Visual processing of complex objects is a feat that the brain accomplishes with remarkable speed – generally in the order of a few hundred milliseconds. Our knowledge with regards to what visual information the brain uses to categorise objects, and how early the first object-sensitive responses occur in the brain, remains fragmented. It seems that neuronal processing speed slows down with age due to a variety of physiological changes occurring in the aging brain, including myelin degeneration, a decrease in the selectivity of neuronal responses and a reduced efficiency of cortical networks. There are also considerable individual differences in age-related alterations of processing speed, the origins of which remain unclear. Neural processing speed in humans can be studied using electroencephalogram (EEG), which records the activity of neurons contained in Event-Related-Potentials (ERPs) with millisecond precision. Research presented in this thesis had several goals. First, it aimed to measure the sensitivity of object-related ERPs to visual information contained in the Fourier phase and amplitude spectra of images. The second goal was to measure age-related changes in ERP visual processing speed and to find out if their individual variability is due to individual differences in optical factors, such as senile miosis (reduction in pupil size with age), which affects retinal illuminance. The final aim was to quantify the onsets of ERP sensitivity to objects (in particular faces) in the human brain. To answer these questions, parametric experimental designs, novel approaches to EEG data pre-processing and analyses on a single-subject and group basis, robust statistics and large samples of subjects were employed. The results show that object-related ERPs are highly sensitive to phase spectrum and minimally to amplitude spectrum. Furthermore, when age-related changes in the whole shape of ERP waveform between 0-500 ms were considered, a 1 ms/year delay in visual processing speed has been revealed. This delay could not be explained by individual variability in pupil size or retinal illuminance. In addition, a new benchmark for the onset of ERP sensitivity to faces has been found at ~90 ms post-stimulus in a sample of 120 subjects age 18-81. The onsets did not change with age and aging started to affect object-related ERP activity ~125-130 ms after stimulus presentation. Taken together, this thesis presents novel findings with regards to the speed of visual processing in the human brain and outlines a range of robust methods for application in ERP vision research.

Vascular smooth muscle cell (VSMC) activation leading to proliferation, migration and extracellular matrix (ECM) production is a major cause of neointimal formation after stenting and coronary artery bypass grafting. Drug-eluting stents have reduced clinical incidence of in-stent restenosis by inhibiting this proliferative response but they can also delay vessel re-endothelialisation after injury leading to an increased thrombotic risk. MicroRNAs (miRNAs) are short (~22 nt) non-protein-coding RNAs which act as regulators of gene expression largely through binding to the 3’ untranslated region of target genes and causing degradation or repression of expression. MiR-143 and miR-145 are a miRNA family that are enriched in VSMCs and have been previously shown to influence VSMC phenotype through regulation of their gene targets. Consequently, the aim of this study was to investigate the role that miR-143 and miR-145 play in the neointimal response to stenting. Initial experiments investigated whether modulation of miR-143 or miR-145 expression was capable of significantly altering VSMC phenotype. It was found that modulation of miRNA levels in human saphenous vein (HSV) SMC or endothelial cells (EC) using adenoviruses was not ideal due to transduction and toxicity issues. Use of antimiR miRNA inhibitors and premiR miRNA mimics revealed that modulation of miR-143 or miR-145 levels alone was not sufficient to alter proliferation or migration of HSV SMCs in vitro. Knockdown of miR-143 expression in cells resulted in de-repression of target genes kruppel-like factor 4 (KLF4) and KLF5 but expression levels of other previously identified target genes were unaltered by miRNA modulation. Pre-clinical stenting studies are largely performed in porcine models due to similarities in vessel structure and neointimal formation, however large animal models are not ideal for early investigative studies. In order to examine the role of miR-143 and miR-145 in stent-induced vascular injury we utilised a mouse model where a bare metal stent is deployed in the thoracic aorta of a donor mouse and interposition grafted into the carotid artery of a recipient. This model resulted in the development of a defined neointima over 28 days which consisted largely of VSMCs and ECM, similar to that of the human in-stent restenosis. Vessel expression of miR-143 and miR-145 has been previously shown to be reduced following vascular injury and furthermore overexpression of these miRNA can reduce neointimal formation. MiR-143 and miR-145 knockout (KO) mice have previously been shown to have abnormal VSMC phenotype in their vessel walls including perturbed stress fiber formation, increased presence synthetic machinery and an increased number of synthetic versus contractile VSMCs. MiR-143 and miR-145 KO mice were found to develop significantly less neointimal formation in response to stenting than WT mice indicating that these miRNA are essential for normal vessel response to injury. The reduced neointimal formation following genetic ablation of miR-143 or miR-145 led to the investigation of whether pharmacological knockdown of these miRNA was able to mimic this effect. An antimiR consisting of DNA and locked nucleic acid bases targeted against mature miR-143 expression was used to knockdown miR-143 expression in mice prior to stenting. AntimiR-143-mediated knockdown of miR-143 expression did not significantly alter the degree of neointimal formation seen 28 days following stent deployment when compared to mice that received a control non-targeted antimiR (antimiR-ctl). The neointima of both antimiR-143 and antimiR-ctl mice were comparable and consisted largely of VSMCs and ECM. Re-endothelialisation had occurred by day 28 post-injury in antimiR-143 and antimiR-ctl treated mice indicating that knockdown of miR-143 did not significantly delay EC repopulation in this model. Expression of miRNA and mRNA after vascular injury can be both spatial and temporal. Target de-repression was not detected in the aorta or heart of miRNA KO or antimiR-143 treated mice. This is likely to reflect the complex nature of miRNA gene regulation in vivo which is governed by many different factors including the relative expression of the miRNA and its target, cell stress, and transcriptional activation or inhibition by growth and transcription factors. In summary, the influence of miR-143 and miR-145 on VSMC biology was investigated in vitro using a range of molecular biology techniques and in vivo in a mouse model of in-stent stenosis. Results have extended current knowledge of the degree of influence these miRNA exert over VSMC phenotype and identified that miR-143 and miR-145 are involved in neointimal formation after stenting.

