It is clear that sub-clinical inflammation is a key factor that triggers type 2 diabetes mellitus (T2DM) and is directly influenced by weight gain. Current studies highlight that obesity, particularly central obesity, heightens the pathogenesis of T2DM. Additionally, factors produced by adipose tissue (AT), referred to as adipocytokines, can influence the degree of insulin resistance as well as inflammation, due to their duality of function. A recently implicated adipocytokine, visfatin, has been identified as a potential insulin mimetic, an enzyme associated with mitochondrial biogenesis and an inflammatory factor. However, current studies lack a clear understanding as to the role and influence of visfatin in human AT and insulin resistant states. Therefore, this thesis examined visfatin expression within specific human AT depots and the influence of adiposity - with specific consideration given to insulin resistant states including T2DM, non-alcoholic fatty liver disease (NAFLD) and human immunodeficiency virus (HIV). Further, serum studies addressed how different insulin resistant states influenced circulating visfatin levels, whilst mechanistic studies explored how the role of visfatin was altered by insulin, an insulin sensitiser, inflammation and/or disease. This current thesis identified that visfatin was abundant in both abdominal depots, with highest expression in the omental (Om) AT, and isolated adipocytes, with an apparent relationship with insulin resistance. Subsequent in vivo and in vitro analysis further identified that, whilst insulin appeared to increase visfatin protein expression in isolated abdominal subcutaneous (Abd Sc) adipocytes, the use of an insulin sensitiser - either used in cultured Abd Sc adipocytes or as part of an oral therapeutic treatment in subjects with T2DM - decreased circulating visfatin levels. In addition, intracellular signalling studies highlighted that visfatin regulation within AT appeared to be dependent upon both nuclear factor (NF)-B and c-Jun N-terminal kinase (JNK) activation, influencing interleukin (IL)-6 as part of a visfatin regulatory feedback mechanism. Following the potential influence of visfatin in T2DM, this thesis explored its’ potential role in disease associated with other insulin resistant phenotypes, such as liver disease and HIV. From serum assessment of visfatin in subjects with NAFLD, it was identified that progression of liver disease was accompanied by a reduction in circulating visfatin levels – a finding that occurred independently of diabetic status. However, circulating visfatin still remained significantly higher in NAFLD with T2DM than those without. Finally, this thesis examined a potentially severe insulin resistant phenotype noted in HIV patients, due to an apparent lipodystrophy - a condition which alters fat oxidation and mitochondrial activation or regulation. In such a condition, circulating visfatin levels and visfatin mRNA AT expression remained unaltered by HIV status or drug therapy. Due to the capacity of visfatin to act as an enzyme involved in nicotinamide adenine dinucleotide (NAD) biosynthesis, essential for mitochondrial function and oxidative phosphorylation (OXPHOS), its role in AT remained unchanged by disease status, whilst other mitochondrial and fat metabolism factors were altered by both disease state and drug treatment. Taken together, these current data suggest a duality of function of which visfatin appears to be regulated by insulin, in addition to inflammation, in different disease states and therefore expands our current understanding of this multi-functional adipocytokine.

Session ID: SA-B1-2 / SCIS & ISIS 2010, Joint 5th International Conference on Soft Computing and Intelligent Systems and 11th International Symposium on Advanced Intelligent Systems. December 8-12, 2010, Okayama Convention Center, Okayama, Japan

While the genetics of retinoblastoma and the implications of the retinoblastoma susceptibility gene, RB1, are well described, there is still scarce evidence to suggest why RB1 acts in such a cell-type specific manner. Using the murine cortex as a model, we examined the effects of RB1 deletion of cycling neural progenitors and post-mitotic neurons, in order to ascertain cell-type specific functions in the central nervous system. Using the previously identified cell-cycle independent role for Rb in tangential migration, we validated Rb/E2f regulation of neogenin and implicated it in this process. In quiescent cortical neurons, we identified a pivotal role for Rb in neuronal survival. Unlike in cycling progenitors, in post-mitotic neurons Rb specifically represses the expression of cell-cycle associated genes in an E2f-dependent manner. Finally, in cortical neurons in the absence of Rb, we observe an activation of chromatin at E2f associated promoters. To determine the role of direct interaction between Rb and chromatin modifying enzymes, we utilized an acute LXCXE-binding deficient mutant paradigm. We report that the LXCXE binding motif is dispensable in establishment and maintenance of cortical neuron quiescence and survival. The activation state of E2f-responsive promoters appears to be dependent on E2f-activity and not simply Rb-mediated repression. Taken as a whole, this thesis serves to support the hypothesis that Rb plays a diverse role in different cell-types by regulation of unique gene targets and regulatory mechanisms. Characterizing specific cancer-initiating populations and understanding the specific function of Rb will help in the treatment of many cancers resulting from RB1 mutation or mutation within the Rb/E2f pathway.

