COPD is a leading cause of morbidity and mortality worldwide and it is believed that the neutrophil is key to the pathology. Evidence to date suggests that neutrophils migrate less accurately in patients with COPD, although the precise mechanisms by which this occurs have not been defined. We have shown that COPD neutrophils migrate faster (chemokinesis) but less accurately (chemotaxis) in various chemokine gradients. It appears to be an intrinsic cell defect, as incubation of healthy neutrophils in COPD plasma did not alter migratory dynamics. This phenomenon does not occur in other respiratory diseases and is unrelated to age, disease severity, smoking status or the presence of emphysema. Furthermore, there were no differences in markers of neutrophil activation or maturity, although degranulation markers were higher in COPD. Expression of certain chemokine receptors was lower on quiescent COPD neutrophils, but these differences were abolished after stimulation. Receptors localised to the leading edge effectively in COPD neutrophils and there were no differences in receptor shedding. PI3K phosphorylation was increased in COPD and inhibition of γ and δ isoforms improved chemotaxis. Neither Akt phosphorylation nor intracellular calcium signalling was altered. Simvastatin also improved chemotaxis of COPD neutrophils but CXCR1/2 inhibitors did not.

Human adipose tissue acts as a multi-functional endocrine organ. It has the capacity to control energy homeostasis through changes in peripheral and central mechanisms. However, with increasing weight gain, adipose tissue can more than double its original mass, leading to pathogenic consequences including chronic low grade inflammation and insulin resistance mediated by adipocytokines. These changes within adipose tissue can induce secondary detrimental effects on other organs, such as muscle and liver; which also produce hormones that can affect energy homeostasis. The inflammatory response appears modulated by dietary factors and nutrients, including lipids and glucose, as well as gut derived bacteria, in the form of endotoxin. This thesis sought to investigate the role of nutrients, such as lipids (fatty acids) and glucose, on inflammatory responses, energy expenditure and insulin resistance, in vitro and in whole body human physiology. Specifically, studies examined the impact of duration and frequency of nutrient exposure as well as the impact of novel hormones on central and peripheral metabolism in healthy and insulin resistant states. Nutrients, such as fatty acids, were noted to have an immediate impact on the inflammatory response in both murine and human differentiated pre-adipocytes in vitro. Follow-on whole body physiology studies examined lean and overweight/obese (OW/OB) subjects, given isocaloric high fat meals as either 2 large meals or 5 smaller meals over 24 hours. These studies showed minimal effects on blood profiles in the lean subjects post-meal, whilst the OW/OB subjects took much longer to regain their normal lipid and glucose profiles. However, the frequency of the meals in both cohorts had no specific or significant impact on energy expenditure, inflammation, glucose/insulin levels or lipid profile. Analysis of factors that could affect adipose tissue metabolism and whole body physiology, such as irisin in muscle and FGF21 in liver, highlighted that insulin resistance status, BMI and lipid profiles could, at times, influence their circulating levels. This further highlighted the impact of adipose tissue mass, function and cross-talk on health. In summary, both in vitro and in vivo findings highlighted that ‘over-nutrition’ with lipids and glucose could trigger an inflammatory response, which was more pronounced with weight gain. Secondly, this thesis provided evidence that the frequency or duration of exposure to nutrients was less important than the total amount of nutrients consumed. Taken together, these studies emphasise the importance of focusing on interventions to examine nutrient composition, and to reduce overall nutrient intake, rather than the frequency of meals to improve metabolic health in obesity.

