The best characterised collagen IV disease is Alport syndrome, caused by α3.α4.α5(IV) mutations. Mutations affecting α1(IV)2α2(IV) can cause diseases, such as haemorrhagic stroke, aneurysm, haematuria, proteinuria, cataracts, vascular tortuosity and iris hypoplasia. Recent data has shown that α and COL4A2 mutations may be more common in the population than previously anticipated. However, the mechanisms of disease of all collagen IV mutations are poorly understood. The aim of this thesis was to investigate the disease mechanism in a selection of COL4A1, COL4A2 and COL4A5 mutations. Collagen IV is folded in the endoplasmic reticulum (ER) then secreted to provide the major structural component of the basement membrane (BM). As such, a combination of ex situ biopsies, in vitro cell cultures and an in vivo mouse model provided an excellent platform for investigating the intracellular and extracellular effects of mutant trimers. Primary dermal fibroblasts containing COL4A2 and COL4A5 mutations, and a Col4a1 mouse model were treated with the FDA approved chemical chaperone phenyl-4-butyric acid (PBA) to determine its effects on intracellular ER retention of trimers (which can activate ER stress), extracellular trimer incorporation into the BM, and disease phenotypes. In vitro and ex situ investigation of a COL4A2 mutation suggested that intracellular α1(IV)2α2(IV) accumulation and ER stress associated with severe BM defects and the disease. Intracellular accumulation and ER stress were reduced by PBA treatment, and treatment of a Col4a1 mouse model displayed elevated α1(IV)2α2(IV) incorporation onto the BM. In vivo PBA treatment did not exacerbate any defects, rescued some disease phenotypes, and had no effect on others. In addition, one of five COL4A5 mutations analysed in vitro displayed intracellular α3.α4.α5(IV) accumulation and ER stress that were ameliorated by PBA. Collectively these data suggest that ER stress and/or reduced α1(IV)2α2(IV) BM incorporation underlie some collagen IV pathologies, and provide novel evidence that they may be amenable to chemical chaperone therapy. More investigation is now needed to further dissect the relative contributions of ER stress and BM defects to collagen IV diseases.

A polymerase chain reaction (PCR) multiplex assay capable of amplifying 13 rapidly mutating Y chromosome short tandem repeats (RM Y-STRs) simultaneously was developed and optimised. This multiplex assay which was termed RM-Yplex is the first to include all 13 RM Y-STRs including DYF387S1, DYF399S1, DYF403S1a/b, DYF404S1, DYS449, DYS518, DYS526a/b DYS547, DYS570, DYS576, DYS612, DYS626 and DYS627. A developmental validation was performed following the Scientific Working Group for DNA Analysis Methods (SWGDAM) revised guidelines. Robustness and limitations of the assay were demonstrated through a range of studies including reproducibility, sensitivity, specificity, stability and mixture studies. Appropriate controls were used during the studies that included a number of male and female commercial controls including, 2800M, 9948 and Taqman male controls and 9947A female control. An allelic ladder was developed for the assignment of the alleles. This was done by choosing samples with different alleles, amplifying them and then adjusting the volumes of amplified products in a mixture. The developed mixtures were used to balance the composite ladder. Multiple alleles of the various loci included in the ladder were sequenced. Reference haplotypes were developed for the 5 male samples included in the Y chromosome Standard Reference Material 2395 (SRM2395) using RM-Yplex. The International Society of Forensic Genetics (ISFG) recommendations were followed for adopting allele nomenclature. As part of developmental validation, the assay was included in an external proficiency trial which was concluded successfully. An internal validation of RM-Yplex was carried out at the Department of Forensic Sciences and Criminology Laboratory, Dubai where apart from other studies; application of the assay was demonstrated using non-probative forensic casework samples. The value of RM-Yplex was demonstrated for differentiating close male relatives in a case where a previously used Y-STR multiplex assay had shown identical haplotypes for those individuals. 1160 male individual samples were analysed in this study including UAE, other Arabian Peninsula populations as well as two South Asian populations residing in United Arab Emirates. RM-Yplex haplotypes have extremely high power of discrimination. The haplotype diversity for RM-Yplex haplotype is much more than the existing commercial Y-STR assays. Population studies have been carried out for the Arab, Indian and Pakistani populations. AMOVA was conducted for determining the apportionment of diversity and pairwise FST’s were estimated between populations. These have shown a marked homogeneity within the UAE Arab sub-populations. MDS plots of pairwise FST’s indicated that populations were not grouped significantly in accordance with the geographical locations. A network analysis showed the extent of distribution of haplotypes of various populations and their relationships. A highly sensitive and reliable RM-Yplex multiplex assay has been thus developed, which is expected to help genetic populations studies and forensic casework.

