NF-κB is a transcription factor which is central to the regulation of genes involved in inflammatory and immune responses. Understanding the operation of NF-κB and its associated intracellular signalling pathway is essential in order to control a wide range of chronic inflammatory diseases, including asthma and rheumatoid arthritis. Abnormalities in the pathway are present in a variety of human cancers and may also affect the pathogenesis of Alzheimer's disease. Computational modelling of the signalling pathway is necessary to overcome the practical limitations of biological experiments and to facilitate a more comprehensive understanding of the system. The thesis begins by outlining existing understanding of the NF-κB signalling pathway, which in conjunction with a review of modelling methods is used to inform a different approach to model the pathway, using computational agents to represent individual molecules and receptors. The agent-based model is tested with well-understood chemical reactions before being used to describe the pathway. This provides a good appreciation of the system as a whole, offering a detailed description of events at every step in the pathway and allowing investigation of stochastic, spatial and structural parameters. The modelling process and simulation help to provide a prediction about the role of actin filaments of the cytoskeleton in regulating the unstimulated pathway; this is quantitatively validated by biological experiment. The effect of cell shape on the pathway is also investigated.

In recent years there has been a dramatic increase in the number of nanomaterials being developed, thus increasing the need for hazard assessment methods beyond the capacity of toxicological screening methods using animals. Current in vitro assays have a number of shortcomings, which were addressed in this thesis. These include: (i) high dependence on immortalized cell lines, (ii) inaccurate dosimetry descriptors, (iii) poor robustness of assay systems, and (iv) hyperoxic culture conditions. A method for harvesting viable human nasal epithelial cells using a washout technique was developed to provide squamous epithelial cells for use in culture assays. However, poor proliferation of these cells in vitro limited their use in toxicological assays. A particokinetic model was developed to relate the ’delivered dose’, i.e. the number of nanoparticles reaching immortalized airway cell layers by gravitational force and diffusional mechanisms, to toxicological endpoints measured in vitro. This model was applied to a panel of rigorously characterized nanoparticles (CuO, TiC>2, polystyrene and in-house manufactured lipid nanocapsules) and the results provided compelling evidence that the delivered dose is a more appropriate dose descriptor for cell-based toxicity assays than the widely used nominal dose, as it reflects the number of particles (or their equivalent surface area) available to interact with the cell layer over a given exposure time. The results were confirmed in two airway epithelial cell lines RPMI 2650 and A549 after 6 and 24 h using two standard toxicological end-points. However, when cells were cultured in normoxic (for the respiratory tract) oxygen concentration, 13%, as opposed to the standard culture conditions of 21% they were found to be more responsive to nanoparticle exposure in terms of both production of reactive oxygen species and reduced cell viability. This suggests that standard incubation conditions of 21% oxygen provide a baseline of oxidative stress within a cell culture system that induces adaptive mechanisms and reduces their sensitivity to materials that exert adverse effects through oxidative stress.

The role of adiponectin, a cytokine produced in adipose tissue and its relationship to risk factors for the metabolic syndrome, and dietary influences on its production are reviewed. The thesis examines the hypothesis that the amount and type of fatty acids in the diet affect serum adiponectin concentrations. The effects of ADIPOQ gene locus on serum adiponectin concentrations were examined in participants of the RISCK study at baseline and following dietary intervention. Major effects of age, BMI, gender and ethnicity on serum adiponectin were found. At baseline the ADIPOQ +276 G/T T-allele was associated with higher serum adiponectin. The ADIPOQ -10066 G/A A-allele was associated with lower serum adiponectin. No influences were observed for the other SNPs -7734 C/A or -11391. Participants with the -10066 GG genotype showed a 3.8% increase and -10066 A-allele carriers showed a 2.6% decrease in adiponectin (n=360; P=0.006) after a high mononounsaturated fat diet. In-10066 GG homozygotes, adiponectin increased progressively with age after a high monounsaturated fatty acid diet but decreased after a low fat diet. A randomised single-blind parallel study in healthy males (n=48) was conducted to investigate the effects on serum adiponectin concentration of two formulations of DHA and EPA (3 g/d each) versus placebo (refined olive oil) for 6 weeks. Compared to the placebo, neither EPA nor DHA changed serum adiponectin concentration. It was concluded that a high monounsaturated fatty acid diet has a moderate effect on adiponectin concentration and that dietary supplements of long-chain n-3 polyunsaturated fatty acids in healthy subjects have no effect on adiponectin concentration. Further investigations were conducted to examine the interaction of dietary fatty acid intake with peroxisome proliferator-activated receptor (PPAR) genes. A significant interaction was found between the habitual dietary polyunsaturated/saturated fatty acid ratio and PPARG Pro12Ala genotype on total and LDL cholesterol and plasma triacylglycerol concentrations. After a high monounsaturated fat diet, carriages of both PPARA Val162 and PPARG Ala12 alleles were associated with a greater reduction in plasma LDL-C and its proportion as small dense LDL, than after low fat diet. A significant interactions between n-3 LCP treatment and genotypes of PPARA Leu162Val SNP were found among 310 participants in the MARINA trial. These contributed to a reduction in plasma triacylglycerol concentration with n-3 treatment in subjects homozygous for the more transcriptionally active Leu162 allele. The findings reported in the thesis are discussed in the context of other research in the area.

