The healthy intestinal mucosa is home to the largest population of macrophages in body. Like all tissue macrophages, intestinal macrophages play vital roles in maintaining tissue homeostasis by removing apoptotic cells and any other cellular debris. In addition they maintain the integrity of the epithelial barrier and support the differentiation and maintenance of regulatory T cells in the mucosa. By virtue of their high phagocytic and bactericidal activity, these macrophages are also vital members of the innate immune system and are strategically positioned adjacent to the epithelium so that they can capture and eliminate any invading organism(s). However unlike other tissue macrophages, those found in the normal gut have several functional adaptations, such as hyporesponsiveness to toll-like receptor (TLR) ligands, which allow them to function without provoking overt inflammation. Macrophages are also abundant during intestinal inflammation, where they show increased TLR responsiveness, pro- inflammatory cytokine and chemokine production and enhanced phagocytic ability. Under these conditions, macrophages perpetuate inflammation. It remains unclear whether these distinct roles in healthy and inflamed intestine roles are carried out by discrete populations of macrophages, or if the resident macrophages alter their behaviour and become pro-inflammatory. One of the main obstacles to gaining a better understanding of the immunobiology of intestinal macrophages during steady state and inflammatory conditions is discriminating them from other mononuclear phagocytes (MP) in the mucosa, such as dendritic cells (DC). At the time of starting my project, it was becoming clear that markers such as F4/80 and CD11c were insufficient for distinguishing between macrophages and DC when used in isolation. Therefore, the aims of this thesis were to first establish reliable multi-parameter flow cytometry staining protocols to allow precise phenotypic and functional characterisation of macrophages in the healthy and inflamed mouse colon, and secondly, to explore the origins of these macrophage populations to assess whether they were derived from distinct precursors, or whether a relationship existed between them. Lastly, I examined the potential mechanisms underlying the characteristic TLR hyporesponsiveness that intestinal macrophages exhibit, focusing on the role of the inhibitory CD200R1-CD200 axis. In Chapter 3, I first set out to characterise phenotypically the macrophage populations present in the steady state mouse colon using multi-parameter flow cytometry. These studies revealed that expression of the chemokine receptor CX3CR1 could be used to identify two main populations of myeloid cells, the bigger of which was a homogeneous population of CX3CR1highCD11b+ macrophages that dominated the resting mucosa. A smaller population of CD11b+ cells expressing intermediate levels of CX3CR1 (CX3CR1int) was also present in the steady state mucosa, but this was remarkably heterogeneous, with at least 4 subsets distinguishable on the basis of Ly6C, class II MHC, F4/80 and CD11c expression. These included F4/80+Ly6ChighMHCIIneg CD11cneg cells that were phenotypically indistinguishable from blood monocytes, F4/80+Ly6C+MHCII+CD11c+/neg cells and F4/80+Ly6CnegMHCII+CD11c+/int cells that were phenotypically and morphologically similar to CX3CR1high macrophages except for their lower level of CX3CR1. Finally there was a minor subset of F4/80negLy6Cneg MHCII+CD11chigh cells that expanded markedly in response to in vivo flt3L treatment and appeared to be genuine DC. CX3CR1neg cells were also found within the CD11b+ population in the healthy mucosa, most of which were Siglec F+ eosinophils, together with a few neutrophils. In the second half of Chapter 3, I examined how these populations changed during acute colitis induced by feeding dextran sodium sulphate (DSS). These experiments demonstrated that the CX3CR1int compartment expanded dramatically during acute inflammation, with preferential accumulation of the Ly6Chigh subsets and relative loss of the CX3CR1high population as colitis progressed. Together these studies suggested that CX3CR1high and CX3CR1int cells represent resident and pro-inflammatory macrophages respectively. I next set out to explore the in vivo origin of the CX3CR1int and CX3CR1high populations, to address whether they were derived from independent precursors as would be predicted by current theories of monocyte heterogeneity, or if a relationship existed between them. By using adoptive transfer of purified BM monocytes, the studies described in Chapter 4 show that 'inflammatory' Ly6Chigh, but not Ly6Clow 'resident' monocytes replenished the CX3CR1high resident macrophage population in the steady state mucosa. This appeared to involve local differentiation of Ly6Chigh monocytes through CX3CR1int intermediary stages, which was accompanied by the acquisition of class II MHC, loss of Ly6C and upregulation of F4/80 and CX3CR1. In vivo BrdU incorporation studies