The one-dimensional pyrolysis computational tool ThermaKin was used to predict the thermal decomposition behaviour of widely used synthetic polymers (polypropylene (PP) and polyethylene (PE)) with and without additives, in order to investigate the suitability of ThermaKin for novel fire retarded samples, under different thermal and fire conditions. The thermal decomposition of materials was investigated using simultaneous thermal analysis technique (STA) coupled with Fourier Transform Infrared Spectrometry (FTIR) at different heating rates and atmospheres. The results show that thermal decomposition of PP follows single mass-loss step, without formation of residue in nitrogen. It was also found that the pyrolysis shifted towards higher temperature with increase of heating rate at different atmospheres. ThermaKin fitted the TGA curves very well. The thermal decomposition behaviour of polypropylene grafted with 5wt% of maleic anhydride (MA), and reinforced with 5wt% of closite 20A as nanoclay (PP-gMA/NC) was also investigated. The main conclusions from this data are that during the thermal decomposition in different atmospheres, TGA curves showed a single step of decomposition process for all samples. The effect of clay is more pronounced during thermal oxidation. In N2 and air, a two-step reaction mechanism was fitted the experimental curves fairly well. The thermal decomposition of PE, pure and reinforced with different types of carbon fillers (single/multi wall carbon nanotubes, carbon fibres, carbon black and single/few layers of graphene nanosheets), at different loadings (0.1, 0.5 and 1 wt%) and atmospheres were investigated, to determine their suitability as potential fire retardant additives. Results showed that thermal decomposition of PE and its composites/nanocomposites followed a single mass-loss step at a range of temperatures, with no residue formation in N2. The DTG curve in air showed two mass loss rate peaks. The experimental results showed that all loadings of these different additives made no improvement to the thermal stability of PE/MA. In air, the compatibilising agent (MA) improved the thermal stability of pure PE, compared to these composites/nanocomposites at the selected loadings. Mechanisms of single or two-step reaction in N2, and three-step reaction in air for the thermal decomposition of PE with and without additives predicted fairly well the experimental curves. Finally, the work was extended to investigate the performance of ThermaKin to establish a model that is able to predict cone calorimetry results. ThermaKin predicted the burning rate of PE/MA, as a good agreement between the experimental and simulated curves was achieved. Sensitivity analysis was performed to investigate the influence of the variation of the material properties on the modelling results. It was found that the heat of decomposition is the most important parameter of those investigated and needs to be determined most accurately. Heat capacity and thermal conductivity are somewhat important. The absorption coefficient and the reflectivity are of lesser importance. In conclusion, this work shows that the combination of pyrolysis modelling, thermal and chemical analysis techniques provides a strong and powerful tool for generating a comprehensive understanding of the thermal decomposition of novel fire retardant materials. However, further work is needed to study the influence of the changes of the material properties in polymeric material while reinforced with different additives and how this will be reflected on the modelling parameters and mechanism.

No description available.

