Introduction: Type 2 Diabetes (T2DM) and sleep-related disorders share common risk factors such as obesity; but the interrelationships between T2DM and sleep disorders are not well examined. Aims: In this thesis I aimed to assess: 1. The longitudinal impact of obstructive sleep apnoea (OSA) on micro vascular complications in patient with T2DM. 2. The relationship between sleep quality, sleep duration and adiposity in patients with T2DM Methods: To examine the first aim, I utilized the data collected from a previous project that examined the cross-sectional associations between OSA and micro vascular complications in patients with T2DM and followed up the study participants longitudinally using 1-2-1 interviews and electronic health records. For aim 2, I conducted a crosssectional study in patients with young-onset T2DM who were recruited from Heart of England NHS Foundation Trust and primary care. Result: For Aim 1: Depending on the micro vascular outcome examined, we had approximately 200 patients in the analysis. Patients were followed up for 2.5 years for renal outcomes, and 4-4.5 years for retinopathy and neuropathy outcomes. The prevalence of OSA was 63%. I found that baseline OSA was significantly associated with greater decline of eGFR and greater progression to pre-proliferative and proliferative retinopathy. I also found that OSA was associated with progression to a combined outcome of foot insensitivity or diabetic foot ulceration but this was a non-significant trend (p=0.06). In addition, I found that patients who received and were compliant with continuous positive airway pressure (CPAP) treatment (delivered during routine care) had improvements in heart rate variability parameters by study end. For Aim 2: Poor sleep quality and shorter sleep duration were associated with increased total body fat% after adjustment for potential confounders. Conclusion: I found that OSA plays an important role in the progression of micro vascular complications in patients with T2DM. Whether treatment with CPAP has a favourable impact on micro vascular complications is currently being examined in a randomized controlled trial. I also found that sleep duration and quality are associated with increased adiposity. The direction of this relationship need to be examined in longitudinal studies and interventional trials.

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Normal mechanical loading potently induces bone formation mediated by osteocyte effects on osteoblasts. Current in vitro bone models do not reflect these cellular interactions, either focusing on mechanical loading of osteoblasts in monolayers or in 3D and therefore not elucidating the osteocyte-osteoblast interactions that regulate mechanically-induced bone formation. Adenosine, calcium-sensing and glutamate signalling have been shown to influence bone biology, with both adenosine precursors and glutamate having been implicated in mechanotransduction. The aims were to develop a novel in vitro 3D co-culture model of bone to investigate mechanically-induced signalling, and to determine the expression of adenosine, calcium-sensing and glutamate signalling components within the 3D model and their contribution to the regulation of mechanically-induced bone formation markers. A 3D model was developed as a two-phase culture system where MLO-Y4 osteocytes were embedded within type I collagen gels and MC3T3-E1osteoblasts were layered on top. In this model, cells were viable over 7 days (100 % osteoblasts, 87 % osteocytes), maintained appropriate morphology and contacted neighbouring cells through CX43 labelled projections. RT-qPCR revealed Runx2, OCN and E11 mRNA expression in both osteoblasts and osteocytes. COL1A1 mRNA expression was significantly higher in the osteoblasts (P=0.0001), whereas ALP mRNA was higher in the osteocytes (P=0.001). RT-PCR revealed expression of adenosine receptors A2A and A2B and glutamate transporter GLAST1 in osteoblasts and osteocytes, as well as glutamate receptors AMPAR2 and KA1 in osteocytes. Immunostaining confirmed expression of A2A, GLAST1 and KA1, and revealed expression of CaSR, in both osteoblasts and osteocytes. A novel mechanical loading device was developed which was used to apply osteogenic loads (5 min, 10 Hz, 2.5 N) to 3D osteocyte mono-cultures and 3D osteocyte-osteoblast co-cultures. A minimum of 48 hr pre-load time was required for a reliable load response. 3D osteocyte mono-cultures cultured for 48-72 hr or 7 days pre-load, remained viable, significantly increased PGE2 0.5 hr after load (48-72 hr: P=0.0249, 7 days: P=0.041) and decreased their IL-6 synthesis. RT-qPCR revealed a load-induced decrease in E11 (P=0.018) and RANKL (P=0.0486) mRNA, in 48-72 hr cultures. In 7 day cultures, E11 mRNA (P=0.041) increased as a result of loading. Preliminary data showed that the same loading conditions increased PINP synthesis, a bone formation marker, in 3D co-cultures (P=0.022). The AMPA/KA receptors antagonist NBQX increased PINP synthesis by 2-fold over 5 days, similar levels induced by loading in untreated cultures, suggesting that NBQX has similar anabolic effects as mechanical stimuli. Similarly, the A2A receptor antagonist SCH 442416 increased osteoblast ALP mRNA expression by 3.5-fold at day 1 post-load and increased PINP synthesis by 1.9-fold, in co-cultures after 5 days. This 3D osteocyte-osteoblast co-culture model represents a useful in vitro model for the investigation of the osteocyte-osteoblast interactions that lead to mechanically-induced signalling and regulation of bone formation markers. Adenosine, calcium-sensing and glutamate signalling components are expressed within the model, facilitating future investigations of their roles in mechanically-induced signalling. Preliminary experiments indicated that adenosine and glutamate signalling may each contribute individually to the regulation of mechanically-induced bone formation markers.