supported the idea that the majority of CX3CR1int cells in the resting intestine represented short-lived intermediaries on their way to becoming CX3CR1high macrophages. Together these studies suggested that rather than representing independent macrophage subsets, the CX3CR1int and CX3CR1high cells in the resting colonic mucosa comprise a differentiation continuum from Ly6Chigh monocytes to mature CX3CR1high macrophages. Analysis of BM chimeric mice confirmed that BM-derived monocytes were the source of the vast majority of colonic LP macrophages. These findings were supported by the fact that CCR2 KO mice, in whom Ly6Chigh monocyte egress from the BM is blocked, lack Ly6Chigh colonic monocytes and have markedly reduced numbers of mature colonic macrophages. In Chapter 4, I also explored whether factors present in the normal mucosa, such as colony stimulating factor (CSF)-1, TGFbeta and the chemokine CX3CL1, could direct monocytes to acquire the phenotype of mucosal macrophages. Although initial in vitro studies suggest that none of these were effective on their own, in vivo administration of recombinant CSF-1 appeared to promote in situ monocyte differentiation in the gut. Taken together, the results in this chapter highlight that the CX3CR1high macrophage population is maintained by Ly6Chigh blood monocytes and that their differentiation is controlled by local factors in the mucosa. In Chapter 5, I went on to investigate whether the phenotypically identifiable differentiation of mucosal macrophages was accompanied by alterations in their functional capacity. Intracellular cytokine staining, qRT-PCR and reporter gene expression revealed that as Ly6Chigh monocytes differentiate locally through the CX3CR1int stages into CX3CR1high macrophages, they progressively acquired the ability to produce IL10 and have reduced production of pro-inflammatory mediators. In addition, the maturation of monocytes was accompanied by an increased ability to phagocytose and kill bacteria. Their response to exogenous stimulation by TLR ligands also altered as differentiation proceeded, with the Ly6Chigh monocytes responding robustly to TLR2 and TLR4 ligation in a TNF-alpha dominated manner, whereas the CX3CR1high macrophages responded less vigorously and their TNF-alpha production was balanced by IL10. This pattern was retained during experimental colitis, where the CX3CR1int cells showed enhanced spontaneous TNF-alpha production, whereas IL10 remained the dominant product of CX3CR1high macrophages. Adoptive transfer experiments in Chapter 6 then showed that donor Ly6Chigh monocytes were recruited to the mucosa of colitic mice, but unlike in resting mice, they failed to acquire the CX3CR1high phenotype.

The kynurenine pathway is thought to be involved in neurological disorders but its precise role and the mechanisms involved have yet to be established. Tryptophan can be metabolised via this pathway to produce the neurotoxic N-methyl-D-aspartate (NMDA) receptor agonist, quinolinic acid (QUIN), and the direct generators of reactive oxygen species, 3-hydroxykynurenine (3HKYN) and 3-hydroxyanthranilic acid (3HANA), as well as the neuroprotective NMDA receptor antagonist, kynurenic acid (KYNA). High performance liquid chromatography (HPLC) methods were successfully developed and validated for measuring tryptophan, kynurenine, KYNA, 3HANA and anthranilic acid (ANA) in blood samples, using absorbance and fluorescence detection. The method for determining 3HKYN using electrochemical detection was more problematic and was only used for tryptophan loaded samples and their respective baseline samples. Using HPLC, the concentrations of tryptophan, kynurenine, KYNA, 3HKYN and 3HANA were measured in the blood of Huntington's disease (HD) patients and patients with chronic brain injury, where the injury had occurred at least one year previously. QUIN was also determined for these patients using gas chromatography-mass spectrometry (GC-MS). In addition, the dynamics of the kynurenine pathway were investigated following oral tryptophan depletion and loading. In contrast to these chronic conditions, patients with acute stroke were also studied. The concentrations of tryptophan, kynurenine, KYNA, ANA and 3HANA were determined in the blood of the stroke patients, examining any changes in these concentrations during the two weeks after the stroke. The extent of inflammation and oxidative stress were also assessed for all patients, by measuring the levels of neopterin and the lipid peroxidation products, malondialdehyde and 4-hydroxynonenal. Patients with late stage HD showed abnormal tryptophan metabolism via the kynurenine pathway, together with increased inflammation and oxidative stress. Increased levels of kynurenine together with increased kynurenine: tryptophan (K:T) ratios, indicating greater indoleamine 2,3-dioxygenase (IDO) activity, were observed in blood samples from HD patients in comparison with healthy control subjects. In conjunction with this increased IDO activity, there was