Section 1. A correlation technique is developed which enables the identification of systems belonging to a restricted class of non- linear systems. The method is applicable to a system which may be represented as a single-valued, instantaneous, time-invariant non-linearity followed by linear dynamics. The characteristic of the non-linearity and the impulse response of the linear element are found simultaneously in a single experiment. A class of pseudo-random test signals is studied, and results are derived for some situations in which the results are contaminated by noise. Further work is required to extend the applicability of the ·technique, to compare its performance with other methods of identification, and to investigate alternative test signals. Section 2. A simple eye-position transducer is described, which measures eyeball rotation about two axes.· Depending upon the characteristics of the subject's eye, and the operating conditions, an accuracy of as good as + 5% may be obtained over a range of 12º. The user wears a light-weight infra-red optical-electronic device on a spectacle frame. The transducer exploits the variation in infra-red reflectivity over the surface of the eyeball, and therefore varies in its performance from one subject to another. It may be used by some subjects in all lighting conditions except direct sunlight. Further work is needed to eliminatethe deficiencies of the device, but an investigation into television techniques, which are now feasible, should be made first. Section 3. The experimental determination of the region of asymptotic stability of a second order time invariant system may be considerably simplified by taking advantage of the nature of the trajectories wwhich form the boundary of the region. These trajectories are found easily by reverse time simulation. Further work is possible to investigate the extension of the method to higher order systems, but useful results seem unlikely.

In this work we study the properties of topological dynamical systems under a positive continuous change of velocity. In §l we define a flow obtained from a flow by a positive continuous change of velocity. We then prove that the time change flow is reversible so that we can recover the original flow. In §2 we define, following Kirillov, the first cohomology group of a dynamical system. A time change flow is then seen to be related to this first cohomology group. We now prove that there exists a group homomorphism between the first Čech cohomology group with integer coefficients and the first cohomology group of a compact dynamical system with coefficients in the reals. Winding numbers, due to Sol Schwartzman, are introduced and are shown to have an equivalent interpretation in terms of the first cohomology of a compact dynamical system. In §3 we show there is a natural invariant measure of a time change system in terms of the invariant measure of the original compact dynamical system. We now prove that ergodicity and unique ergodicity are preserved under a positive continuous change of velocity. Finally we relate the winding numbers of a time change system to the winding numbers of the original system, and show that under certain conditions they are invariant. In §4, we show that a compact dynamical system admits a Global Cross-Section if and only there exists an Eigen function, with non-zero eigenvalue, of a time change system. Lastly we show that, under certain conditions, a non-zero winding number is an eigenvalue associated to an eigen function of a time change dynamical system. In §5 we show that it is possible to eliminate eigen functions with non-zero eigenvalue under a positive continuous change of velocity of a compact dynamical system, if there exists at least one orbit homeomorphic to the real numbers : if, in addition, the original dynamical is ergodic we prove that weak-mixing is not invariant under a change velocity.

The perceptron and the hologram are two dissimilar devices which have been advanced as neurological models. It :is shown that there are other and perhaps more plausible models which have properties common to both of these devices. The performance of these intermediate models which are termed Associative Nets is described and analysed statistically. The main similarities and differences between perceptron theory and holography can also be demonstrated; it is possible to demonstrate formal links between the translation invariance in certain types of holography and group invariance in perception. theory. Some single proofs of certain theorems in the latter are also given and some other learning procedures are formulated. It is shown that the important difference between these various models resides in the method used to accomplish a modification. If this modification is an analogue of a neurological change a~ a synaptic level, then it should be possible to qualify the relevance of those models by determining what types of synaptic change can take place in different parts of the nervous system. -Although the evidence is far from complete, it suggests that the neocortex is limited to having one type of synaptic change. Finally, each model is discussed in respect of its neurological plausibility.

