The mechanisms responsible for maintenance of AF remain poorly understood. This thesis examines the frequency domain characteristics of AF in order to gain further insights into this arrhythmia. Through a series of studies involving patients undergoing catheter ablation for atrial fibrillation, intra-cardiac electrograms of AF were collected and analysed using Fast Fourier Transform to derive frequency domain parameters of dominant frequency (DF) and organization index (OI). It was found that intravenous flecainide reduced DF of AF, but only an associated increase in OI was predictive of successful return to sinus rhythm. In another study of patients having catheter ablation for persistent AF, a higher OI post-ablation was found to be associated with medium-term freedom of AF, suggesting that OI may be a useful guide to determine the extent of radiofrequency ablation needed. The effects of vagal blockade with atropine were also studied and compared with that of catheter ablation using a stepwise strategy of isolating the pulmonary veins, linear ablation and complex fractionated electrogram ablation, without deliberately targeting ganglionated plexi. This showed that atropine reduced DF and increased OI of AF electrograms, while decreasing mean RR intervals, standard deviation of RR intervals and 5th percentile of RR intervals. The directional changes of all the above parameters mirrored that of catheter ablation, suggesting that vagal blockade and catheter ablation not deliberately aimed at autonomic tissue can have similar effects on the frequency spectrum of AF, probably mediated through modulation of the autonomic tone. The relationship of regional DF and electrogram complexity as assessed by automated measurement of complex fractionated electrogram – mean (CFE-mean) were also compared, pre and post-ablation of the left atrium. There appeared to be only a modest correlation between the two and this was further weakened following ablation, suggesting that these are possibly separate substrate entities.

Procoagulant microparticles (MPs) in the circulation are increasingly recognized as playing a role in haemostasis and inflammation and may prove useful biomarkers for clinical studies. Platelets are known to generate MPs in response to stimulation, and platelet-derived MPs (PDMPs) form the majority of the MPs found in the normal circulation and can be elevated in a number of disease states. The current study has focused on the procoagulant activity of platelets and PDMPs following stimulation with the collagen-mimetic peptide CRP-XL. Their activity in accelerating thrombin generation can be accurately measured by the Calibrated Automated Thrombogram (CAT) assay using 1pM TF reagent to initiate the reaction, with the finding that plasma from patients with chronic renal disease has significant thrombin generation due to increased procoagulant activity of MPs. Conversely premature MI patients in a stable condition have thrombin generation comparable to matched healthy control. In all subjects, removal of MPs from plasma by filtration (>0.2 μm) eliminates this procoagulant activity, indicating the importance of MP size in driving the procoagulant response. The procoagulant activity of activated platelets and PDMPs showed a strong correlation with annexin-V binding measured using flow cytometry. Comparison of the regulatory mechanism of the procoagulant activity of platelet and PDMPs upon activation with platelets undergoing apoptosis showed that although both activation and apoptosis resulted in exposure of the procoagulant surface, apoptotic platelets did not release procoagulant MPs or show any markers of activation such as P-selectin expression, fibrinogen binding or aggregation. Reactive oxygen species (ROS) are generated during platelet activation or apoptosis mainly through the NADP(P)H oxidase pathway. The procoagulant activity of platelets and PDMPs was significantly attenuated (as measured by thrombin generation and annexin-V binding) by inhibition of the NAD(P)H oxidase pathway by apocynin, which had similar inhibition on other platelet responses including on 12-HETE generation, TxB2 production and aggregation. However antioxidants only inhibited apoptosis-induced platelet procoagulant activity. These data demonstrate significant involvement of ROS in platelet procoagulant activity induced by both activation and apoptosis and suggest the involvement of lipid peroxidation during apoptosis.

This research work explores the feasibility of using frequency domain analysis in the study of arrhythmias. The research involves the application of spectrum analysis to obtain the dominant frequency (DF) of atrial electrograms (AE) at different sites in the atria. It is an alternative way of interpreting the chaotic electrical activity seen during AF and reveals critical sites to guide ablation. As longer ablation procedure time implies higher risk to the patient, DF estimation needs to be obtained as quickly as possible. Four techniques (FFT, Blackman-Tukey, Autoregressive and Multiple Signal Classification) were used to compare the computation times taken for spectrum estimation analysis. The FFT technique produces an accurate DF result with the shortest time. DF analysis was first used for ventricular fibrillation with data from the surface of the left ventricle (in animal studies). It was found that spectrograms show the DF drifting along time and with significant changes in power. This approach was then applied for bipolar AF signals (in human studies). The changes of the frequency along time were observed when the stimulation was given, either using high frequency stimulation or drug infusion. We have developed a novel technique for the removal of ventricular signals from virtual AE. The surface ECG is used to identify ventricular activity. A band pass filter (8 Hz to 20 Hz) followed by rectification and then a low pass filter (6 Hz) are used for QRS detection. QRST subtraction was performed using three different approaches: flat, linear and spline interpolation. QRST subtraction affects the power of the signals but not the DF. We also developed an adaptive power threshold tool to observe the distribution of the DFs with an adjustable power threshold setting. Using this tool the 3D maps can display the evolution of the DFs within a chosen threshold power bracket.

