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Tissue factor (TF; CD142) is the principal initiator of the coagulation cascade. This 45kDa protein is constitutively expressed in extravascular cells and as such forms a protective haemostatic envelope surrounding the vasculature ready to initiate coagulation on vascular injury. When bound to its cofactor factor VII/VIIa in the normal bloodstream, it forms a highly catalytic complex (TF/VIIa) which triggers the activation of factors IX and X and ultimately the formation of a fibrin clot. Peripheral blood cells do not, in a resting state, express TF allowing maintenance of the fluidity of the blood. However, agonists such as bacterial lipopolysaccharide (LPS) and immune complexes, if present in the bloodstream can induce the expression of TF on monocytes resulting in activation of the clotting process. This induction of Monocyte TF has been clearly implicated in the pathology of various disease processes such as disseminated intravascular coagulation (DIC) following endotoxaemia, thromboembolic disease and atherosclerosis. We have characterised a whole blood model to investigate the induction of TF on monocytes, and have developed a highly specific and sensitive flow cytometric technique to measure the TF protein on the cell surface of activated monocytes. We have shown that there is a reproducible dose dependent induction of TF on monocytes in whole blood by LPS (10pg/ml-100μg/ml) after a 2 h incubation. Antisense therapy is aimed at the specific inhibition of a particular target protein by preventing the translation of its mRNA. Antisense (AS) oligodeoxynucleotides (ODNs) are short single stranded lengths of reverse complementary DNA which can be designed to specifically hybridise to a target mRNA, through Watson and Crick base pairing laws, arresting translation of the protein. We have investigated the potential of antisense therapy to inhibit specifically the nascent induction of TF in monocytes in response to LPS. We have characterised the uptake kinetics of a fluorescently labelled 25 base phosphorothioate oligonucleotide into peripheral blood monouclear cell (PBMC) subtypes. We have shown that the uptake of GEM<SUP>(R)</SUP>91 is dose and time dependent and is not saturated at concentrations of up to 10μM for 4 hours.

Plasma Membrane Calcium-ATPases (PMCA’s) extrude calcium from a variety of cell types and have recently been shown to regulate signalling events and function in the cardiovascular system. PMCA (isoform 4) is expressed in platelets, however its functional role remains undefined. The aims of this project were to investigate the roles of PMCA in regulating calcium homeostasis and human platelet function during the various stages of platelet activation by evaluating the effects of a pharmacological inhibitor of PMCA, carboxyeosin, upon a range of in vitro and in vivo assays. Additional experiments were performed to determine the functional consequences of PMCA4 ablation using platelets from PMCA4 knock-out mice and the roles of PMCA were contrasted with other calcium transporters. Finally, the mechanism of action of PMCA in platelets was investigated by integrating molecular studies with the functional assays. The findings presented in this thesis suggest that PMCA plays an important role in regulating calcium both basally and during platelet activation. PMCA positively regulates platelet aggregation through the modulation of negative signalling pathways but negatively regulates the earlier and later stages of activation measured as adhesion and clot retraction respectively. PMCA also regulates platelet function in vivo. Experiments in PMCA4 knock-out mice implicated the PMCA4 isoform in the regulation of platelet function. Comparison of PMCA, SERCA (sarcoendoplasmic reticulum ATPase) and NCX (Na+/Ca2+-exchanger) pumps indicated that all three calcium transporters play pivotal but contrasting roles in platelet function and calcium homeostasis. In conclusion, PMCA is a key regulator of calcium homeostasis and differentially regulates the various stages of platelet activation. PMCA regulates intracellular signalling events in platelets although the mechanisms remain to be fully determined. PMCA is therefore a key modulator of haemostasis and thrombosis and may be a potential target in the treatment of conditions such as arterial thrombosis and stroke.

Chronic mechanical load variation triggers a wide range of responses in the heart, a part of which includes cellular remodelling. Over the past 15 years, evidence has amassed that a part of this remodelling process involves changes to a sophisticated structure in the cell membrane, called the transverse (t)-tubule system. The t-tubules are a series of regular membrane invaginations, which contain a high density of ion channels responsible for local Ca2+ induced Ca2+ release (CICR). This thesis addresses the question of whether the t-tubule system can be said to be specifically load sensitive, the nature of that load sensitivity and its molecular regulators. Using surgical models, the influence of mechanical load variation of different durations, degrees and settings are studied. Local CICR and t-tubule structure are investigated. First, it was found that prolonged mechanical unloading induces subtle changes to the t-tubule system, which functionally uncouples the Ryanodine receptors (RyR) and L-type Ca2+ channels (LTCC) and induces a loss of whole cell Ca2+ release synchrony. Second, heart failure was found to be associated with loss of t-tubule structure and Ca2+ handling abnormalities. Following mechanical unloading, the t-tubule system recovered with enhanced LTCC-RyR uncoupling, resulting in improved Ca2+ handling. Third, the t-tubules were found to be unchanged initially during graded mechanical load variation. Prolonged myocardial unloading or overloading impaired t-tubule structure, with loss of normal CICR. Telethonin (Tcap), a member of the cardiomyocyte stretch sensing complex, is a candidate regulator of the t-tubules. In a Tcap knock-out (KO), cardiomyocytes show a primary t-tubule defect, which becomes more pronounced following mechanical overload. These results support the notion that the t-tubule system is dynamically regulated by mechanical overload and unloading, via a molecular pathway including Tcap.

