were assessed in a low-dose mouse model. This extreme immunogenicity model was chosen to highlight any subtle differences in the immune response to individual antigens which may have been overlooked by previously high-dose immunisation studies. The recombinant vaccine components are synthetic homologues of proteins naturally present on the bacterial cell and changes in their structure can induce loss of functional responses. By deliberately stressing the antigens to lose functional immune responses, it was possible to study the structural changes at the molecular level. Using molecular modeling and protein-specific monoclonal and polyclonal antibody binding in parallel with UV circular dichroism (CD) spectroscopy, it was possible to assess at which point changes in the structure of proteins affected their ability to induce functional antibody responses in vivo. The two fusion proteins present unique biophysical characteristics derived from their component parts. As a result, they were shown to possess distinctive profiles by CD. When combined with fluorescence unfolding studies, in which the tryptophan residues were used as reporters for integrity, CD uncovered significant structural differences among the vaccine antigens. The NHBA-FP comprises two domains having different thermodynamics while in the fHbp-fusion both halves cooperate in the unfolding process. The choice of including a β-barrel protein in the chimeric constructs proved successful as both the NHBA and fHbp exhibited excellent solvent accessibility profiles due to the numerous inter- and intra-strand hydrogen bonds stabilising the structure. This had a direct effect on their immunogenicity which could only be adversely affected by prolonged harsh treatments. Maintenance of the α-helical profile was found to be critical for the immunogenicity of NadAΔ351–405 component as was the maintenance of its trimeric organisation. Overall, results confirmed the requirement and importance of the three additional protein antigens to the bactericidal response, even when directed against the homologous OMV bacterial strain. Structural studies confirmed the quality of the vaccines antigens and supported their critical contribution to the vaccine.

Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by chronic inflammation within the synovial tissues in multiple joints leading to progressive, erosive destruction of cartilage and underlying joints. The severity of RA is associated with the overexpression of proinflammatory cytokines within the synovial tissue, such as TNF-, IL-1 and IL-6. Recently, IL-17 is thought to play a critical role in maintaining the inflammatory processes within the arthritic joints. Although the etiology and pathogenesis of RA has not been completely elucidated, neutralizing Antibodies against the inflammatory components have been shown to successfully suppress joint inflammation, reduce the relapse rate and delay disease onset in RA patients. Therefore, the expression and regulation of cytokines produced during the disease progression has been the centre of interest in therapeutic studies. Cytokines are important mediators of immune functions in humans and animals. In this thesis, a murine model of RA has been used to investigate the roles of new cytokines Interleukin (IL)-27 and Interleukin (IL)-35. Interleukin (IL)-27, is a heterodimeric cytokine comprised of an IL-12p40 related protein, Epstein-Barr virus-induced gene 3 (EBI3) and a unique IL-12p35 like protein p28. IL-27 is a member of IL-12 family, mainly generated by activated macrophages and dendritic cells. IL-27 binds a receptor composed of WSX-1/TCCR, a ligand-specific chain, and gp130, a signal-transducing molecule shared with other cytokines such as IL-6. IL-27 can promote both pro- and anti-inflammatory immune responses. A novel role of IL-27 regulating autoimmunity has been suggested by experiments on experimental autoimmune encephalomyelitis (EAE) and central nervous system (CNS) inflammation when infected with Toxoplasma gondii. IL-27 suppresses these chronic diseases through inhibiting Th17 activity. Thus, IL-27 may have an important therapeutic potential for treatment of RA in humans. A major aim of this project has been to clone and express a recombinant murine IL-27 in sufficient quantities to study the role of IL-27 in a murine model of RA closely related to the human disease, collagen-induced arthritis (CIA). A short term administration of IL-27 to mice at the onset of the disease had a significantly suppressive effect on disease severity and incidence compared with untreated controls. Mice treated with the recombinant IL-27 also showed reduced serum IL-6, IL-17 and collagen-specific IgG2a. Spleen