Type 1 diabetes mellitus (T1DM) results from auto-immune destruction of the insulin-secreting β-cells of the pancreas. The most common treatment is injection of exogenous insulin, but this allows only partial control over blood glucose levels, so other therapies are needed. Pancreatic islet transplantation has shown proof of principle for cell replacement therapy to treat T1DM. There are several sources of cells which could be used, but much of the focus has been on pluripotent stem cells, which are able to self-renew indefinitely in culture and give rise to any cell in the body. Insulin-expressing cells have successfully been produced from embryonic stem cells (ESCs) by recapitulating embryonic development in vitro. However, problems associated with the use of ESCs mean that an alternative cell source is needed. In 2006, it was discovered that 4 transcription factors can reprogram somatic cells into induced pluripotent stem cells (iPSCs). iPSCs provide an alternative source of pluripotent stem cells and can be derived in a patient-specific manner. iPSCs have been shown to differentiate in vitro into insulin-expressing cells, but it is unknown whether iPSCs are truly equivalent to ESCs. Important differences have been shown to exist between iPSCs and ESCs which may affect the ability of iPSCs to give rise to cells of a pancreatic lineage and therefore limit their usefulness for the treatment of T1DM. The aim of this project is to identify whether iPSCs are a viable alternative to ESCs for generating β-cells in vitro for cell replacement therapy to treat type 1 diabetes. The differentiation potential of iPSCs and ESCs to give rise to first definitive endoderm (the first stage in differentiation towards a pancreatic lineage) in vitro will be compared, and the involvement of miRNAs in differentiation of ESCs and iPSCs to definitive endoderm will be investigated.

Obesity is a growing public health problem with multiple aetiological factors. Behavioural determinants are likely to be key contributors to obesity, with a need for applied research in this field. Recently the obesity has been compared to food addiction with the connotation that obese individuals are impulsive in their behaviour. Impulsivity is a trait that is closely linked to addiction and has been studied in personality, psychiatry and more recently in the neurocognitive arena. A conceptual review of the construct of impulsivity identified inhibitory control (SST) and temporal discounting (TD) as two key behavioural constructs universal to all the key fields of impulsivity research. A systematic review of the literature supported their use to profile participants based on their Body Mass Index. The validity of the tools were proven by endophenotyping participants (N=202) of both normal weight and those seeking weight loss intervention. Both measures could successfully differentiate between obese and normal weight adolescents (N=85). The SST was also prognostic for short-term weight reduction in adolescents attending a lifestyle intervention, with the TD being able to predict weight loss maintenance at 6 months. The tasks could not differentiate significantly between adults of different weights but the TD was able to predict weight reduction after surgery (N=90). The modifiability of obesity through neuronal dopamine pathways was supported by a randomised controlled trial testing neurocognitive enhancement agents (N=40) against a placebo (N=40) in normal weight adults. Weight was also controlled by a commitment intervention targeting automatic impulsive behaviours (N=27). These findings support an association between impulsivity, obesity and weight reduction. The experimental inferences have been described in terms of a novel interconnected neuronal network, which leaves itself open to testing using functional brain imaging.

The link between early life and development of metabolic disorders and obesity in later life has been the focus of many studies over the past decades. Fetal life and early infancy are two of the most critical periods of physiological and metabolic development and plasticity, and thus, are periods whereby a stimulus can cause long term consequences on the health of an individual (developmental programming). Accelerated weight gain during early life per se or in combination with extreme birth weights (low/high birth weight) have been postulated as factors that can affect the development of individuals with later consequences in health. However, there is not a clear understanding of the specific contribution at different ages to impaired health neither of the mechanisms involved in these alterations. In this thesis, I used an isogenic murine model with natural birth weight variation within a normal range to investigate the effects of extreme birth weights on body composition and appetite regulation at different stages of life. I compared low and high birth weights phenotypes during lactation, at the time of weaning, and a young adulthood as well as early matured age. Mice were challenged to a moderate high fat diet for 12 weeks after weaning in order to assess the effects of both birth weight and a hypercaloric feed on body composition and hypothalamic neural activity. At weaning, adiposity was positively related to birth weight and weight gain but negatively related to growth rate. Low birth weight male mice (LBWm) had a lower plasma glucose concentration but similar levels of insulin to High birth weight male mice (HBWm), indicating a degree of hyperinsulinemia. Low birth weight females (LBWf) were hyperinsulinemic and hyperglycemic compared to High birth weight females (HBWf). There was an upregulation in the expression of genes related to insulin signaling, adipogenesis/lipid metabolism and thermoregulation in Subcutaneous Adipose Tissue (SAT) of LBWm mice compared to HBWm mice but the contrary was seen in LBWf mice in respect to their counterpart. LBWm mice caught up in weight with HBWm mice at younger age than the equivalent catch up in female mice. Birth weight and diet impacted body fat patterning and appetite regulation differently in both young males and females. However, at week 51 of age (early matured age), diet seemed to override the effects of birth weight on total body fat. LBWm mice tended to have smaller adipocytes than young and matured HBWm mice, especially when fed a HF diet, and this pattern was independent of fat mass. In conclusion, the current study suggests that extreme birth weights in an isogenic mouse model (within natural birth weight variation), as well as postnatal nutrition influenced growth, glucose / lipid metabolism, body fat patterning and appetite regulation in an age-gender dependent manner.