A major action of the hormone insulin is to increase glucose uptake into muscle and adipose tissues. This occurs through the insulin-regulated translocation of the glucose transporter GLUT4 from intracellular deposits to the cell surface. The dysregulation of this process is a major facet of insulin-resistance and Type-2 diabetes. In the absence of insulin, GLUT4 is sequestered intracellularly in two interlinked pools. These pools are the endosomal recycling compartment and a specialised insulin-sensitive compartment. GLUT4 vesicles within the specialised insulin-sensitive compartment are termed GSVs. GSVs provide a readily available store of GLUT4 that can be rapidly mobilised to the cell surface in response to insulin stimulation. GLUT4 within the cell is continually recycled through multiple compartments, including the endosomal recycling compartment and GSVs. Perturbation of flux through any of these compartments disrupts GLUT4 traffic and thus disrupts the insulin-response. Consequently, understanding these trafficking steps is an important research goal. The formation of GSVs and their insulin-stimulated translocation to, and fusion with, the plasma membrane are examples of regulated and specific membrane trafficking events. As such these events require SNARE proteins. This study has examined the functions of each of the members of the post-Golgi vSNAREs that make up the VAMP subfamily in the trafficking of GLUT4 into the insulin-sensitive compartment, as well as examining the contribution each of these vSNAREs plays in the SNARE complex involved in the insulin-stimulated translocation of GLUT4 to the cell surface. GLUT4 traffics through the endosomal system and the trans Golgi network (TGN) en route into GSVs in a process known to involve the t-SNARE syntaxin 16. In order to dissect the roles VAMP proteins play in the intracellular recycling and trafficking of GLUT4, the interactions of VAMP proteins with syntaxin 16 and its regulatory Sec-1/Munc-18 protein, mVps45 have been examined. Further, the effects of depletion of these VAMPs in GLUT4 distribution has been examined in HeLa cells expressing HA-GLUT4-GFP as a surrogate for adipose and muscle tissue. The fusion of GLUT4-containing vesicles with the cell surface in response to insulin stimulation is a key component of the insulin-response. Although there is consensus that tSNAREs involved in this process are syntaxin 4 and SNAP23, there is discord in the literature regarding the role each vSNARE plays in this fusion event. Many studies support a role for VAMP2, however recently it has been suggested that VAMPs 3 and 8 may also be involved. The roles of these VAMPs, as well as the other post-Golgi vSNAREs expressed in adipocytes, in the fusion of GLUT4-containing vesicles with the cell surface have been examined. This has been achieved through the characterisation of the expression level, subcellular distribution of the VAMPs and determination of associations that take place between each VAMP protein and the tSNARE complex made up of syntaxin 4 and SNAP23. The results from these experiments show that despite all VAMPs being capable of forming SDS-resistant SNARE complexes with either syntaxin 4 and SNAP23 or syntaxin 16 and SNAP23, in the cellular environment only VAMP2 interacts with syntaxin 4 and only VAMP4 interacts with syntaxin 16. This, alongside the finding that each VAMP protein is expressed at varying levels and localises with different pools of GLUT4, suggests that VAMP2, not VAMPs 3 or 8, is the major vSNARE involved in the trafficking of GLUT4 to the cell surface. The finding that VAMP4 is the only VAMP that interacts with syntaxin 16, alongside the results of VAMP depletion in HeLa cells expressing HA-GLUT4-GFP, support the hypothesis that VAMP4 is involved in sorting GLUT4 out of the endosomal pool and into GSVs.