and lymph node cells from the IL-27-treated mice produced significantly less IFN- and IL-17 compared with cells from the control mice when cultured with collagen in vitro. In contrast, administration of IL-27 to mice during the late phase of CIA significantly exacerbated disease severity. IL-27-treated mice also showed elevated IFN-γ and IL-6 production by the lymphoid cells when compared to untreated mice. However, IL-17 synthesis was not affected between IL-27-treated mice and untreated mice. Consistent with this finding, in vitro IL-27 markedly inhibited the development of Th17 from naïve CD4+, CD4+CD25- and CD4+CD25+ T cells, but had little or no effect on differentiated Th17 cells. Together, these results demonstrated that IL-27 had both pro-inflammatory and anti-inflammatory effects on chronic articular inflammation, mainly associated with Th17 functions. Interleukin (IL)-35, another novel heterodimeric cytokine belonging to IL-12 family, is composed of EBI3 and the IL-12p35 subunit. Little is known about the biological function of IL-35. To study the role of IL-35 in immune responses, murine recombinant IL-35 was cloned and expressed in a mammalian GS system to produce sufficient quantities. IL-35 induced proliferation of murine CD4+CD25+ and CD4+CD25- T cells when stimulated with immobilized anti-CD3 and anti-CD28 antibodies in vitro. The IL-35-expanded CD4+CD25+ T cell population expressed Foxp3 and produced elevated levels of IL-10, whereas the IL-35-induced CD4+CD25- T cells produced IFN-γ but not IL-4. The IL-35-expanded CD4+CD25+ T cells maintained their suppressive functions against CD4+CD25- effector cells. Furthermore, when cultured with soluble anti-CD3 and antigen-presenting cells, IL-35 directly suppressed the proliferation of CD4+CD25- effector cells. Moreover, IL-35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively suppressed established collagen-induced arthritis in mice with the suppression of IL-17 production but enhanced IFN-γ synthesis. Therefore, IL-35 is a novel cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development. For the future study of human IL-35, human EBI3 and p35 were cloned and linked together with an Fc fusion part. Human IL-35 was expressed in GS system and the function of the recombinant protein needs further study. These data in this thesis provide direct evidence that IL-27 and IL-35 are important mediators in murine collagen-induced arthritis disease. This implicated that IL-27 and IL-35 could represent potential new targets for novel therapeutic agents in human RA. However, the findings on the dual role of IL-27 at the different disease process suggested that the involvement of IL-27 in the pathogenesis of human RA should be carefully investigated before clinical therapy application.

Vigabatrin, a GABA (γ-aminobutyric acid) agonist is a drug widely prescribed in Europe and Asia between 1989 and 1997 for drug resistant, partial epilepsy and has been associated with visual field defects. Uncertainity in the effect of Vigabatrin on vision resulted in decreased prescriptions. However, there has been poor reproducibility in previous studies due to many factors, especially poor sensitivity and specificity of tests for Vigabatrin associated visual dysfunction. The wide field multifocal electroretinogram (WF-mfERG) can objectively measure discrete areas of retinal function up to 90 degrees. The results of 204 patients with epilepsy divided into four groups are presented. A subgroup of 89 patients had repeat investigations. The patients were divided into four groups. Group 1.The Vigabatrin group comprised patients who had been taking Vigabatrin for at least 1 year (56 patients). Group 2. Forty nine patients who had previously taken Vigabatrin for at least 1 year but had stopped taking this treatment for at least 2 years comprised the ex-Vigabatrin group. Group 3.The GABA group had 46 patients who used other anti-epileptic drugs (AED) with GABA action other than Vigabatrin. Group 4. Fifty three patients who had never used an AED with GABA action including Vigabatrin made up the non-GABA group. Surprisingly, the percentage of patients with visual field defects were high in all groups investigated (Vigabatrin group 59%, ex-Vigabatrin group 46%, GABA group 30.2% and non-GABA group 21.2%). However, abnormal bilateral WF-mfERG responses were only found in the Vigabatrin group (48%) and the ex-Vigabatrin group (22%). The study suggests that there are probably different causes of visual field abnormalities in patients with epilepsy not related to Vigabatrin. We propose that the most sensitive and specific tests that can be used to detect visual dysfunction associated with Vigabatrin is the WF-mfERG