During the past few decades a new approach has emerged for the treatment of human disease. In that short period, the concepts and techniques of gene therapy have progressed from being entirely fanciful to experimental clinical application. A major stumbling block for gene therapy is the inefficiency of gene transfer and the transient nature of therapeutic gene expression. Attempts to deliver therapeutic genes using replication-defective adenoviruses have been hampered by a strong host immune response to the vector, leading to clearance of transduced cells and loss of transgene expression. Current adenovirus vectors have an additional disadvantage of being able to carry only approximately 10 kb of exogenous DNA. The work here describes attempts to improve upon existing adenoviral vectors by addressing these limitations In order to reduce the host immune response to the vector, additional deletions in the residual viral coding regions are required to prevent expression of immunogenic proteins. Deletion of the major late transcription unit (MLTU), which encodes virtually all of the viral structural proteins, would achieve this and would also increase the transgene carrying capacity of the vector. In order to create such a vector, a cell line would be required to complement the growth of the deleted vector by providing late gene functions in trans. The work presented here describes the successful cloning and subsequent analysis of the Ad5 MLTU with expression driven by the major late promoter (MLP). The expression plasmids constructed express one or more late proteins from each late gene segment in a transient assay. The plasmids also carry the EBNA-I and oriP sequences from Epstein-Barr virus which allow the plasmids to be stably maintained in eukaryotic cells. Stable cell lines were constructed using these plasmids but no late protein expression from the MLTU could be detected. Attempts to activate expression from the major late promoter by providing viral transactivating factors in trans also proved to be ineffective. To address these problems, an alternative inducible promoter was chosen. The sheep metallothionein Ia promoter was cloned upstream of the MLTU and this construct was then cloned into an episomal expression vector. This plasmid was also used successfully to regulate the expression of late proteins during transient transfection studies. However, a stable cell line constructed using the same plasmid did not show any expression of late proteins. The reasons for the inability of any cell line constructed to express late proteins are still undetermined. Possible reasons discussed are plasmid rearrangement, promoter down-regulation and possible blocks to post-transcriptional processing and translation of the complex MLTU transcript. Suggested future studies include testing of these possibilities in order to gain further insight into the regulation of expression from the MLTU construct, ultimately leading to the construction of a cell line capable of complementing the growth of adenovirus vectors with late gene deficiencies.

Gene therapy is a novel approach to the treatment of human disease that is in its very early stages of development. Its purpose is to add to and/or alter the pattern of gene expression in cells so as to achieve a therapeutic benefit and is being developed for application to such diverse conditions as simple inherited diseases, cancer, AIDS, and cardiovascular disease. Poor uptake of DNA by cells in vivo is a significant technical barrier for gene therapy. Viruses have evolved to carry their nucleic acid contents into cells very efficiently and are thus considered as potential vectors for gene therapy, provided their pathogenicity and other adverse features can be overcome. Adenovirus is a virus type, which is a prime candidate for development as a vector for efficient transfer of genes into human tissues. In recent years, there have been many studies in order to develop replication deficient adenoviral vectors as gene therapy vectors. Current disadvantages of these vectors include their limited capacity to accommodate exogenous DNA and elucidation of a host immune response against these vectors. Currently they have a capacity for about 9 kilobasepairs of DNA, which is not sufficient for some therapeutic purposes. The work described in this thesis aimed to provide a way to produce highly deleted adenoviral vectors containing deletions in their L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD and IX coding regions. These genes encode proteins, which contribute to virus particles, and their assembly and some of which have been reported to increase expression of the viral late (structural protein) genes. Together with the deletions available in currently used vectors, these new deletions would create vectors with an increased capacity to accommodate exogenous DNA. Furthermore, these deletions were expected to make the vectors more replication-deficient and less immunogenic by decreasing the expression of residual viral genes. Initially, cell lines expressing adenovirus L1 52/55-kD, L1 52/55-kD and IVa2, or L1 52/55-kD and IX proteins were constructed for use as complementing cell lines. These were designed to supply in trans the relevant proteins that would be missing during attempts to construct recombinant viruses with deletion mutations in these gene(s). 293-L1 cells were proven in their ability to complement missing L1 52/55-kD protein function using existing adenovirus L1 52/55-kD point mutants ts369 and H5pmSOO1. However, attempts to isolate an L1 52/55-kD coding region deleted recombinant adenovirus using this cell line were unsuccessful, possibly due to the nature of sequences missing in the deleted L1 52/55-kD coding region which might have currently undefined cis acting regulatory functions. L1 52/55-kD protein is known to form a complex with IVa2 and the latter protein has been reported to activate the major late promoter. The effects of L1 52/55-kD protein on the adenovirus major late promoter transactivation were therefore investigated by transient expression experiments carried out by transfection of COS cells with a major late promoter-dependant reporter gene (CAT) and expression vectors for L1 52/55-kD and/or IVa2. These experiments did not reveal any role for the L1 52/55-kD protein in the activation of adenovirus major late promoter.