a decrease in the ratios of KYNA: kynurenine, suggesting decreased kynurenine aminotransferase (KAT) activity. Inflammation, which may be stimulating IDO activity, could also be decreasing KAT activity, suggested by negativecorrelations between the KYNA: kynurenine ratios and the inflammatory marker, neopterin. The inactivity of KAT suggests a small deficiency in KYNA over a long period of time which could cause a reduction in NMDA receptor antagonism, resulting in slow progressive excitotoxicity contributing to the neurodegeneration in HD. Low KYNA: kynurenine ratios were observed in baseline and tryptophan depleted samples, but after tryptophan loading, HD patients showed similar ratios compared with control subjects. This suggests that loading may be beneficial for HD patients, as more of the neuroprotectant, KYNA can potentially be produced. However, the results suggest that concentrations of the neurotoxin, QUIN, may also be increasing after tryptophan loading. Low concentrations of 3HKYN and 3HANA, with no change in QUIN levels, were also observed in the blood of HD patients. 3HANA levels continued to be decreased for the HD patients after loading. This may suggest degradation of 3HKYN and 3HANA by autoxidation producing reactive oxygen species which could contribute to the high levels of oxidative stress found in these patients. Tryptophan loading in healthy control subjects showed significant increases in the inflammatory marker, neopterin, and in the lipid peroxidation products. These results should be considered when tryptophan loading is used in psychiatric practice and in diets high in tryptophan, such as the Atkins diet. Patients with severe chronic brain injury showed similar alterations in kynurenine pathway metabolism as HD patients, at baseline and throughout the loading and depletion protocols. Although the brain injury had occurred at least one year previously, these patients showed persistent inflammation and oxidative stress, demonstrated by their increased levels of neopterin and lipid peroxidation products compared with healthy controls. In baseline blood samples, there were increased K:T ratios indicating greater IDO activity in the patients. Patients with chronic brain injury showed decreased concentrations of the neuroprotectant, KYNA, as well as low KAT activity, indicated by the decreased KYNA: kynurenine ratios. After tryptophan loading, K:T ratios decreased and the KYNA: kynurenine ratios increased in patients in comparison with controls, suggesting a reversal in the activities of the enzymes IDO and KAT. Similar levels of the inflammatory marker, neopterin, were observed in patients and controls after tryptophan loading. This suggests that these changes in IDO and KAT activities may be related to inflammation. As for the HD patients, patients with chronic brain injury showed lower levels of 3HKYN and 3HANA in their blood, with no change in QUIN levels. These metabolites may be undergoing autoxidation, producing reactive oxygen species which contribute to the ongoing oxidative stress in these patients.The kynurenine pathway was activated following an acute stroke, as indicated by the increased K:T ratios, suggesting greater IDO activity. Stroke patients also had raised levels of neopterin and lipid peroxidation products, indicating inflammation and oxidative stress. There were no changes in the blood concentrations of kynurenines, neopterin or lipid peroxidation products during the fourteen days after a stroke. Stroke patients had reduced levels of 3HANA in their blood, as observed for the HD and chronic brain injury patients. There were negative correlations between the concentration of 3HANA and the volume of the brain lesion, measured by computed tomography (CT) scan, demonstrating the importance of the decreased concentrations of 3HANA. In addition, there were increased levels of ANA in the blood of the stroke patients and the ratios of 3HANA: ANA also correlated with brain lesion volume. Another measurement which correlated with lesion volume was lipid peroxidation, suggesting that oxidative stress contributes to the extent of the brain damage after a stroke. This may suggest that the role of 3HANA in stroke is related to its autoxidation and the generation of reactive oxygen species. Increased concentrations of KYNA were observed in patients who died within three weeks of having a stroke. These high levels of KYNA may have been produced following excitotoxicity and the generation of free radicals, and may cause excessive NMDA receptor blockade or reduced mitochondrial adenosine triphosphate (ATP) synthesis, thus contributing to cell death. The kynurenine pathway was activated and showed abnormal metabolism in all the patient groups, suggesting a potential role for these metabolites in neuronal dysfunction in HD, chronic brain injury and acute stroke. Further work is required to elucidate the role of tryptophan metabolites and whether they may have a direct contribution to neuronal damage in neurological disorders.