Pain is the foremost clinical symptom of osteoarthritis (OA), however the processes that lead to pain in the joint are poorly understood. I have used two different murine models of OA, induced by surgical destabilisation of the joint. Upon destabilisation, two episodes of painful behaviour are observed, an initial post-operative phase, lasting a few days, and a late phase that starts between 8-11 weeks post-surgery, detected by differences in weight bearing measured by the Linton incapacitance tester. The later phase is associated with significant cartilage damage, but no overt synovitis. A screen of pain sensitising molecules expressed in the whole joint at the time animals developed late pain was performed. Statistically significantly regulated molecules included nerve growth factor (Ngf), bradykinin receptors 1 and 2 (Bdkrb1 and Bdkrb2), Tachykinin 1 (Tac1) and its receptor (Tacr1), determined by real-time PCR. When a similar analysis was performed in the microdissected tissues of the joint (cartilage, bone and meniscus), all but one (Bdkrb2), of the previously regulated molecules were significantly regulated in the articular cartilage. Ngf, Bdkrb1, Bdkrb2 and Tac1 were also statistically significantly regulated upon in vitro cartilage injury, using the murine hip and porcine metacarpophalangeal explantation models, suggesting that in vivo mechanical damage may be sufficient to drive pain molecules. One mechanism previously described by our group, by which chondrocytes respond to injury, is via release of FGF2 from the pericellular matrix. When in vivo cartilage injury studies were performed in Fgf2-/- tissues, or in the presence of an FGF2 receptor inhibitor, induction of Ngf was significantly blunted, indicating that FGF2 drives Ngf induction upon in vitro cartilage injury. Induction of Bdkrb1, Bdkrb2 and Tac1 were not FGF-dependent in vitro. To test the significance of FGF2-driven Ngf induction in vivo, pain was assessed in Fgf2-/- mice following joint destabilisation. Rather than reducing or delaying the development of pain in these animals, painful behaviour developed earlier than wild type mice, and correlated with cartilage damage scores. Pain could also be significantly neutralised by anti NGF antibody, indicating that FGF2 is unlikely to be driving Ngf expression in vivo. Female mice are relatively protected from OA following joint destabilisation, a phenomenon that may be related to differences in their molecular response to acute joint destabilisation. Despite having little in the way of cartilage damage, they develop painful behaviour at a similar stage as male mice, associated with the same pain sensitising molecules.