High mobility group nucleosome binding (HMGN) proteins belong to the superfamily of high mobility group (HMG) proteins. HMGN1 and HMGN2 are ubiquitously expressed in all vertebrates, and are most highly expressed in embryonic tissue. Moreover, HMGN1 and HMGN2 were found to be highly expressed in neural stem/progenitor cells in the mouse brain. Here, mouse embryonal carcinoma cells (P19 EC) were used as a model system to study the role of HMGN proteins in pluripotent stem cells. Previously, experiments using short interfering RNA (siRNA) technology to knockdown HMGN1 and HMGN2 have suggested that HMGN proteins are important for the expression of key pluripotent genes, Oct4, Sox2 and Nanog, in P19 EC cells (Mohan, 2012). The aim of this thesis was to develop a lentiviral system for the long term knockdown of Hmgn2, in order to investigate more fully the role of this protein in stem cell pluripotency and differentiation. Constitutive and inducible lentiviral shRNAmir systems were tested and optimized, and a constitutive system was chosen for further work. HMGN2 knockdown in undifferentiated P19 EC cells resulted in the down-regulation of Oct4 protein levels. ChIP assays showed that HMGN2 binding over the Oct4 gene was absent in HMGN2 knockdown cells. Furthermore, binding of HMGN1 at this locus was increased in the absence of HMGN2. Consistent with the reduction in Oct4 expression, levels of the active histone modification, H3K4me3, were also decreased at the Oct4 gene. These results support a role for HMGN2 in the regulation of Oct4 expression in P19 cells, and imply that HMGN2 may be important for maintaining stem cell pluripotency.

Liver fibrosis has a range of aetiologies and is a global cause of mortality. A critical effect of liver fibrosis which also increases mortality is portal hypertension. The hepatic stellate cell is accepted as a major progenitor of liver myofibroblasts, which have been shown to be a major source of collagen and extracellular matrix proteins that disrupt liver architecture and function. Relaxin is a hormone involved in remodelling of extracellular matrix in the uterus and cervix and is known to increase renal blood flow in pregnancy. It has been implicated in the regulation of fibrosis in animal models and to modify the cell biology of hepatic stellate cells in vitro. I have demonstrated the profile of expression of relaxin receptors in primary human stellate cells (HSC), showing them to express RXFP-1, 3 and 4. Using a cAMP assay I confirm these receptors to be functional, with RXFP-1 positively and RXFP-3 and 4 negatively coupling to cAMP. The expression of RXFP-1 is coupled with the level of activation, demonstrating a possible role for H2-relaxin in the regulation of HSC. I have established a dynamic regulation of fibrotic mediators and HSC activation markers, including a reduction in α-SMA, TIMP-1 and TGF-β with increases in MMP-1 and MMP-2, consistent with H2-relaxin having potentially therapeutic antifibrotic effects by increasing the fibrolytic phenotype. In addition through the use of gel contraction assays I demonstrate that H2-relaxin reduces serum or endothelin-1 induced HSC contraction. Through the use of siRNA I have confirmed that H2-relaxin mediates its regulation of fibrotic mediators and HSC activation markers as well as the inhibition of gel contraction through the relaxin receptor RXFP-1. I have evidence to suggest that the inhibition of contraction may in part be via nitric oxide release in HSC. In conclusion I propose that RXFP-1 is a potential therapeutic target in end stage human liver disease, targeting fibrosis and portal blood hypertension via both resolution of the phenotypic collagen deposition and vascular constriction associated with the human hepatic stellate cell.

Interleukin 6 (IL6) is associated with arterial disease development, progression and surgical outcome. Raised levels of IL6 may play a causal role in disease development or may be the effect of pathology. An IL6 single nucleotide polymorphism (SNP) G- 174C has been identified and reported to associate with IL6 expression. However, conflicting results have emerged and both the relationship between IL6 and vascular disease and the precise effect of SNP G-174C in vivo in humans remains obscure. The aim of this study was to establish the effect of SNP G-174C in humans, in vivo in different tissues. Varicose vein surgery patients donated adipose tissue, skeletal muscle, vein and blood samples. Patients were genotyped for SNP G-174C. A new pre-mRNA assay was developed, using gel electrophoresis, restriction digest and fluorescence quantification, to measure the ratio of heterozygous allelic pre-mRNA transcription. IL6 mRNA expression in different tissues was also measured using relative real time PCR (RT-PCR) to assess effect of tissue type on expression profiles. mRNA expression within tissues was compared between G-174C genotypes, to further quantifying the association of SNP G-174C with transcript levels. The pre-mRNA assay showed higher expression for the C-allele, though not statistically significant. The pre-mRNA assay needed to detect low levels of intron retaining allelic pre-mRNA isoforms. Replicates and controls for residual genomic DNA were used to monitor assay precision. Adipose tissue gave the greatest precision in the pre-mRNA assay. In the RT PCR assay adipose tissue expressed significantly more IL6 mRNA than all other tissues examined. In vein and leukocytes subjects with the CC genotype expressed significantly higher levels of IL6 mRNA than subjects with GC or GG genotypes. There was a trend towards higher expression for the CC genotype in all tissue types. A significant though weak correlation between IL6 mRNA expression and age was demonstrated for vein and leukocytes. Adipose tissue may be an important source of IL6 compared to other tissues. This may be relevant for obesity associated diseases. Subjects with G-174C genotype CC showed a trend towards higher IL6 RNA expression. Further studies are necessary to clarify the effect of genotype on IL6 expression.