This project assessed the use of short chain peptide (F2/S) hydrogel biomaterial substrates as an instructional tool for driving stem cell differentiation through fine-tuning of the substrate mechanical properties (altered elasticity or stiffness) to mimic that of naturally occurring tissue types. By doing this, differentiation of mesenchymal stem cells (MSCs) into neuronal cells on a 2 kPa (soft) substrate, chondrocytes on 6 kPa (medium) substrate and osteoblasts on 38 kPa (rigid) substrates was achieved. This non-invasive procedure of influencing stem cell behaviour allows a means of exploring innate cell behaviour as they adopt different cell lineages on differentiation. As such, an LC-MS based metabolomics study was used to profile differences in cell behaviour. Stem cells were observed as having increased metabolic activity when undergoing differentiation compared to their ‘resting’ state when they are observed as metabolically quiescent or relatively inactive. As such, the metabolome, as a reflection of the current state of cell metabolism, was used to illustrate the observed divergence of phenotypes as differentiation occurs on each substrate F2/S type. The project further investigated the potential of endogenous small molecules (metabolites) identified using metabolomics, as effective compounds in driving or supporting cell differentiation in vitro. From this, the compounds cholesterol sulphate and sphinganine were found to induce MSC differentiation along the osteogenic and neurogenic routes respectively. A third compound, GP18:0, was observed to have influence on promoting both osteo- and chondrogenic development. These results highlight the potential role a broad based metabolomics study plays in the identification of endogenous metabolites and ascertaining the role(s) they play in cellular differentiation and subsequent tissue development. Lastly, the use of F2/S substrates as a potential clinical scaffold for the regeneration of cartilage tissue was explored. Long term differentiation of pericytes into chondrocytes cultured in 20 kPa F2/S substrates was assessed and the cellular phenotype of the resultant chondrocytes compared to the more conventionally used induction media method. Pericytes cultured within the biomaterial alone showed a balanced expressed of type II collagen and aggrecan with lessened type X collagen expression compared to the coupled use of induction media which showed a bias towards collagen (both type II and type X) gene expression. This observation suggests that in order to mimic native hyaline cartilage tissue in vitro, the use of biomaterial mechanics is potentially a better approach in guiding stem cell differentiation than the use of chemical cues.