Recent advances in genome sequencing are improving our better understanding of genetic variation. However, the investigation of the genotype-phenotype relationship is still challenging, especially for the interpretation of the myriad of discovered genetic variants that weakly relate to disease. Recently, researchers have confirmed that disease causing genetic variants typically occur at functional sites, such as protein-protein or protein-ligand interaction sites. Giving this observation, several bioinformatics tools have been developed. This thesis first details VarMod (Variant Modeller), an algorithm that predicts whether nonsynonymous single nucleotide variants (nsSNVs) affect protein function. The recent Ebola virus outbreak in West Africa demonstrated the potential for the virus to cause edipdemics and highlighted our limited understanding of Ebola virus biology. The second part of this thesis focuses on the investigation of the molecular determinants of Ebolavirus pathogenicity. In two related analyses knowledge of differing pathogenicity of Ebolavirus species is used. Firstly, comparison of the sequences of Reston viruses (the only Ebolavirus species that is not pathogenic in humans) with the four pathogenic Ebolavirus species, enabled the identification of Specificity Determining Positions (SDPs) that are differentially conserved between these two groups. These SDPs were further investigated using analysis of protein structure and identified variation in the Ebola virus VP24 as likely to have a role in determining species-specific pathogenicity. The second approach investigated rodent-adapted Ebola virus. Ebola virus is not pathogenic in rodents but it can be passaged to induce pathogenicity. Analysis of the mutations identified in four adaption studies identified that very few mutations are required for adaptation to a new species and once again the VP24 is likely to have a central role. Subsequent molecular dynamics simulations compared the interaction of Ebola and Reston virus VP24 with human karyopherin alpha5. The analysis suggests that Reston virus VP24 has weaker binding with karyopherins and we propose that this change in binding may reduce the ability of Reston VP24 to inhibit human interferon signaling.

A STUDY OF THE ROLE OF MICRORNAS IN INFLAMMATORY BOWEL DISEASE: THE EFFECT OF MIR-31 DYSREGULATION IN THE EXPRESSION OF TSLP IN ULCERATIVE COLITIS By Andrew Cia ridge Our current understanding suggests that Inflammatory Bowel Disease (lBD) is an immunological mediated disease triggered by unknown environmental factors in those who are genetically susceptible. MicroRNAs (miRNAs) are a class of small non coding RNAs that play a critical role in many immunological pathways by regulating gene expression. To date over 70 miRNAs have been identified as having a differential expression in IBD although their roles remain unlmown. Using microarray and RT-qPCR we have identified the differential expression of 7 miRNAs; miR-31, -146b, -194, -200b, -223, -375 and -422a, in the sigmoid mucosa of treatment na'ive Ulcerative Colitis. Of these, the expressions of miR-31 and -223 are also increased in mucosal Iymphocytes and the CD4+CD25in •• rm.dia •• subfamily. Using in silica analysis, thymic stromal Iymphopoietin (TSLP) is identified as a target for 4 of the 7 miRNAs; miR-31, -223, -194 and -422a. TSLP has been shown to be increased in TH2 mediated disorders of the lung and skin and implicated in epithelial barrier homeostasis and Iymphocyte function in response to luminal flora. Here we show for the first time the expression of TSLP by healthy Iymphocytes and a decreased expression in mucosal biopsies and mucosal Iymphocytes in active uc. In addition, using a human Iymphocyte cell model we demonstrate an increased expression of miR-31 in stimulated Iymphocytes which is inversely related to a decreased expression of TSLP mRNA and an even larger Abstract effect on the expression of TSLP protein in the same cells. Selective inhibition of miR-31 prevents the decrease in TSLP in stimulated Iymphocytes. The increased expression of miR-31 in UC actively decreases the expression of TSLP, a factor associated ' with epithelial homeostasis and less destructive inflammation. Manipulation of the miR-31 / TSLP pathway offers a potential therapeutic strategy for uc.