The postoperative period is characterized by massive shifts of fluid between body compartments and accumulation of fluid in the extracellular space, which may manifest clinically as central and or peripheral oedema. The incidence of oedema in patients undergoing routine major abdominal surgery (MAS) is unknown and there are no objective means of quantifying or monitoring its presence. Furthermore, the aetiological factors responsible for post-surgical oedema formation in patients with no overt signs of cardiovascular disturbance are poorly understood and the relationship between the development of oedema and clinical outcomes such as the recovery of gastrointestinal function, postoperative complications and duration of hospital stay is unclear. Observational studies were therefore conducted on patients undergoing MAS. The presence of oedema was related to changes whole-body impedance (Z), obtained at four frequencies (5, 50, 100 and 200 kilohertz (kHz)) using bioelectrical impedance analysis (BIA) and to clinical outcomes. The fluid intake and output and changes in plasma concentration of albumin, total protein, C-reactive protein (CRP) and reduced glutathione in whole blood (GSH) were compared before and after surgery in patients who subsequently developed oedema (OD group) and patients who consistently remained free of oedema (NOD group) Oedema occurred in 40 per cent of the patients observed prospectively and was significantly related to age (odds ratio 1.087 (95 per cent confidence interval (c.i), 1.016 -1.163; P =0.016). The preoperative ratio of Z at 200 kHz to 5 kHz (Z200/Z5) was higher in patients who subsequently developed oedema than those who did not (0.809 v 0.799; P = 0.015), suggesting that it may be possible to identify patients who are prone to abnormal fluid shifts preoperatively. The change in (Z) was greater in the oedematous than non-oedematous groups (at all frequencies (P < 0.001)), and more so at lower frequencies (5kHz) than higher frequencies (100 kHz) (P < 0.001). The impedance quotient (ht2/Z) in the whole group changed in a similar direction at each frequency but to a greater extent in the OD compared to NOD groups. The total volumes of administered fluids in both groups of patients were similar but the average urine output per kg body weight was significantly lower in the OD compared to NOD patients (29.4(2.3) versus 40.5(3.7) mls/kg, P = 0.023). There were no significant differences before and after surgery in the concentrations of albumin, total proteins and GSH in both patient groups. Preoperative CRP concentration in the OD and NOD patients were similar but the mean (s.d) CRP concentration over duration of observation in the OD compared to the NOD patients was significantly greater (148 (54.1) versus 89.6 (43.8) mg/L, P = 0.006). Oedema was associated with a significant delay in the recovery of gut function (median (range) 6(3-17) versus 5(1-13) days, P = 0.020) and prolonged hospital stay (17(8-59) versus 9(4-27) days, P = 0.001) and increased incidence of postoperative complications (65 versus 22%, P = 0.011). This study shows that the incidence of early postoperative oedema is high and preoperative identification and monitoring of surgical patients vulnerable to abnormal fluid shifts may be possible with non-invasive techniques. Age, impaired ability to excrete administered fluid load and an exaggerated inflammatory response to surgical trauma rather than hypoalbuminaemia and hypoproteinaemia were significant factors for oedema formation. Postoperative oedema was associated with a significant increase in postoperative morbidity.