Mass spectrometry is an analytical technique which is used extensively in the fields of chemistry and physics. Developments in the field over the last two decades have permitted the analysis of a wide variety of biological molecules from a range of sources. The term proteomics relates to the study of the protein complement of a cell or organism with particular interest in the identification and quantification of these analytes. A biomarker is a characteristic that can be measured and evaluated to give an indication of normal, biological processes, or pharmacological responses to a therapeutic intervention. Bodily fluids are a rich source of potential biomarkers as they can be obtained in reasonable quantity and their extraction is generally minimally invasive. The plasma proteome, which contains many thousands of analytes spanning a dynamic range greater than 10 orders of magnitude, reflects the status of the many tissues and organs in the body serving as an ideal medium for potential biomarker discovery. The analytical challenges posed by the plasma proteome are significant. Depletion of the highly abundant proteins is usually a prerequisite of any biomarker study and no technique has the dynamic range to study all of the proteins present. Comprehensive characterisation of the plasma proteome requires significant experimental effort and cost. Use of pooled samples in biomarker studies is widespread and the majority of biomarkers, which have been identified in the discovery phase, have not passed clinical validation. A data independent, label-free quantitative approach has been evaluated for the study of depleted maternal plasma proteomes taken in the first trimester. Plasma was characterised from individual and groups of patients from three obstetric conditions using single and multi-dimensional chromatography. Potential biomarkers from each source were identified and evaluated. Multi-dimensional chromatography was used to simplify the complexity of the analytes introduced to the mass spectrometer and the benefits and limitations of the approach in terms of biomarker discovery have been demonstrated.

Insulin stimulates glucose transport in fat cells by inducing the movement of glucose transporters (Glucose transporter-4) from specialised storage vesicles to the plasma membrane. Insulin resistant individuals and those with Type II Diabetes exhibit impairment in the ability of insulin to stimulate glucose transport. The molecular mechanisms of glucose transporter-4 trafficking in adipocytes are an important focus in understanding the underlying etiology of this disease. Glucose transporter-4 (GLUT4) recycles between the plasma membrane and intracellular stores in the absence of insulin using a complex intracellular pathway. This involves two intracellular cycles: one is the prototypical endosomal system, the other a specialised cycle involving the trans-Golgi network and a sub-set of intracellular vesicles called GSVs (the slow cycle). Understanding the control of the entry into this second cycle is the subject of this thesis. In particular, the work in this thesis will examine the role of Syntaxin 16 and its cognate Sec1/Munc18 protein mammalian Vps45 (mVps45). The regulation of Syntaxin 16 has not been fully elucidated and understanding the role of Syntaxin 16 in SNARE complex regulation and subsequent control of GLUT4 traffic into the slow cycle requires an understanding of its cognate binding partner Sec1/Munc18 (SM) protein, mammalian Vps45 (mVps45). The absolute levels of both Syntaxin 16 and mVps45 were quantified and found to be present in 3T3-L1 adipocytes in roughly stoichiomeric amounts. IP experiments also showed the ability of mVps45 to interact with Syntaxin 16 in the absence of insulin. Using the model eukaryote Saccharomyces cerevisiae, we found that mVps45 could complement for the deletion of Vps45p. Assays for CPY secretion showed that mVps45 is able to complement for the loss of Vps45p function in the trafficking of carboxypeptidase Y (CPY). Additionally, mVps45 mutants were made that correspond to yeast mutants made previously in the lab and were tested for homology of function. Depleting 3T3-L1 adipocytes of mVps45 showed alterations in the levels of GLUT4 protein as well as the protein levels of Syntaxin 16, IRAP, and Rabenosyn. Insulin-stimulated deoxyglucose uptake was also profoundly decreased upon depletion of mVps45. Further experiments using mVps45 depleted cells show that these cells lose their sensitivity to insulin and that the loss of mVps45 in these cells causes GLUT4 to have the inability to enter the slow cycle. Taken together, these findings demonstrate that mVps45 has a role in allowing GLUT4 entry into the slow cycle.

Having identified differentially regulated genes, the final and most labour intensive part of the analysis process is drawing biological conclusions and hypothesises about the data. A novel solution is presented which combines experimental data with a curated annotation sources along with analysis tools to assist the researcher in exploring the information contained within their dataset.