Essential hypertension (EH) is considered one of the major contributors to the present pandemic of cardiovascular disease (CVD). EH has a largely obscure aetiology, which lies upon both environmental risk factors and underlying genetic traits. The stroke-prone spontaneously hypertensive rat (SHRSP) is an excellent model of human EH and exhibits salt sensitivity. Two quantitative trait loci (QTL) for blood pressure (BP) regulation have been identified on rat chromosome 2 (chr.2). On this basis, previous work in our laboratory focused on construction of chr.2 congenic strains, on both the SHRSP and Wistar-Kyoto (WKY) genetic backgrounds. In combination with microarray gene expression profiling in kidney from salt-loaded rats, two positional candidate genes for salt-sensitive hypertension were identified. Sphingosine-1-phosphate receptor 1 (S1pr1) and vascular adhesion molecule (Vcam1) lie on the chr.2 congenic interval implicated in salt-sensitivity. Additionally, studies on vascular smooth muscle cells (VSMC) demonstrated enhanced S1PR1-mediated sphingosine signalling in SHRSP compared to WKY. Finally, glutathione S-transferase mu 1 (Gstm1) was identified as another chr.2 candidate gene for BP regulation, lying outside the region implicated in salt-sensitivity. This project attempts to comprehensively investigate the potential role of altered S1PR1 signalling in BP regulation and salt-sensitivity, through comparative proteomic and metabolomic profiling in WKY, SHRSP and chromosome 2 congenic and transgenic stains (WKY.SPGla2a, SP.WKYGla2a, SP.WKYGla2k and Gstm1-transgenic). Characterisation of S1PR1 expression in renal and vascular tissue from 21 week-old salt-loaded rats, demonstrated below detection protein levels across parental and congenic strains. To further investigate the effect of the congenic interval and Gstm1 on salt-sensitivity and BP regulation and identify putative biomarkers, high-throughput metabolomic screening of urine and plasma was conducted in parental, SP.WKYGla2k congenic and Gstm1-transgenic strains, on a normal-salt and high-salt diet. In both urine and plasma, salt-loading affected processes implicated in CVD, including inflammatory response, free radical scavenging and lipid metabolism. In urine, oleic acid, implicated in regulation of renin levels, was increased in the SHRSP and transgenic salt-sensitive strains compared to the WKY and 2k congenic salt-resistant strains, upon salt-loading. In plasma, known biomarkers of CVD were altered in SHRSP compared to the other three strains, at normal-salt, including L-proline and linoleic acid. Upon salt-loading, glutathione disulfide and sphingosine-1-phosphate (S1P) were identified in high levels in the salt-sensitive strains. However, at normal-salt S1P was decreased in SHRSP compared to WKY and 2k congenic strains. Therefore, characterisation of the impact of S1P/S1PR1 signalling in the vasculature across the different strains was further investigated. Initially, structure, mechanical properties and vascular reactivity of mesenteric resistance arteries (MRA) were studied in 16 week-old parental and reciprocal 2a congenic strains (WKY.SPGla2a and SP.WKYGla2a). There was no significant remodelling observed across the strains. However, SHRSP vessels were stiffer and this phenotype was under the control of the congenic segment. SHRSP exhibited hypercontractility, which was mediated by RhoA/Rock signalling pathway and was corrected by the transfer of the congenic interval in SP.WKYGla2a. SHRSP also displayed endothelial dysfunction, which was related to reduced nitric oxide (NO) bioavailability and was not improved by the congenic interval. The predominant regulatory mechanisms of contraction and relaxation in MRAs from WKY and WKY.SPGla2a were demonstrated to be different compared to SHRSP. Subsequently, representation of these physiological differences in MRAs, at the molecular level, was investigated along with the effect of S1P-signalling in HTN. Comprehensive, high-throughput proteome profiling of S1P-stimulated primary mesenteric VSMCs from parental and 2a-reciprocal congenic strains, was achieved through triple stable isotope labelling (SILAC), LC-MS/MS analysis and MaxQuant quantification. Detection of few abundant phosphorylated proteins was