Atherosclerosis occurs in a spatially heterogeneous fashion within the arterial system. The patchy nature of the disease is thought to reflect spatial variation in haemodynamic factors such as wall shear stress (WSS) and in the concentration of vascular species. This thesis numerically investigates blood flow and vascular transport of the atheroprotective agent nitric oxide (NO) using a spectral/hp element method. Firstly, NO distribution is analysed within a parallel-plate flow chamber coated with endothelial cells. Contrary to the accepted hypothesis that NO concentration increases with WSS (as does NO production), it is observed that NO concentration depends on WSS in a non-monotonic fashion. Furthermore, these results emphasise the effect of convection on in vitro NO transport, which has been overlooked or misinterpreted in most previous computational studies. Secondly, blood flow and NO transport are investigated within a detailed representation of the rabbit thoracic aorta and its branches, which was reconstructed using high resolution computed tomography (CT) scan data of a vascular corrosion cast. The computed WSS distribution exhibits significant spatial heterogeneity, reflecting the complexity of the blood flow. In particular it is observed that two Dean-type vortices (associated with a skewed velocity profile) form in the aortic arch and propagate along the descending aorta. This results in streaks of WSS similar in nature to the fatty streaks of early stage atherosclerosis observed in mature rabbits. This finding provides further support for the hypothesis that blood flow mediates atherogenesis. The vascular distribution of NO is found to depend significantly on the relationship between NO production and WSS. Furthermore, it is concluded that mechanisms preventing NO consumption by haemoglobin (such as diffusional barriers) must exist in order for NO to exert its atheroprotective action. This study represents the first effort to model NO transport in a realistic representation of the major arteries.

Thin filaments, through interaction with thick filaments, form the contractile apparatus of striated muscle. Therefore, the length and arrangement of the thin filaments are of key importance to the function of the muscle. The thin filaments from adjacent sarcomeres are anchored at the Z-disc. In 1968 Pringle predicted that thin filament are organised in the Z-disc in a rhomboid lattice rather than a square lattice. Previous experimental evidence has been insufficient to verify Pringle’s suggestion. In the A-band the thin filaments interdigitate with the thick filaments on a hexagonal lattice, hence from the Z-disc to the A-band, there is a transition of the lattice from square to hexagonal. In this project, I have firstly used Fourier analysis and electron tomography to investigate the thin filament lattice in the Z-disc. I have used electron tomography to determine how the lattice transition occurs between the Z-disc and the A-band. Electron tomography of these samples also allowed me to determine the lengths of thin filaments, showing unequivocally that they are of variable lengths in cardiac muscle.

Endothelin-1 (ET-1) promotes changes in gene/protein expression in cardiomyocytes leading to hypertrophy. This results from activation of intracellular signalling pathways including small G proteins that activate protein kinases. Thus, ET-1 activates RhoA that stimulates ROCK and PKN, and Ras that promotes activation of extracellular signal-regulated kinases 1/2 (ERK1/2). Microarrays were used to dissect the roles of ERK1/2 vs RhoA in the cardiomyocyte transcriptomic response to ET-1 using PD184352 and C3 endotoxin from C. botulinum (C3T) for selective inhibition of the ERK1/2 cascade and RhoA, respectively. Microarray data were analysed using GeneSpring and data were validated by qPCR. ERK1/2 signalling positively regulated ~65% of the early gene expression response to ET-1 with a small (~2%) negative effect, whereas RhoA signalling positively regulated ~11% of the early gene expression response to ET-1 with a greater (~14%) negative contribution. Of RNAs non-responsive to ET-1, 66 or 448 were regulated by PD184352 or C3T, respectively, indicating that RhoA had a more significant effect on baseline RNA expression. mRNAs upregulated by ET-1 encoded several receptor ligands (e.g. Ereg, Areg) and transcription factors (e.g. Abra/STARS, Srf) that potentially propagate the response. Published studies suggest that PKN1 (activated by RhoA) is important in cardiomyocyte gene expression. Adenoviruses were generated to overexpress FLAG-tagged PKN1 in cardiomyocytes for protein kinase studies. Unexpectedly, PKN1 was not activated by ET-1, but was activated by oxidative stress, insulin, or hyperosmotic shock, stimuli that do not activate RhoA. Thus, PKN1 is not necessarily an effector of RhoA in cardiomyocytes. In conclusion, ERK1/2 dominates over RhoA in the early transcriptomic response to ET-1. RhoA plays a major role in maintaining baseline RNA expression but, as with upregulation of Abra/Srf by ET-1, RhoA may regulate changes in RNA expression over longer times. However, the effects of RhoA on cardiomyocyte gene expression are unlikely to be mediated through PKN1.