MLL2 is a H3K4 specific HMT which vital for normal embryonic development in the mouse. Little is known on how MLL2 is recruited to its target genes and activates transcription. To gain insight into the molecular mechanism underlying MLL2 function, we focused on a known MLL2 target gene: Magoh2. This gene is controlled by a CpG island promoter and is ubiquitously expressed. Our results demonstrate that in the absence of MLL2, the Magoh2 promoter is methylated and Magoh2 is transcriptionally silenced. The Magoh2 promoter adopts the active conformation only in the presence of MLL2. Pol II is lost from the Magoh2 promoter in the absence of MLL2, resulting in Magoh2 down-regulation. We observed loss of H3K4me\(_3\)and H3K9ac and relocation of a nucleosome over the promoter, coinciding with the onset of DNA methylation. Use of ORB and a-amanitin demonstrated that neither transcription nor the presence of Pol II are required for the maintenance of H3K4me\(_3\). Magoh2 silencing can be overcome by re-introducing full-length MLL2. We investigated the role of MLL2 in haemopoiesis and demonstrated that MLL2 is vital for macrophage differentiation from embryoid bodies. MLL2 may be required for correct upregulation of Flk1 and generation of haem angioblast cells. When M//2 was deleted in haemangioblasts, the haemopoietic transcriptional program was perturbed suggesting that MLL2 may also play a role at this later developmental stage.

Genome-Wide Association Study (GWAS) has recently been proposed as a powerful strategy for detecting the many subtle genetic variants that underlie phenotypic variation of complex polygenic traits in population-based samples. One of the main obstacles to successfully using the linkage disequilibrium based methods is knowledge of any underlying population structure. The presence of subgroups within a population can result in spurious association. A robust statistical method is developed to remove the population structure interference in GWAS by incorporating single control marker into testing for significance of genetic association of a polymorphic marker (SNP) with phenotypic variance of a complex trait. The novel approach avoids the need of structure prediction which could be infeasible or inadequate in practice and accounts properly for a varying effect of population stratification on different regions of the genome under study. Both intensive computer simulation study and eQTL analysis in genetically divergent human populations show that the new method confers an improved statistical power for detecting genuine genetic association in subpopulations and an effective control of spurious associations stemmed from population structure.

Replacement of the prototype promoter recognition factor (PRF) TFIID by alternative PRFs or changes in its subunit composition have recently been implicated in differentiation processes during development. TFIID is composed of TBP (TATA-binding protein) and 13 TAFs (TBP-associated factors). I comprehensively studied the roles of Tafs during vertebrate development using zebrafish model. Taf knock down (kd) phenotypes generated in an antisense morpholino oligonucleotide (MO) screen suggest differential functions of Tafs during zebrafish development. The kd phenotypes also propose a special requirement of DNA-binding Tafs during zebrafish development. In conjunction with zygotic mutant phenotype analysis of zebrafish taf8-mutants compared to taf6-mutants I investigated a potential coactivator function of Taf8 for Pparγ, which has been suggested by in vitro data. Oil Red O (ORO) stainings of 5 dpf (days post fertilisation) taf8- and taf6-mutant larvae revealed a specific lipogenesis defect in liver and intestine of taf8-mutant larvae. The results from treatments with a PPARγ inhibitor suggest that this lipogenic process is Pparγ-dependent. To convincingly establish a coactivator function of Taf8 for Pparγ during early zebrafish development, future work has to link the 5 dpf lipogenesis phenotype of taf8-mutants to defects in Pparγ-dependent transcription.

Peptide inhibitor of trans-endothelial migration (PEPITEM) is a B cell-secreted peptide which inhibits T cell trafficking across cytokine-stimulated endothelium. This homeostatic mechanism is lost in autoimmune and chronic inflammatory diseases, leading to inappropriate T cell trafficking with pathological consequences, e.g. in type-1 diabetes and rheumatoid arthritis. We aimed to investigate the structure and function of PEPITEM in vitro and in vivo to establish its pharmacokinetics, therapeutic potential, and underlying molecular mechanisms. The efficacy of PEPITEM was verified, showing the same regulatory control of T cell trafficking as previously reported. Nuclear magnetic resonance (NMR) studies revealed PEPITEM to be a linear peptide lacking secondary structure. Intravenous administration of radiolabelled native PEPTEIM in wild-type mice showed rapid clearance by the renal circulation, conferring a circulatory half-life of < 2 minutes. Common conjugation strategies employed to modify PEPITEM, e.g. PEGylation, did not affect peptide function, demonstrating its potential for therapeutic development. The evolution of PEPITEM from its parent protein, 14-3-3ζ was investigated. PEPITEM is probably cleaved from 14-3-3ζ by matrix metalloproteinase 9 extracellularly after exocytosis from B cells. Interestingly, this generates a 17aa peptide which requires additional proteolytic processing to evolve a smaller biologically active pharmacophore. These observations add to our current knowledge of the PEPITEM paradigm, which appears central to regulated trafficking of T cells. Future work will formulate new PEPITEM versions with suitable pharmacological profiles, and screen them for function in vitro and in vivo to develop suitable therapeutic agents.