attributed to lack of enrichment for phosphoproteome. Therefore, focus was placed on proteins whose differential expression between SHRSP and WKY was genetically regulated. These proteins mapped to pathways implicated in BP-regulation, including oxidative stress, vascular tone regulation and vascular remodelling. Glutathione S-transferase mu 1 (GSTM1) was upregulated in SHRSP, as opposed to down-regulated NAD(P)H oxidase quinone 1 (NQO1) and heme oxygenase 1 (HMOX1), suggesting different antioxidant mechanisms in health and disease. Natriuretic peptide receptor C (NPR3) which is implicated in vascular relaxation was increased in SHRSP, along with activators of RhoA contractile mechanism, such as caveolin1 (CAV1). Furthermore, RhoA/Rock signalling pathway was highly altered in SHRSP. Finally, differentially expressed proteins were related to sphingosine signalling, including superoxide dismutase 2 (SOD2) and collagen type III, alpha 1 (COL3A1). To further investigate the metabolic effect of sphingosine signalling across the strains, and assess the contribution of the congenic interval, metabolomic profiling of primary mesenteric VSMCs from parental and SP.WKYGla2a congenic strains, was performed at basal conditions and upon S1P-stimulation. A labelling-free, untargeted approach was employed, using HILIC-MS analysis and data processing through IDEOM. The effect of the congenic interval on the metabolic profile of SP.WKYGla2a was more profound under basal conditions. S1P-stimulation induced greater responses in SHRSP than WKY, indicating altered signalling. Furthermore the responses were different in each strain, suggesting a combined effect of the genetic background and the congenic interval on S1P signalling regulation. Inosine, which is implicated in purine metabolism, was significantly decreased in SHRSP compared to SP.WKYGla2a, at basal conditions, but was increased upon-S1P stimulation, implying that this S1P effect depends on the congenic interval. Moreover, tyramine, which has vasodilatory properties, was increased in stimulated SHRSP compared to basal conditions, indicating potential relation of sphingosine signalling with BP-regulation. This study has combined high-throughput proteomic and metabolomic screenings with congenic and transgenic strains to capture a clearer picture of the pathophysiological processes that underlie HTN in SHRSP. Individual metabolites and proteins or pathways and processes identified to be altered in HTN, through this work, can be used for generation of new testable hypothesis towards the development of new therapeutic approaches against HTN.

It is now accepted that there is a link between obesity and several diseases such as cardiovascular disease (CVD), diabetes, rheumatoid arthritis (RA), and atherosclerosis with the common initiating factor in pathogenesis being a state of low grade, chronic inflammation. This state, characterised by elevated levels of pro-inflammatory cytokines such as interleukin (IL) 6, leads to sustained activation of inflammatory signalling pathways such as the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and subsequently pathogenesis. Suppressor of cytokine signalling (SOCS) 3 is inducible by several stimuli including IL6 and 3'-5'-cyclic adenosine monophosphate (cAMP), and via these routes has been demonstrated to terminate IL6 signalling thus quenching JAK/STAT signalling and an inflammatory response. While SOCS3 was primarily characterised as a competitive inhibitor of intracellular signalling, it also functions as specificity factor for an elongin-cullin-SOCS (ECS)-type E3 ubiquitin ligase. In this role, it has been demonstrated to direct ubiquitin-mediated proteasomal degradation of several substrates and lysosomal routing. However, the full spectrum of SOCS3-dependently ubiquitinated substrates is unknown. Given that JAK/STAT signalling is critical in the development of chronic inflammatory disorders, delineating the role of SOCS3 as an E3 ligase might be therapeutically beneficial. However, given the broad range of SOCS3 stimuli, the availability of certain SOCS3 substrates might be conditional on the route of SOCS3 induction. Using a global proteomics approach, this study aimed to identify SOCS3-dependently ubiquitinated substrates in response