Serum Amyloid P Component (SAP), a putative molecular chaperone, is a homopentameric plasma protein of 25kDa subunits. It binds to the amyloid fibrils of misfolded proteins, which cause amyloidosis in humans. SAP not only stabilizes amyloid fibrils but also protects them from proteolytic and cell mediated degradation. SAP has been co-crystallized with three different aminoalkyl phosphonates that bind at the amyloid recognition site of SAP, and the X-ray crystal structures were determined at atomic resolution. A secondary aim of this work was to understand the enhanced amyloidogenic potential of L55P and V30M transthyretin (TTR) protein. TTR misfolding has been implicated in number of human diseases such as senile systemic amyloidosis, familial amyloid polyneuropathy and familial amyloid cardiopathy. TTR protein is a thyroxine binding protein (14kDa) existing as a tetramer in vivo. L55P and V30M mutant TTR are the most aggressive and most common mutants, respectively, in causing FAP. L55P and V30M mutant TTR protein were expressed in E.coli and purified using anion-exchange chromatography and gel filtration. L55P and V30M TTR were co-crystallised with MDS84, a compound that has been demonstrated to stabilize the tetramer in vitro. The X-ray structures of L55P and V30M, TTR mutant proteins have been determined at 1.5Å and 2.1Å resolution. In addition, research was carried out on the bacterial protein Burkholderia invasion protein D (BipD). BipD (33kDa) belongs to type III secretion system of Burkholderia pseudomallei. It creates a pore in the host cell membrane to help the B. pseudomallei invasion. This bacterial infection causes melioidosis disease in humans. To determine the ligand recognition site of BipD, its high-resolution crystal structure has been determined at 1.5Å. This high resolution BipD structure is more complete than its previously solved structures and is in the new space group C2. The BipD structure presented in this research may help to design potential chemical inhibitors of BipD to prevent bacterial invasion into human body. The molecular chaperones play an important role in the protein refolding and assembly. ATJ11 is of 14kDa protein belongs to DnaJ co-chaperone family. The protein has been expressed in E.coli and purified by affinity and cation exchange chromatography. The biophysical study of ATJ11 (CD spectrum) has been conducted showing its predominant α-helical structure and attempts were made to crystallise it but no protein crystals have been obtained.

The bacterial pathogen Chlamydia trachomatis causes Trachoma, the worlds leading cause of preventable blindness and is also responsible for the most common curable sexually transmitted disease in the UK and United States. C. trachomatis is an obligate intracellular organism characterised by a unique and complex growth cycle. Its study presents many challenges since it has historically been recalcitrant to genetic manipulation and growth in the absence of a host cell. Nevertheless, the sequencing of the C. trachomatis genome and its relatively small size by comparison to genomes from other bacterial pathogens, has paved the way for studies at the proteomic level. This thesis describes the development and application of proteomic approaches to study C. trachomatis L2. To survey the expressed chlamydial proteome, a combination of the qualitative approaches, 2-DGE, MudPIT and GeLC-MS/MS; and the quantitative approaches AQUA, iTRAQ and LC-MSE were used. Collectively, the approaches efficiently identified 648 expressed proteins, representing ~72% of the predicted proteome of C. trachomatis L2, from both the infectious (elementary body, EB) and replicating (reticulate body, RB) form of the pathogen. In the infectious EB, the entire set of predicted glycolytic enzymes were detected, indicating that metabolite flux rather than de novo synthesis of this pathway is triggered upon infection of host cells. Further, proteomic analysis of the RB form also uncovered biosynthetic enzymes for chlamydial cell wall synthesis, indicating that peptidoglycan is produced in some form during growth in host cells. Comparison of the quantitative approaches iTRAQ and LC-MSE demonstrated that LC-MSE quantitative data was significantly more robust and extensive relative to iTRAQ data. In addition to information on relative amounts of these proteins between the two forms, LC-MSE data also yielded the cellular concentration (molecules per cell) for 489 proteins. This extensive set of absolute quantitation data permits estimates of the energy invested in the synthesis of various classes of proteins. The results indicate that C. trachomatis devotes most of its energy into maintenance of the translational machinery. However, it also expends significant amounts of energy into making cell envelope components and a set of hitherto hypothetical proteins. These proteins, which account for the bulk of the energy invested by the intracellular RB form of the pathogen as it converts to the extracellular EB form, highlight the importance of absolute quantitation data for understanding the biological processing status of the cell. The datasets also revealed a large number of proteins that were differentially expressed between replicating RBs and infectious EBs, ranging from 8.4-fold down-regulation to 3.5-fold up-regulation. Consistent with transcriptomic studies (Belland et al., 2003), proteins involved in protein synthesis, ATP generation, central metabolism, secretion and nutrient uptake were predominant in the metabolically active RB at 15 h PI. Although many of the proteins in these functional categories were down-regulated in EBs, proteins required for glycolysis, central metabolism, protein synthesis, and type III secretion were present in significant amounts in EBs suggesting that the infectious EB is primed ‘ready-to-go’ upon contact with the host cell.