Following repeated exposure to vestibular stimulation, the vestibular response as measured by the vestibular-ocular reflex and perception of self-motion is reduced. Similarly, prolonged viewing of a visual motion stimulus results in a reduced sensitivity to the adapting motion stimulus. These phenomena of visual-vestibular desensitisation are utilised as part of treatment of patients with peripheral or central vestibular disorders. Patients typically receive vestibular rehabilitation therapy, which involves exposure to repeated visual and/or vestibular stimulation. Whilst the effects of vestibular rehabilitation at the behavioural level have been widely studied, the neural mechanism of how it works is unclear. In this thesis, asymptomatic subjects were recruited to investigate the neural mechanisms underlying visual and vestibular desensitisation. In the first study, the effect of long term vestibular training was investigated on the vestibular psychophysical measures and on the structure of the brain. To this aim, a group of high level dancers and a group of non-dancers were recruited with both groups undergoing a battery of vestibular tests and neuroimaging brain scans. Compared to controls, dancers showed a significant reduction in both vestibular ocularmotor response and perception of self-motion. Moreover, in controls a significant correlation was found between ocularmotor and perceptual measures, which was absent in dancers. This uncoupling of the vestibular measures was also seen at the neuroanatomical level in the locus of the vestibular-cerebellum, as revealed by voxel based morphometry (VBM) analysis of the dancers' brain grey matter. Using diffusion tensor imaging (DTI), a widespread cortical white matter (WM) network was found to correlate with vestibular perception in the control group only. The findings suggest that in dancers, a cerebellar gating of perceptual signals to cortical regions takes place that may mediate the training-related resistance to vertigo. The second study of the thesis looked at the effect of a single prolonged exposure to unilateral visual motion stimulus in healthy untrained subjects. This involved using transcranial magnetic stimulation (TMS) induced phosphenes to assess early visual cortical excitability. Following visual motion adaptation, excitability of visual cortex (V1) was significantly reduced when viewing motion in the adapted direction and significantly increased when viewing motion in the non-adapted direction. This suggests that reciprocal inhibition takes place between oppositely tuned directionally selective neurones in V1 to facilitate motion perception. The visual cortical excitability returned to its prior-adaptation state after five minutes suggesting that a single exposure to visual motion stimulus is not sufficient to cause a long-term adaptive effect. The final study of the thesis investigated potential neural mechanisms involved in suppressing visual symptom of oscillopsia (perception of the world oscillating/moving), a potentially distressing condition that occurs in some vestibular and ocularmotor disorders. The study recruited participants with nystagmus and they were divided into two groups according to their experience of oscillopsia: symptomatic (with oscillopsia) and asymptomatic (no oscillopsia). TMS induced phosphenes were used to assess (1) whether visual cortical spatial updating takes place according to the eye position and (2) whether modulation of visual cortical excitability takes place during nystagmus. In the asymptomatic group only, evidence for both visual cortical updating and modulation of visual cortical excitability was found, which was absent in the symptomatic group. The findings suggest that spatial updating of eye position and changes in visual cortical excitability are implicated in the suppression of oscillopsia. In particular, the work presented in the thesis provides neuroanatomical imaging basis for vestibular adaptation and provides evidence for a direct cortical involvement in visual motion adaptation. Both of these mechanisms are likely to be involved in the clinical recovery process of patients with vestibular and ocularmotor disorders. Greater understanding of the neural mechanisms involved in long lasting visual-vestibular desensitisation will bring us closer to developing personalised treatments that are more effective in improving symptoms of patients with visuo-vestibular disorders.

Crescentic glomerulonephritis (CRGN) is a significant cause of renal failure in humans and often has an autoimmune aetiology. The Wistar-Kyoto (WKY) rat has a marked susceptibility to CRGN, whilst the Lewis (Lew) rat is resistant, making this a useful model system to study molecular mechanisms underlying the disease. Previous work has demonstrated the importance of macrophages in CRGN and defined some of the genetic determinants underlying CRGN susceptibility. Given emerging data linking epigenetic modifications to both macrophage activation and autoimmune diseases, I hypothesized that variation in DNA methylation in macrophages could contribute to susceptibility to glomerulonephritis in the WKY rat. This thesis investigated this hypothesis using both experimental and bioinformatic approaches. Experimental methods including locus-specific sequencing of differentially expressed genes and unbiased whole genome sequencing allowed investigation of variability in DNA methylation between the two rat strains. Further analyses of methylation in the pathogenesis of CRGN was achieved by both in vivo experiments in WKY rats and in vitro work on cultured macrophages using an inhibitor of DNA methylation. Bioinformatic analysis of sequencing and transcriptional data allowed examination of the affect of DNA methylation on gene expression in rat macrophages, and histone and transcription factor binding site data were used to explore the interaction of methylation and distant gene regulatory sequences. Whole genome shotgun bisulfite sequencing revealed that WKY bone marrow-derived macrophages were generally less methylated than Lew, and more than 13,000 CpG dinucleotides were robustly differentially methylated between the two strains. Multiplexed PCR sequencing was used to verify whole genome methylation signatures. At the genome-wide level, the relationship between methylation and expression was complex but my analyses suggested that differentially methylated CpGs might be enriched at distant gene regulatory elements. These data offer evidence of an association between DNA methylation and susceptibility to CRGN and therefore impart a basis for understanding the distinct genes, pathways and gene regulatory elements that could underlie this association.