Leishmania species are protozoan parasites which have a complex life cycle, which is coordinated with its cell cycle. There are 11 cyclin dependent kinases (CDKs) and 11 cyclins present in the Leishmania genome reflecting the complexity of cell cycle control in this parasite, perhaps due to the requirement for synchronisation with the life cycle. Leishmania mexicana CRK3, a cdc2-related serine/threonine protein kinase of the CDK family, is essential for transition through the G2-M-phase checkpoint of the Leishmania cell cycle. The Trypanosoma brucei homologue of CRK3, with 78% identity to L. mexicana CRK3, has been shown to form an active kinase complex with the CYC6 cyclin. Using this knowledge a putative mitotic cyclin, CYC6, from Leishmania major was identified. Monomeric CRK3 does not have protein kinase activity, but was activated in vitro with CYC6 to produce a protein kinase complex with histone H1 kinase activity. CRK3his and CYC6his were co-expressed and co-purified from Escherichia coli via metal affinity and gel filtration chromatography to obtain a 1:1 ratio of CRK3:CYC6 proteins, which formed a stable protein kinase complex. Using histone H1 as a substrate, active CRK3:CYC6 was used to develop a radiometric assay suitable for low to medium throughput compound screening and then an assay suitable for high throughput screening (HTS) using IMAPTM fluorescence polarization technology. This HTS assay was used to screen a 25,000 compound chemical library to identify hits which significantly reduced CRK3:CYC6 protein kinase activity. Two main pharmacophores with the highest potency towards CRK3:CYC6 protein kinase activity were identified from the high throughput screen. Structure Activity Relationship (SAR) analysis of the hits identified the chemical groups attached to the scaffold structures which are essential for the inhibition of CRK3:CYC6 protein kinase activity. The CRK3:CYC6 hits were subsequently counter-screened against a panel of 11 mammalian kinases including human CDK1:CYCB (the functional orthologue of CRK3:CYC6), human CDK2:CYCA and human CDK4:CYCD1 to determine their selectivity. Compound hits that were selective towards CRK3:CYC6, were tested against Leishmania in vitro. Progress towards synthesising potent and selective derivatives of the HTS hits will be discussed, with the view to evaluating their potential for the development of novel therapeutics against leishmaniasis.