Connective tissue growth factor (CTGF) has been implicated in the pathogenesis of diabetic nephropathy and has been proposed as a possible specific biomarker for the early detection of fibrosis. CTGF is reported to be a 349-amino acid cysteine-rich, heparin binding monomeric polypeptide that is secreted as a 36-38 kDa protein. To date, the natural form of CTGF has not been successfully purified from clinical samples. Commercial test kits for detecting CTGF by ELISA utilize antibodies raised against recombinant human (rh) CTGF. In the present study, assessment of specific analytical methods for the reliable detection of naturally occurring CTGF has been deployed. In order to achieve this, a peptide sequence to CTGF was synthesized and an antibody raised against this peptide. This antibody was characterized using novel chemistry to improve the specificity of the final assay. The antibody raised against a unique peptide sequence in CTGF, and another to rhCTGF, were used, alongside a commercially available anti-rhCTGF antibody to detect this protein in diabetic urine and peritoneal dialysis fluid. The synthetic peptide antibody was also coupled to Sepharose 4B for the purification of CTGF from clinical samples. ELISA performed on the diabetic urine and peritoneal dialysis fluid showed that the response of the antibody against the synthetic peptide was the highest when compared to the rhCTGF and commercial antibodies. Five peritoneal dialysis fluid and five diabetic urine samples were assayed for CTGF. Diabetic urine was found to be a richer source of CTGF. 2-dimemsional electrophoresis and Western blot analyses on the peritoneal dialysis fluid showed various immunoreactive species to the synthetic peptide antibody at isoelectric point 8.0. It can be concluded that these immunoreactive species may be CTGF bound to other proteins or CTGF complexes that have been formed from the oxidation of the cysteine residues.

No description available.

No description available.

In my thesis I have investigated the role of the two different mechanism of epigenetic silencing: Methyl-CpG binding protein-dependent repression and chromatin remodelling activity in the neural stem cell system. I hypothesised firstly, that MeCPs may have a redundant role in neural stem cell function. Secondly, that the effect of lacking Mdb3 in neural stem cells may be different and more severe that the absence of the methyl-CpG binding proteins. The approach to investigate these hypothesis consisted on a first stage in the study of the neuroectoderm differentiation capacity of ES cells lacking one MeCP, Kaiso, following by study the function of neural stem cell (NS) lines derived from the cortex of mice lacking the MeCPs Kaiso, MeCP2 and Mbd2. I have found no defects in proliferation or self-renewal of triple null NS cells. Additionally, although triple null NS cells present a normal astrocyte differentiation, they present a delay in neuronal differentiation, defect that is only visible at early differentiation stages. Therefore, these proteins are dispensable for viability and differentiation of NS cells <i> ex vivo. </i>In contrast, I have found that Mbd3 is important for differentiation of neuroectoderm in culture, as Mbd3 null ES cells differentiate into neurons at very low frequency but do not differentiate into astrocytes. Moreover, Mdb3 is essential for establishment and/or maintenance of ES cell-derived NS cell lines. Thus while there is no evidence for a role of three MeCPs in NS cell maintenance or differentiation, the activity of the NuRD co-repressor complex is important for both properties.

Exploiting the ability of ES cells to differentiate toward haematopoietic lineages, the project sought to investigate gene expression with the view of identifying novel genes/proteins involved in the early stages of ES cell commitment to haematopoietic lineages. In the culture system used, it has been shown that under standard differentiation conditions 30-50% of embryoid bodies are able to give rise to haematopoietic colonies. This level of haematopoietic commitment was modulated with the addition of chemical inducers of the differentiation environment of embryoid bodies. Using the CFU-A assay to assess haematopoietic commitment and differentiation, we have found that the number of CFU-A colonies can be increased 2-fold after treatment of embryoid bodies with 1% DMSO during the first 48 hours of differentiation, and conversely reduced to below 5% after treatment with 10<SUP>-8</SUP> all <I>trans</I>-retinoic acid (atRA) for the same period. Lineage commitment in untreated, DMSO and atRA embryoid bodies were further investigated using reverse transcription-polymerase chain reaction techniques. The mesoderm marker <I>brachyury</I> was found to be expressed in both DMSO and atRA treated samples to similar extents, suggesting neither compound to be exclusively exerting its effect by enhancing or inhibiting mesoderm commitment. However, the level of transcripts of various haematopoietic markers such as β-globin, PU-1 and CD45 were found to be raised in DMSO treated embryoid bodies compared to either untreated or atRA treated samples. Studies investigating the ability of cells from treated and untreated embryoid bodies, to reconstitute the haematopoietic system of lethally irradiated mice were also carried out. Overall these showed that mice injected with cells from DMSO treated embryoid bodies survived for longer periods of time than those injected with cells from either untreated or atRA treated embryoid bodies. It has been clearly demonstrated that the haematopoietic commitment of ES cells and their differentiated aggregates can be successfully modulated by the addition of chemical inducers. Consequently these effects will prove a useful means of screening for genes and proteins involved in haematopoietic commitment and differentiation.

No description available.

No description available.