Background: The identification of the vulnerable carotid plaque is of paramount importance in order to prevent the significant stroke-related mortality and morbidity. Currently the clinical decision-making around this condition is based on the traditional ultrasound evaluation of the degree of stenosis. However, there is emerging evidence supporting that this is not sufficient for all patients. Aim of this thesis: The evaluation of novel carotid plaque features for the characterisation of plaque composition, volume and motion using 2 and 3 dimensional ultrasound technology. The ultimate goal is to identify novel sensitive imaging markers for carotid plaque characterisation and stroke-risk stratification. Methods: The Asymptomatic Carotid Stenosis and Risk of Stroke (ACSRS) Study was a large prospective multicentre trial that was recently completed. A post-hoc analysis of the sonographic and clinical data from this study was performed in order to evaluate the effectiveness of novel ultrasound texture features, such as second order statics, on stroke-risk prediction. In addition, the change of specific texture features and degree of stenosis during the ACSRS follow-up time (8 years) and their importance for stroke prediction was evaluated. In order to assess the potential of 3D ultrasound carotid imaging we also developed a special methodology using a 3D broadband, linear array probe and the Q-lab software. This methodology was then applied in a clinical, cross-sectional study of patients with symptomatic and asymptomatic carotid disease. Finally we developed a carotid plaque motion analysis methodology that we tested on a feasibility study. Results: The post-hoc analysis of more than 1,000 patients from the ACSRS database showed that there are novel ultrasound features of plaque homogeneity that can contribute to plaque characterisation and improve stroke-risk prediction. Similarly our results suggest that the change of degree of stenosis or plaque's composition through time might have significant predictive value when combined with the above novel features. The study in 3D ultrasound prospectively assessed more than 80 people with symptomatic and asymptomatic carotid disease with both 2 and 3D carotid ultrasound without, though, revealing any significant benefit from the use of 3D imaging in terms of stroke-risk prediction. Finally, our feasibility study on plaque motion analysis showed that it is possible to objectively characterise plaque motion, using ultrasound and dedicated software without complicated reconstructions. Conclusion: The use of novel 2D ultrasound texture features in combination with traditional ones can improve the stroke-risk stratification. 3D ultrasound is a promising new approach, however, the current technology does not appear to offer a significant benefit in comparison to cheaper traditional 2D ultrasound for carotid plaque evaluation. Further research is warranted on this issue.

Background: Epidemiologic evidence is sparse on the role of dietary patterns that may be important drivers of high blood pressure (BP) and body mass index (BMI) levels. Additionally, dietary fibre intake in association with BP and BMI yielded inconsistent results. Objective: Investigate the relationships of eating frequency, dietary energy density, diet quality, evening energy intake, GI, GL, and dietary fibre to BP, BMI using cross-sectional data from the INTERnational study on MAcro/micronutrients and blood Pressure (INTERMAP) of 4680 men and women aged 40-59 y from Japan, China, the United Kingdom, and the United States of America. Methods: During 4 visits, eight BP, four weight and height measures, four 24-hour dietary recalls, and two 24-hour urine samples were collected. Consumption of all solid foods was aggregated into eating occasions. Nutrient density is expressed using the Nutrient Rich Food index. Multivariable adjusted linear regression models were used to estimate BP and BMI differences per 2SD higher intakes of eating occasions, dietary energy density, Nutrient Rich Food index, evening energy intake, GI, GL, and dietary fibre. Results: Compared to participants with <4 eating occasions/24-hours, those with ≥6 eating occasions/24-hours had lower average: systolic BP: 116.4 vs. 121.4 mm Hg; BMI: 27.3 vs. 29.0 kg/m2; total energy: 2127 vs. 2521 kcal/24-hours; dietary energy density: 1.5 vs. 2.2 kcal/g; and higher Nutrient Rich Food index score: 35.1 vs. 26.8. Additionally, insoluble fibre higher by 4 g/1000 was inversely associated with systolic BP (p<0.05), while soluble fibre and GI, GL showed no associations with BP and BMI. Conclusions: Results suggest that higher meal frequency may be associated with improved diet quality and lower BP and BMI. Higher intakes of insoluble fibre may contribute to lower BP and BMI. This may have implications for behavioural approaches to controlling high BP levels and the obesity epidemic.