Abstract Human cytomegalovirus (HCMV) is a highly host-specific, ubiquitous herpesvirus that results in asymptomatic infection for the majority of those infected. However, it produces serious clinical disease in neonates and immunocompromised individuals such as transplant recipients and AIDS patients. The majority of the 236 kbp genome is highly conserved, but there are a number of highly variable regions, coding and non-coding, scattered throughout the genome. Numerous studies have been published investigating the genotypes of hypervariable genes, most focussed on potential associations between genotype and clinical disease or tropism. In general, no convincing connections between genotype and disease have been found. The present study investigated two hypervariable HCMV genes, UL146 and UL139, in a large number of clinical samples (179) from a number of locations worldwide in Europe, Africa, Asia and Australia. A total of 14 UL146 genotypes (G1-G14) were detected, which agrees with previous findings based on many fewer samples. For UL139, eight genotypes were detected, three of them (G5, G7 and G8) novel. The genotypes of both genes appear to have evolved under constraint rather than positive selection. Possible bias in the geographical distribution of the UL146 and UL139 genotypes was investigated. In general, all genotypes were found in all areas and any variation from the expected distribution was probably a result of small sample numbers from certain regions, specifically Asia and Australia. This general finding is in agreement with that of a previously published study on gene UL73. No evidence for linkage disequilibrium between UL146 and UL139 genotypes was found. This is in accordance with a previously published study of linkage disequilibrium among six other genes (UL55, UL74, UL75, UL115, US9 and US28), and is consistent with the theory that recombination has played a role in HCMV evolution. The absence of linkage between highly variable genes complicates attempts to examine associations between genotype and disease, as many combinations of genotypes are possible. Investigation of transcriptional expression of UL146 and UL139 from HCMV strain Merlin in fibroblast cell culture revealed that UL146 is expressed with late kinetics and UL139 with early-late kinetics. Northern blot and RACE data suggested that UL146 is 3’-coterminally expressed with UL147, UL147A, UL148 and UL132, and that UL139 is 3’-coterminally expressed with UL140 and UL141. To determine whether the high degree of sequence divergence corresponds to structural divergence, the UL146 genotypes were homology modelled on the related human chemokines IL-8, gro-a and IF9S. All 14 genotypes were predicted to be structurally very similar, which suggests they may also be functionally similar. However, small differences between the structures of human chemokines are known to result in slightly differing binding affinities for cellular receptors, and therefore even small differences between UL146 genotypes could conceivably confer functional differences. UL139 has been predicted to encode a type 1 membrane glycoprotein. No information has been published regarding UL139 function, although a short region of similarity with the cellular signal transducer CD24 has been noted previously, tentatively suggesting an immunomodulatory role. Preliminary experiments to characterise UL139 were performed utilising recombinant adenovirus vectors expressing tagged UL139 variants from three genotypes (G1, G5 and G7). The tagged UL139 variants expressed proteins that were considerably larger in mass than predicted from amino acid sequences. This extra mass may be attributable to glycosylation as well as other forms of post translational modification. Mixed infections of HCMV strains in immunocompromised individuals, such as transplant recipients, have been associated with enhanced pathogenesis and increased risk of transplant rejection. The presence of mixed infections also further complicates attempts to establish connections between genotype and disease outcome. In the analysis of UL146 and UL139 genotypes, multiple genotypes were detected in 14% of samples and in 29% when repeated experimental results were included, and even these values may be underestimations. The utility of a QPCR-based assay using genotype-specific primers was assessed as a means of more accurately determining the occurrence of mixed infections, and showed promise. Passage of HCMV strains in cell culture has been shown to result in various mutations. AD169, a commonly used laboratory strain, lacks 15 kbp sequence that includes UL146 and UL139. An alternative stock of AD169 (AD169varUC) was obtained that was thought to contain most or all of the deleted region and, indeed, both UL146 and UL139 were detected. Further sequencing confirmed that this stock is derived from AD169 and revealed that it contains all but 3.2 kbp of the 15 kbp absent from commonly used AD169 stocks. The 3.2 kbp deletion affects UL144, UL142, UL141 and UL140. This propensity of HCMV to undergo mutation during cell culture highlights the importance of studying characterised strains that are as close to wild type virus as possible.