In the myocardium action potentials are transmitted from cell-to-cell through gap junctions. These specialised junctions play a pivotal role in regulating the speed and safety of impulse propagation by controlling the amount of depolarised current that is passed from excited to non-excited regions of the heart. In mammalian hearts gap junction proteins connexin43, connexin40 and connexin45 are co-expressed in distinctive combinations and relative quantities in functionally specialised subsets of cardiac myocyte. The functional consequences of these connexin expression/co-expression patterns in modulating impulse propagation are poorly understood. To study the relative importance of membrane excitability and electrical coupling in relation to propagation velocities, clones of the HL-1 mouse atrial myocyte tumour line were used as an in vitro cell model. Five clones were characterised for expression of myocytic markers, calcium handling proteins and connexins, two of which (#2 and #6) displayed large differences in conduction velocities using microelectrode arrays. To ascertain which factor(s) were the main determinants of speed of conduction, the membrane excitability (voltage-gated channels) and electrical coupling (gap junctions) between the two clones were compared. Sodium, L- and T-type calcium channels were present in both clones but no significant differences were found in the current densities. However, large differences were seen in expression levels of connexin43, connexin40 and connexin45. RNA interference combined with microelectrode arrays was employed to establish the relative importance of each connexin in impulse propagation. The results indicate that electrical coupling by gap junctions is a major determinant of conduction velocities in HL-1 cell lines. Further experiments using RNA interference to suppress the expression of proteins thought to play a role in the action potential parameters should help in defining the part played by either the active or passive electrical properties in action potential propagation.

Dielectrophoresis (DEP) is an electrostatic technique which can be used to examine cellular electrophysiology. This method offers many advantages in characterising a cell population over conventional methods; however, it has yet to see mainstream pharmacological application. This work demonstrates a DEP assay, 3DEP, as a viable method in the electrophysiological measurement of cells, its application for measuring drug effects, and more specifically, its use as a molecular targeting tool in circadian research. The reliability of the assay was investigated measuring DEP response of several cell types with high resolution spectra and the operational limits of automatic fitting algorithms were determined for single and multiple populations. It was found that reliable readouts from 3DEP could be achieved in as little as 6 s. The ability of 3DEP to test drug cytotoxicity on Jurkat cells was tested with doxorubicin. DEP measured IC50 values compared well with previoiusly measured colorimetric assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) experimental results, suggesting 3DEP could provide an alternative way to measure molecular events due to drug intervention. With 3DEP having been appropriately optimised, it was then applied to the study of RBC circadian rhythm through both human and vole in vitro and in vivo studies. Disruption of these rhythms can lead to negative health outcomes related to areas such as metabolism, cardiovascular health, and mental health. Since RBCs are anucleated, the mechanism cannot rely on the typically accepted transcription-translation feedback loop found for other cells. 3DEP detected robust rhythmicity in the electrophysiological properties of RBCs in isolated and whole blood samples. These rhythms were altered when introduced to pharmacological intervention. Taken together, these findings suggest a model for the non-transcriptional clock in RBCs. Many applications of the 3DEP system were investigated with respect to molecular targeting highlighting 3DEP’s utility and limitations as a biological tool in research.

Diffusion magnetic resonance imaging (dMRI), tractography and the use of network measures have combined to form an established approach for exploring brain connectivity. When applied to the human brain, a definition of regions of interest (ROIs) which act as network nodes is required. In adults, regions commonly represent brain areas that are assumed to be functionally coherent. During early development however, a complete set and locations of ROIs in the brain is yet to be established. This motivates the use of random parcellation schemes with varying numbers of regions or scales. However, network measures can be scale dependent, making comparisons across multiple scales challenging and hindering group comparisons. To address such scale dependence, network measures are commonly normalised using random surrogate networks which act as a baseline. In this work, the efficacy of commonly used normalisation techniques is determined and new methods for generating randomised surrogate networks are introduced. Furthermore, a subset of measures is derived by investigating inter-measure correlations and the framework is then applied to serial dMRI data of a preterm cohort. It is shown that a new method for generating surrogate networks for normalisation improves on established approaches and eliminates scale dependencies over a local range, allowing for meaningful group comparison. While normalisation may be used for group comparison over a local range, scale dependence can remain over larger ranges. This work shows that the nature of the scale dependence varies between cohorts, and proposes a multiscale framework for group comparison. Using this framework to characterise the scale dependence, it is possible to differentiate the groups of neonates studied. This approach, however, requires the calculation of networks at multiple scales. Therefore the use of a node-merger scheme is also proposed to infer network properties at a coarse scale from a single network estimated at a fine scale. This approach allows for multi-scale group comparison based on a single starting network.