Gene expression is a process that is tightly regulated by many factors. Different genes are transcribed not only in a cell specific manner but are also differentially expressed at different stages of the cell cycle. Cnot3 is part of the CCR4-NOT deadenylation complex, which is involved in the turnover of mRNAs in the cytoplasm and has also been shown to have roles in regulating transcription and cell proliferation and in maintaining ES cell pluripotency. Previous work demonstrated that Cnot3 interacts directly with Aurora B kinase and is phosphorylated by Aurora B in an in vitro assay. Aurora B and Cnot3 co-localise at active gene promoters in resting B cells. Since Aurora B is a cell cycle kinase, I have developed a cell synchronization system to analyse the role of the Cnot3-Aurora B interaction at different stages of the cell cycle in primary B cells. Using this system, I have demonstrated that the interaction between Cnot3 and Aurora B varies during cell cycle progression. In vitro analysis showed that the interaction occurs through the NOT box domain of Cnot3. Mass spectrometry analysis of Cnot3 interactors, performed on nuclear extracts from B cells in the early G1 and G2 phases of the cell cycle, identified interactions with many factors that are known to have roles in transcription regulation and RNA processing. Interaction of Cnot3 with Histone H1 was confirmed using a peptide binding assay, suggesting a potential role in chromatin organization. Cnot3 was also shown to interact with Xrn2, a 5'-3' exoribonuclease that is involved in RNA turnover and termination of transcription. ChIP analysis demonstrated promoter binding of Cnot3 at a number of cell cycle stages. Cnot3 shows cell cycle dependent binding to promoters of a wide range of active genes, including promoters that are not directly involved in cell cycle regulation. Genome wide analysis using ChIP sequencing revealed changes in the binding profiles of Cnot3 at promoters and enhancers during cell cycle progression. A Cnot3 conditional knock out mouse has been generated, which will be used to test the functional importance of these observations.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in man and despite decades of effort, there is still no protective vaccine. Left untreated, genital infection can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility. However, infection-induced immunity in both animal models and humans indicates a strong role for CD4+ Th1-biased immune responses. Using a multi-component vaccine approach we assessed the immunogenicity and protective efficacy of novel plasmid DNA, Adenovirus 5 (HuAd5) and modified vaccinia Ankara (MVA) vectors each containing a major outer membrane protein (MOMP) transgene and recombinant MOMP protein in various heterologous prime-boost regimens in BALB/c and B6C3F1 mice. During the course of the prime-boost regimens, serum and vaginal MOMP-specific antibody titres, subtypes, avidities and neutralisation abilities were assessed, alongside IFN-γ ELISpot and CD4+ and CD8+ T cell polyfunctionality (IFN-γ, TNF-α, and IL-2). Regimens were grouped on the distinct MOMP-specific immune environments they elicited, with these regimens taken through into C. trachomatis vaginal challenge studies in two mouse models to shed light on the relative contribution of each environment to protective immunity. The DNA-HuAd5-MVA-Protein (D.A.M.P.) vaccine regimen resulted in a significant reduction in C. trachomatis vaginal shedding at day 3 post-infection in both BALB/c and B6C3F1 mouse strains. This significant reduction was lost when D.A.M.P. vaccinated mice were depleted of their CD4+ T cells prior to challenge, indicating the protection is CD4+ T cell mediated. C. trachomatis EB serum neutralisation profiles were similar between protective and non-protective vaccine regimens and combined with passive transfer experiments into naïve C57BL/6 mice and IFN-γ knock-out C57BL/6 mice we concluded that the antibody response did not play a significant role in this vaccine-induced protection. As well as infecting the genital tract, Chlamydia trachomatis is also the causal agent of trachoma, the leading cause of infectious blindness in the world. Recently, Kari et al. revealed serum anti-MOMP antibodies correlated with the reduction in chlamydial ocular burden in non-human primates, while anti-PmpD and anti-Pgp3 serum antibodies correlated with chlamydial eradication. We therefore investigated if we could induce such anti-chlamydial antibodies on the murine eye through heterologous prime-boost vaccinations. We uncovered a vaccination regimen that induced significantly greater anti-MOMP ocular antibodies, and employed this regimen for the additional chlamydial antigens, of which all induced ocular antigen-specific IgG antibodies. This is the first investigation into such vaccination regimens to induce chlamydial specific ocular antibodies and provides a new model for the screening of future potential trachoma vaccines.