Mouse mammary gland morphogenesis at puberty is a complex developmental process, regulated by systemic hormones, local growth factors and dependent on the epithelial/epithelial and epithelial/stromal interactions. TEBs which invade the fat pad are important in laying down the epithelial framework of the gland at this time point. The objective of this thesis was to use a combination of ‘pathway-‘ and ‘candidate gene analysis’ of the transcriptome of isolated TEBs and ducts and associated stroma, combined with detailed analyses of selected proteins, to further define the key proteins and processes involved at puberty. Using GeneChip® Mouse Exon 1.0 ST Arrays we identified the epithelial-, epithelial-stromal- and stromal transcriptomes of TEBs and ducts and defined the major functional pathways/biological processes in each compartment. By ranking the transcripts according to their expression levels and known functions in other systems, we identified genes of potential importance for pubertal mammary morphogenesis. We focused our study on Upk3a and Fbln2 and their protein products. Upk3a could only be detected at mRNA level and thus further analysis was based on Fbln2. We demonstrated that Fbln2 V1 and Fbln2 protein are predominantly expressed in the epithelium and stroma of TEBs. Using hormone primed mice we demonstrated that Fbln2 expression and localisation in the mouse mammary gland is positively regulated by E2 and P. Furthermore, by a combination of further in silico analysis, in vitro functional assays, IHC and IF we identified Vcan, Lama1, Fbn1, ColVIαIII, ColIVαI, ColXVIIIαI, Eln, Per, Acan, Nid, Itgb3 and Itga5 as potential binding partners of Fbln2 in mammary gland. Finally, we reported lack of an obvious mammary phenotype in Fbln2 KO-/- mice at puberty but demonstrated that this may be attributed to the over-compensation by Fbln1. This thesis demonstrates the benefit of DNA microarray analysis in studying pubertal development of mouse mammary gland. It identifies Fbln2 as a potential pubertal mammary regulator which by interacting with various ECM proteins at different sites of mammary milieu may contribute to an array of structural and migratory functions during mammary morphogenesis. These data substantially add to the understanding of the development of mammary gland at puberty and reveal many potential avenues for further investigations.

Gangliosides are a family of sialic acid-containing glycosphingolipids that are enriched in the nervous system. They are located in the outer leaflet of the plasma membrane within lipid rafts and are thought to be involved in numerous cellular events, including proliferation, differentiation, migration and neurite outgrowth. Gangliosides have also been shown to have neuroprotective actions and have been considered as candidates for the treatment of several neurodegenerative disorders. In this thesis, the role of gangliosides in glial proliferation, migration and differentiation as well as the regeneration of the olfactory system and myelination were studied using mice lacking enzymes involved in ganglioside biosynthesis. Regeneration of the olfactory system in ganglioside knockout mice was similar to that of wild-type mice. However, proliferation of olfactory ensheathing cells grown on collagen and the migration of Schwann cells grown on laminin or collagen was increased in Sia T -/- mice, which lack b-series gangliosides but have increased levels of a-series gangliosides. These findings suggest that complex gangliosides modulate glial cell function to some extent. However, since the effects observed were subtle, it is possible that simpler gangliosides are able to compensate for the lack of complex gangliosides. Axonal density and myelination were unaffected in ganglioside knockout mice. However, the localisation of sodium channels at the node of Ranvier and potassium channels at juxtaparanode was retarded in GalNAc T -/- mice lacking all complex gangliosides, suggesting that complex gangliosides modulate the formation of nodes of Ranvier. In addition, GalNAc T -/- mice had significantly lower numbers of NG2 positive early oligodendrocyte progenitors compared to wild-type and Sia T -/- mice, suggesting that complex gangliosides may affect early progenitor differentiation, proliferation or survival.

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states.

coli at different temperatures, it was observed that both types of presequence as well as the nature of mature protein affect the level of protein expression. Therefore, it was noticed that both types of presequence had no significant effect on the level of protein expression when they were linked to the mature E3 whereas the negative effect of these presequences was apparent when they were linked to mature E2 or E3BP. Secondly, comparing the solubility of precursors with their mature forms, both extended and standard presequences markedly reduced the solubility of precursor constructs by inducing the production of inclusion bodies although the effect appeared to be more marked with the former. Thirdly, on decreasing the rate of the protein synthesis by growing E. coli cultures at lower temperatures, it was possible to minimise the formation of insoluble protein aggregates and achieve partial or indeed complete solubility of precursor forms in some cases. Fourthly, these precursors appeared to retain the ability to fold correctly or at least to initiate the correct folding pathway. Thus both soluble and insoluble fractions of E2 and E3BP precursors contained lipoylated domains as judged by their ability to cross-react with PD2, an indication that these N-terminally located domains had adopted their native conformations. These observations were consistent with the view that N-terminal mitochondrial targeting sequences markedly reduced the rate of protein folding rather than suppressing the folding process completely. In this scenario, precursors would exist as nascent folding intermediates for longer periods compared to their mature equivalents and so would be more prone to aggregation and degradation as observed in this study. Further experiments are planned to test this hypothesis.