to cAMP and thus elaborate on the already well-established role of cAMP in inflammation. Differentially stable isotope labelling of amino acids in cell culture (SILAC)-labelled, tandem affinity purified ubiquitinomes of wild type (WT) murine embryonic fibroblasts (MEFs) and SOCS3-/- MEFs, each expressing epitope-tagged forms of ubiquitin, were compared using mass spectrometry (MS) following cAMP-mediated SOCS3 induction. Using this approach, proteins modified by SOCS3 with the epitope-tagged form of ubiquitin should be enriched in WT MEFs but not SOCS3-/- MEFs. MaxQuant analysis of raw mass spectromeric data identified several candidate SOCS3 substrates. Of these, SOCS3 was found to interact with PTRF/cavin-1, a regulator of caveolae formation and stability. Other substrates were tested but with limited success. Co-immunoprecipitation studies showed that SOCS3 could precipitate cavin-1 however the interaction was reduced following the inhibition of protein tyrosine phosphatases (PTPs) using sodium orthovanadate and hydrogen peroxide. This was surprising since all known SOCS3 substrates are tyrosine-phosphorylated prior to interacting with SOCS3 via its Src-homology (SH) 2 domain. Consistent with this finding, SOCS3 did not interact with known cavin-1 tyrosine-phosphorylated peptides spotted on a peptide array. However, a full-length cavin-1 peptide array spotted with non-tyrosine-phosphorylated peptides showed specific interactions at multiple sites. It is proposed that this interaction might influence the localisation and stability of either protein. While SOCS3 was demonstrated to impact cavin-1 ubiquitination, the mechanism by which it does so or the functional consequence is still not clear. Immunoprecipitation of cavin-1 following the introduction of SOCS3 was accompanied by a shift in the polyubiquitin signal from a high molecular weight, seen with cavin-1 alone, to a low molecular weight. Furthermore, an enhanced K48-polyubiquitin signal was detectable in this low molecular weight fraction, which was focused around the molecular weight of cavin-1. It is not known if this ubiquitin signal is SOCS3-dependent. In conclusion, the project has identified and validated a novel substrate of SOCS3. However, the mechanism by which SOCS3 regulates cavin-1 ubiquitination or the biological function of the interaction is currently unknown.

The structure and properties of a nascent pectin-xyloglucan complex in etiolated pea epicotyls were investigated. Membrane pellets were prepared and incubated with UDP-[14C] galactose, and were extracted with six different reagents: Tris-HC1 buffer (pH 7.4); 50mM EDTA/50mM PO4 (pH6.8) at 100°C; 50mM EDTA/59mM PO4 (pH6.8) at 25°C; phospholipase C with 100 ml of 0.1M Tris-HC1 pH7.4 at 25°C; trypsin at 25°C and 0.1% Triton X-100 at 25°C. The best extractant used to solubilise the pectin-complex from the pellets was 50nM EDTA/50mM PO4 pH 6.8 at 100°C. Aqueous solutions of pectic polyuronides tend to associate covalently into multichain aggregates. Because of the tendency of pectins to aggregate in solution, the effect of a number of eluents on the behaviour of the complex on gel filtration was studied. 10mM EDTA/10nM PO4/1M NaC1 (pH6.8) was chosen as the best eluent to minimise this aggregate formation. On gel filtration using Sepharose CL-2B with this eluent, the complex eluted with a Kav of around 0.8, corresponding to a molecular size approximately 200 kDa, as judged by dextran standards. In other solvents tested, aggregation appeared to occur. On Sepharose CL-4B and CL-6B columns using the same EDTA/Pi/NaC1 solvent, the apparent molecular size was significantly reduced by a xyloglucan-specific endoglucanase, confirming the presence of a xyloglucan rather that a glucan in the complex. Polygalacturonase caused a greater decrease in apparent molecular size, to 10-20 kDa while endo-1 4-b-galactanase converted the radioactivity to [14C] galactobiose and [14C] galactose. When the [14C] galactose-labelled complex was incubated in solution with 3MM paper, the radioactivity was almost completely absorbed onto the paper over a period of about 6 hours. This confirmed the presence of xyloglucan attached to pectin. A new model for the structure of the nascent pectin-xyloglucan complex is proposed.