3’,5’-cyclic adenosine monophosphate (cAMP) is the best studied intracellular second messenger. Adenylyl cyclase (AC) catalyses the synthesis of cAMP from ATP following the stimulation of a G protein coupled receptor (GPCR), and its degradation is catalysed by cAMP phosphodiesterases (PDEs) to allow cessation of signal. cAMP can act to bring about a multitude of varying and often opposing cellular responses, which depend on the stimulus received by the GPCR, the cell type, the cell cycle stage, and the complement of downstream effector molecules within that cell. The cAMP PDE subfamilies express multiple splice variants, which possess unique N-termini and non-redundant functional roles. By virtue of this, they are targeted to specific and discrete subcellular locations, where they may form highly specific interactions with scaffold proteins and other enzymes. Here, in these discrete locales, PDEs act to hydrolyse local cAMP, thereby underpinning the spatial and temporal compartmentalisation of cAMP gradients. This fine-tuned balance of synthesis and degradation is paramount for the dynamic cellular responses to extracellular stimuli, allowing differing signal transduction cascades to occur simultaneously in the crowded macromolecular environment of the cell. The compartmentalisation of cAMP signalling is, thus, essential for maintaining cellular homeostasis, and is subject to perturbation in various diseases, including prostate cancer (PC). Despite the wealth of literature implicating cAMP signalling in the progression of PC, little work has been done on the expression or function of PDE splice variant in this disease. Our group, in collaboration with Philips Research and the Prostate Cancer and Molecular Medicine (PCMM) group in the Netherlands, set out to investigate the changes in cAMP signalling during PC progression by studying the expression of cAMP PDE isoforms, with the aim of identifying a novel PC biomarker, as the current standard biomarker (PSA) is not disease-specific and leads to much over-diagnosis and over-treatment of otherwise non-life threatening prostate tumours. Interestingly, we found PDE4D7 to be dramatically downregulated as PC progresses from an androgen sensitive (AS) to an androgen insensitive (AI) state, and, indeed, this enzyme is showing promise as a novel, disease-specific PC biomarker. In this thesis, I report my efforts to characterise a function of PDE4D7 within prostate cancer. Firstly, I report the raising of a novel highly specific PDE4D7 antibody and describe the differential expression of this isoform, at the protein level, between AS and AI PC cell models. I present evidence to suggest that PDE4D7 mediates PC cell growth and migration, and that its loss may play a role in PC progression. I propose that an altered epigenome plays a role in the downregulation of PDE4D7 expression. I then report on the raising of a novel phospho-specific antibody and present evidence to show that PDE4D7 is regulated by PKA phosphorylation within its unique N-terminal region, and that this event confers negative regulation on enzyme activity. Finally, I describe my endeavours to elucidate a PDE4D7 protein-protein interaction that may help transduce PDE4D7-specific signals and maintain the enzymes cellular location.

In apicomplexan parasites, the machinery required for gliding motility is located between the plasma membrane and the Inner Membrane Complex (IMC). This type of motility depends on the regulated polymerisation and depolymerisation of actin and a multi-subunit complex, known as the Myosin A motor complex. This complex consists of the myosin heavy chain A (MyoA), the myosin light chain 1 (MLC1), the essential light chain 1 (ELC1) and three gliding-associated proteins (GAP40, GAP45 and GAP50). Gliding motility is thought to be essential for host cell egress and linked to active, parasite driven penetration of the host cell. Many components of this complex are extensively studied using either the ddFKBP system or the tetracycline-inducible knockdown system (Tet-system). Strikingly, while depletion of myoA has no impact on IMC formation, overexpression of the tail domain of MyoA results in a severe IMC biogenesis phenotype. In order to investigate this issue, conditional knockout (KO) mutants of the interacting partners of MyoA-tail were generated using the conditional site-specific DiCre recombination system. Indeed, GAP40 and GAP50 were identified as being essential for parasite replication and having a crucial role during IMC biogenesis. This is the first evidence showing that components of the MyoA motor complex fulfil essential functions during IMC formation and thus are not exclusively important for gliding motility dependant processes. Several components of the MyoA motor complex were characterised using the Tet-system and showed a complete block in gliding motility, but not in host cell invasion. While it is possible that leaky expression of the gene in the knockdown mutants is responsible for this uncoupling of gliding motility and invasion, it remains feasible that different mechanisms are involved in these two processes. In order to shed light on this issue, conditional KOs for the Acto-MyoA motor complex were generated in this study and their functions during gliding dependent processes thoroughly analysed. Intriguingly, while depletion of individual components of this complex caused a severe block in host cell egress, gliding motility and host cell penetration were decreased, but not blocked, demonstrating an important, but not essential role of the Acto-MyoA motor complex during these processes. Altogether, this study raises questions of our current view of what drives gliding motility and invasion and supports the argument for critical revision of the linear motor model.