High concentrations of large very low density lipoproteins (VLDL1) give rise to atherogenic dyslipidaemia, which is usually associated with insulin resistant conditions (e.g. obesity) and increases the risk for cardiovascular disease (CVD). Isotopic tracer methods for determining VLDL1 kinetics are costly, time-consuming, labour intensive and need experience and skill to calculate the kinetic parameters. The aim of this thesis was to develop a simpler and cost-effective method of obtaining triglyceride-rich lipoproteins (TRL) kinetic data, based on the fact that chylomicrons (CM) or CM-like particles (e.g. Intralipid) compete with large VLDL1 for the same lipoprotein lipase (LPL)-mediated catalytic pathway. From this method, it was possible to determine VLDL1-triglyceride (TG) and -apolipoprotein (apo) B production rates and the Intralipid-TG clearance rate (as a surrogate measure of CM clearance). Kinetic data obtained from this method agreed with values and relationships obtained from stable isotope methods. The protocol is relatively quick, inexpensive, and transferable to non-specialist laboratories. As a first application, the ‘Intralipid method’ was used to investigate the effects of hyperinsulinaemia and hyperglycaemia due to glucose ingestion on VLDL1-TG and -apoB production rates and Intralipid-TG clearance rate. This showed that hepatic VLDL1 production is suppressed in response to hyperinsulinaemia and that the change in Intralipid-TG clearance rate with hyperinsulinaemia correlated significantly with HOMA-estimated insulin resistance (HOMA-IR). In addition, alanine aminotranferase (ALT) concentrations (a marker for liver fat), within normal range, predicted the extent of hepatic VLDL1 suppression. Secondly, the Intralipid method was used to investigate the mechanisms responsible for the hypotriglyceridaemic effect of a moderate exercise session (120 min walking at 50% VO2max) in overweight/obese middle-aged men; the section of the population at high risk of CVD in whom exercise-for-health guidelines are targeted. This showed that the exercise-induced reduction in plasma TG was due to increased VLDL1-TG and -apoB clearance, rather than decreased hepatic production. Exercise also increased Intralipid-TG clearance rate, but to a lesser extent than VLDL1, suggesting an increased affinity of VLDL1 for LPL-mediated lipolysis post-exercise. Taken together, the Intralipid method is a relatively simple, safe and cost-effective method to determine in VLDL1-TG and -apoB production rates and Intralipid-TG clearance rates. It is also sensitive enough to detect physiological changes in TRL kinetics.

Small celestial bodies such as Near Earth Objects (NEOs) have become a common subject of study because of their importance in uncovering the mysteries of the composition, formation and evolution of the solar system. Among all asteroids, NEOs have stepped into prominence because of two important aspects: they are among the easiest celestial bodies to reach from Earth, in some cases with less demanding trajectories than a simple Earth-Moon trajectory and, even more meaningful, they may pose a threat to our planet. The purpose of this thesis is to provide a comprehensive insight into the asteroid hazard problem and particularly to its mitigation. Six different concepts are fully described; specifically models for nuclear interceptor, kinetic impactor, low-thrust propulsion, mass driver, solar collector and gravity tug are developed and their efficiency is assessed for a complete set of different types of hazardous celestial objects. A multi-criteria optimization is then used to construct a set of Pareto-optimal asteroid deflection missions. The Pareto-optimality is here achieved not only by maximizing the deflection of the threatening object, but also by minimizing the total mass of the deflection mission at launch and the warning time required to deflect the asteroid. A dominance criterion is also defined and used to compare all the Pareto sets for all the various mitigation strategies. The Technology Readiness Level for each strategy is also accounted for in the comparison. Finally, this thesis will also show that impulsive deflection methods may easily catastrophically disrupt an asteroid if the required energy for a deflection reaches a certain limit threshold. A statistical model is presented to approximate both the number and size of the fragments and their initial dispersion of velocity and then used to assess the potential risk to Earth posed by the fragmentation of an asteroid as a possible outcome of a hazard mitigation mission.