Patients with rheumatoid arthritis (RA) frequently present with pain under their feet. Forefoot bursae can give rise to such symptoms, but are rarely investigated. The aim of this thesis was to use musculoskeletal ultrasound (MSUS) performed by a podiatrist to evaluate the prevalence and natural history of bursae in the forefoot in RA patients. Once reliability of technique was established, a longitudinal study design was used in which a sample of RA patients (N=149) and a comparator group (N=50) of healthy individuals were assessed at baseline. A Diasus MSUS system was used to image the forefeet of all participants to determine prevalence of bursae. 120 patients (98 female, 22 male) with RA (24 seronegative, 93 seropositive, 3 unknown) completed the study at twelve months: mean age 60.7 (SD 12.1) years and disease duration 12.99 (10.4) years. Results confirmed a high prevalence of forefoot bursae (92.6% of patients; mean per individual =3.54, range 0-9) and that these were often missed by clinical examination. Findings that there could be an association between patient reported foot impact scales of impairment/footwear (LFISIF) and activity participation restriction/limitation (LFISAP) and presence of bursae (LFISIF β=0.377, p=0.033; LFISAP β=0.762, p=0.013) independent of disease activity were unique. On examination of prospective data after one year, 25.8% of participants had increases in bursae and 23.3% decreases. There was a significant correlation between changes in bursae with changes in LFISIF (PCC=0.216, p=0.018) and LFISAP (PCC=0.193, p=0.036) and a significant negative correlation with changes in duration of RA (PCC=-0.269, p=0.003). The findings imply that MSUS detectable bursae in the forefeet are highly prevalent, clinically under-reported and change over time. The findings suggest that bursae within the foot in RA deserve increased clinical attention and that further work is required to confirm associations with patient reported foot impact outcome measures.

No description available.

Objective: There is a general agreement that physical activity (PA) has a beneficial effect on health and those who are more active have a reduced risk of developing many chronic diseases, such as coronary heart disease (CHD). However, the amount, type and intensity of PA deemed to be sufficient to achieve good health remains unclear. Different methods have been used to categorise activity behaviour, but the level of agreement, consistency and coherence between methods and how this might influence their relationship with CHD risk factors and estimated CHD (eCHD) risk are poorly understood. This uncertainty is reflected in many different messages communicated to the public as to how active they should be to prevent chronic diseases. The primary objective of this thesis was to determine whether the methods used to categorise PA (as either inactive/active or level of PA) influence the extent to which PA is associated with CHD risk factors and eCHD risk. Methods: This thesis was divided into two parts. The first part was to conduct a secondary analysis of data on activity and CHD risk factors (blood pressure and lipid profile) obtained from the 2004 UK National Diet and Nutrition Survey (NDNS) in 1658 adults aged 19-64 years. Using the information obtained from the NDNS 7-day diary, it was possible to extend the original observations and to re-categorise individuals according to measures of PA in terms of number of days and minutes of at least moderate PA, total activity expressed as metabolic-equivalents (METs) and self-perception of PA. Each of these methods was then used to examine the proportion of the variance in CHD risk factors and the eCHD risk attributable to differences in PA using General Linear Modelling with adjustment for BMI, age and smoking. Partial eta squared a “proportion of variance due to physical activity plus error that is attributed to physical activity alone” was used. In the second part, the concurrent validity of measures of PA derived from the NDNS 7-day diary, using different systems for coding and classifying of different physical activities, was compared against those measures of PA obtained from the International Physical Activity Questionnaire (IPAQ) in a group of medical students (n = 26). Results: Taken together, this thesis revealed: 1) poor agreement across different methods of categorisation of PA level, 2) no support to justify a curvilinear dose-response relationship between PA level and CHD risk factors and eCHD risk and that a linear model was sufficient, 3) the differences in CHD risk factors or eCHD risk that could be directly attributable to differences in PA in men was modest (generally < 5%) although no associations evident in the women, 4) effect was most obviously demonstrable as improvements in lipid profile, no demonstrable effect on blood pressure, 5) a potential problem might arise when using one system and applying its results to different guidelines established by different systems. Conclusion: These findings support the view that being physically active is associated with markers of better health and lower CHD risk; a small but consistent effect that was the same irrespective of which method of categorizing PA was used and even after adjustment for differences in age, BMI and smoking. The effects were most evident in men and largely attributable to improvements in lipid metabolism.