Carbene intermediates can be generated by thermal, photochemical and transition metal catalysed processes from diazoalkanes. The carbene intermediates are very reactive and can add across double bonds to give 3-membered rings (cyclopropanes), insert into -OH bonds to give esters or ethers and insert into neighbouring -C-H bonds to give 4 or 5-membered rings, such as β- and γ-lactams or γ-lactones. Copper salts and complexes were amongst the first catalysts to be used for carbene generation from diazoalkanes. However, current tendencies are to use very expensive, especially, platinum group salts and complexes to generate the carbene intermediates, as yields and specificity tend to be higher. We have found that Cu²⁺-exchanged clay minerals (e.g. Wyoming bentonite) and zeolites (zeolite A), have proven to be very competitive in yield with such transition metal catalysts and they have the added advantage that the restricted reaction space within the zeolite pore or clay interlayer favours the more planar/less bulky product. With the clay minerals, when the layer spacing is kept low by judicious choice of mineral or solvent, the selectivity is improved. Herein we report a wide range of carbene addition (cyclopropane formation) and -C-H insertion reactions (β-lactam, γ-lactam and β-lactone formation) catalysed by the Cu²⁺-exchanged clay minerals and the stereochemical consequences of carrying out the reactions within the clay interlayer. Preliminary studies on the successful formation of aziridines from azides via nitrene intermediates with Cu²⁺-exchanged clay minerals are also reported.

Catalysts have been studied for the enantioselective hydrogenation of prochiral imines. Work has centred on the support of homogeneous catalysts, with the intention of establishing a heterogeneous catalyst without loss of activity. Initially, established heterogeneous systems were investigated. As the methyl pyruvate system of cinchona modified catalysts was to be used, this system was first investigated with a series of platinum catalysts. Although these results provided some interesting results they maintained that the 5% Pt/alumina catalyst was the best for this hydrogenation. Investigations using this system for prochiral imine hydrogenation resulted in the production of chiral amines. Hydrogenation of N-(1 -methylbenzylidene)-2,3-dimethylbutylamine gave encouraging percentage conversions, but unfortunately, enantiomeric excesses were particularly low. These results led to investigations on supporting a suitable homogeneous catalyst. Initial work involving Wilkinson's catalyst investigated different preparations, solvents and supports. Results achieved in this section of the study showed an increase in activity of clay and zeolite supported catalysts when run in 1,1,1-trichloroethane and ethanol, but a drop in activity when benzene was the reaction solvent. Work with imines centred around two homogeneous catalysts. BINAP and DIOP ligands were used to create rhodium and iridium centred catalysts which were used to homogeneously hydrogenate two imines (N-(1,2-dimethylpropylidene)aniline and N-(1 methylbenzylidene)-2,3-dimethylbutylamine). These catalysts were then tethered to a Montmorillonite clay support and NaY zeolite, before repeated hydrogenations of the above imines. Results achieved were very encouraging, with an increase in activity being noted for some of the supported catalysts.

This thesis presents friction and wear rate measurements of the copper based bearing materials used in thrust bearing of aircraft fuel pumps. Highly detailed friction and wear data was collected from 20% and 30% lead in bronze (leaded bronze) substrates and 10% indium in lead (lead/indium) coated leaded bronze substrates in unlubricated and marginally lubricated conditions. Measurements were made under a wide range of load and speed using a thrust washer test apparatus and pin on disc test equipment.

A series of reduced scale emulations of standard fires in a 2 m3 enclosure have been developed for studies at laboratory scale enabling useful comparison and correlation with full scale EN54/7 and UL268 test fires. This makes study of standard test fire conditions and products substantially more accessible. The reduced scale test fire emulations have smoke obscuration characteristics matched to the fire standards and show acceptable matching of experimental CO levels Sensor, fire detector, and analytical studies have been carried out on test fires in the 2 m3 enclosure and in a full scale test room. Protocols were developed for capture of gas and vapours from fires on absorbent media and their subsequently desorption and analysis by GC/MS techniques. A data set of GC chromatograms has been generated for full and reduced scale test fires and for a number of non standard fire or false alarm related process including overheating of cooking oils and toasting bread. Analysis of mass spectrometry ion fragmentation spectra has been carried out and a wide range of products identified. Products occurring for a range of different fires include propene, benzene, and some polyaromatics. The value of the scaled test fire emulations has been demonstrated by monitoring response of a range of sensors, detectors and instruments including electrochemical gas sensor, experimental and conventional light scattering smoke detectors, and ion mobility measurement equipment (FAIMS). The study has provided information on fire characteristics and products to inform future research and development on fire detection technologies.