The utility of breath trace compounds as bio-markers for various physiological conditions has long been exploited for the diagnosis of various diseases. Urea breath tests have been adopted as the gold standard for the detection of Helicobacter pylori which is a primary cause for acute gastritis and peptic ulcers. In these tests, small changes in the ratio of stable CO2 isotopomers, 13CO2 and 12CO2, present in exhaled breath are measured precisely and this is conventionally done by using an Isotope Ratio Mass Spectrometer. However, the huge cost and complexity involved in operating these instruments has restricted their widespread use. A viable and low cost alternative is offered by instruments employing non-dispersive infrared absorption techniques. The feasibility of such an instrument has been explored in this work. The instrument presented here is a two channel isotope ratiometer that performs whole band integrated absorption measurements. Detection is based on a novel feedback mech- anism whereby an imbalance in the channel absorptions causes the pathlength along one of the channels to be altered in order to bring the system back to balance. This change in ratio of pathlengths is directly related to the change in the 13CO2/12CO2 concentration. Signffcant amount of work has already been done to investigate the effects of interferences from coincident absorption bands and other spectral effects that can lead to spurious results. A comprehensive description of the overall system design, development and performance evaluation of the first prototype instrument has been presented here. This involved significant computer modeling and simulations and the results were verified experimentally. These results provided sufficient evidence to suggest the feasibility of such an instrument as a diagnostic tool. It was also concluded that some design improvements were required to circumvent issues related to pathlength variation and a list of recommendations has been provided for this purpose. On the basis of the results obtained as part of this research endeavour, it was concluded that the non-dispersive instrument design presented here can form the basis for a low cost commercial alternative for performing carbon isotope ratio breath tests.

Intrinsic protein folding pathways are modulated by molecular chaperones, such as the diverse group of heat shock proteins (Hsps). Among these is the small heat shock protein (sHsp) family which in the mammalian genome consists of 10 low molecular weight (15-30kDa) members. The sHsps have classical chaperone functions but additionally contribute to pathways that protect against cellular stresses, maintain the cytoskeleton, prevent protein aggregation and regulate apoptosis. They contain a characteristic C-terminal α-crystallin domain, which is exclusive to the sHsp family. In addition to their constitutive expression under physiological (non-disease) conditions, they are also induced under conditions of stress/heat shock which is thought to play a role in response to protein misfolding that underpins disease. There are a wide range of diseases in which the sHsps function or are dysfunctional by mutations, such as neurodegenerative disorders, cataract, and desmin related myopathy. Each of the 10 sHsps is believed to have a unique expression profile. Seven of the sHsps are expressed in heart and muscle, but little is known about their precise expression and/or physiological role in the CNS. In the present study the expression of the mammalian sHsps in various mouse tissues including the brain was investigated. This provided evidence for the constitutive expression of 4 sHsps in the brain. In situ hybridization using naïve adult mice revealed a distinct white matter (oligodendrocyte) specific expression pattern for HspB5 (αBcrystallin). HspB1 (Hsp25) and HspB8 (Hsp22) demonstrated overlapping expression in the lateral and dorsal ventricles of the brain, as well as expression in a distinct set of motor neurons in the ventral horn of the spinal cord. Further, cellular immunostaining and subfractionation of brain tissue supports a distinct cellular and subcellular protein expression of HspB1, HspB5, HspB6 (Hsp20) and HspB8 in the brain. Both HspB5 and HspB6 were enriched in the myelin fraction. In view of the potential for induction of these sHsps by stress and modulation in chronic brain diseases we systematically investigated the sHsp signature in two distinct models of intracellular (R6/2) and extracellular (ME7) proteinopathies. These models recapitulate key features of Huntington’s and prion disease, respectively. Analysis of the sHsps in the R6/2 Huntington’s disease (HD) mouse model showed a specific down-regulation of HspB5 in the white matter at all time points analyzed. All other sHsps investigated did not change in this model of HD. Analysis of the sHsps in ME7 prion disease showed up-regulation of HspB1, HspB5 and HspB8 in the hippocampus. For HspB1, this was selective to an anatomically defined sub-population of astrocytes distributed in the stratum radiatum. In contrast, all GFAP positive astrocytes throughout the hippocampus exhibited induced expression of HspB5 and HspB8. Based on QT-PCR data, the changes in expression of the sHsps in either model was not under transcriptional control, suggesting translation/posttranslational regulation. The differing results in the two models suggest that the presence of intracellular (R6/2) or extracellular (ME7) aggregates may dictate the sHsp response associated with non-neuronal cells. In view of the emerging significance of non-neuronal cells in chronic diseases the data supports adaptive and differential responses that might contribute to and/or provide a route to therapy of distinct aspects of neurodegeneration.