The haemostatic response comprises the interaction of coagulation factors and peripheral blood cells which play a vital role in maintaining vascular integrity. This study begins with the finding that plasma from premature MI subjects has increased endogenous thrombotic potential linked to higher levels of circulating Tissue Factor (TF), suggesting these individuals may have a hypercoagulable phenotype. This study seeks to further understand how cellular interaction, in particular platelet-monocyte interaction, modulates the haemostatic response. Throughout the study, TF and Tissue Factor Pathway Inhibitor (TFPI) were studied as representing the procoagulant and anticoagulant response, respectively. Study of the effects of activated platelets on monocytes found that whereas direct platelet or platelet-microparticle adhesion to monocytes, and release of platelet soluble mediators induced monocyte gene expression of TF, induction of TFPI was driven solely by platelet soluble mediators. Extension of these studies using gene expression microarray technology found that whereas activation of monocytes via PSGL-1 generates a pro-angiogenic expression profile, platelet soluble mediators significantly enhanced this profile, enabling monocytes to interact with ECM components involved in the wound healing environment of the thrombus, and additionally induced anti-inflammatory, and anti-atherothrombotic genes. Whether cells within a thrombus act simply as structural and secretory components, or play a more active role involving gene expression is unclear, therefore gene expression array analysis was carried out on thrombi generated in vitro. Genes demonstrating significant time-dependent increases included those encoding chemotactic proteins (IL8, CCL2, CXCL1, CXCL2), cell adhesion (ITGAV, ITGA5, ITGB1), regulation of coagulation (THBD, PLAU, SERPINE1), wound-healing (ENDG, SPP1, LAMB3), and regulatory transcription factors (FOS, EGR1, PPARG). Whereas initiation of thrombosis is driven by plasma proteins and facilitated by the platelet surface, this study provides evidence that thrombus resolution may be driven by changes in gene expression within the thrombus that regulate the haemostatic response, thrombus growth, and facilitate wound-healing. These findings could have implications for individuals at risk of plaque rupture, where variation in gene expression may affect not just the formation of an occlusive thrombus but also the rate of resolution.

Arrhythmias in Long QT syndrome are known to occur during autonomic activity. The changes in repolarization of the myocardium under autonomic activity are thought to influence arrhythmia mechanisms. Although autonomic modulation of the ventricle is modulated by both humoral and nervous components, yet, due to technical difficulties a detailed understanding of the underlying mechanisms of the neuronal modulation is still elusive. This work is a description of the evolution of a novel model and early results of studies pertaining to the effects of the autonomic nerve stimulation on ventricular repolarization, in the context of long QT pathophysiology. These neuro-cardiology studies were performed using a combination of isolated innervated heart preparation and optical mapping for the first time. Two important repolarization characteristics namely physiological restitution and dispersion of repolarization were studied. The early results suggest that during sympathetic nerve stimulation, physiological restitution curve show unique characteristics of a negative slope at peak heart rates, not reported by classical restitution studies. Also, the action potential duration adaptation at peak heart rates showed heterogeneity over the surface of the myocardium and in addition, exhibited some features of a possible reduction of repolarization reserve, especially during ion channel inhibition. Another set of data obtained using this novel model revealed the autonomic nerve stimulation modulated the dispersion of repolarization in a unique way, and differed from pharmacological autonomic stimulation significantly. Interestingly, preliminary data evaluating the myocardial substrate suggests heterogeneity to be at more than one level i.e. key ion channel distributions, nerve terminal distribution over the myocardium and a possible interaction between them. When the hearts were treated with key repolarization inhibitors, the dispersion of repolarization showed a generically similar response rather than a characteristic response of individual ion currents. In these studies, the substrate changes of repolarization seem to dominate that of neuromodulation of the ventricle.

Dilated and Hypertrophic Cadiomyopathy can be caused by mutations of genes encoding sarcomeric proteins. Mutations in cTnT are of particular interest since they are generally associated with mild or no ventricular hypertrophy but a high incidence of sudden death. Previous investigations have focused on steady state parameters such as maximal activation and inhibition of actomyosin ATPase and force and Ca2+ sensitivity. We have aimed to use transient kinetics to investigate the effects of 7 cTnT mutations on the dynamics of thin filament switching. We have studied two DCM mutations (R141W, ∆K210) and five HCM (∆E160, S179F, K273E, ∆14, ∆28+7) mutations present in two functional domains of TnT (T1 and T2). Overall circular dichroism studies showed that the structure of these mutant proteins is not grossly affected although minor changes in the α–helical content were found for cTnT mutants K273E, ∆14, ∆28+7 and ∆E160. Co-sedimentation with actin suggested that most of cTnT mutations do not interfere with the association between cTn and thin filament except for the truncated mutations. Cooperativity along thin filament was changed for all deletion mutations (∆K210, ∆E160, ∆14 and ∆28+7) but unchanged by the point mutations. In this study we also demonstrated that the equilibrium constant between the blocked and closed states (KB) for DCM mutations were unchanged but increased dramatically for HCM mutations suggesting loss of blocked state specifically for those in the T2 region. We assessed Ca2+ binding of the regulatory site of cardiac TnC using IAANS attached to C35 and C84 of cTnC. Ca2+ binding affinity (pCa50 =6.65) of reconstituted Tn complex was unaffected by all mutations with the exception of ∆28+7 which caused a decrease (pCa50 0.34). In contrast when incorporated into thin filament, all HCM mutations and DCM ∆K210 showed increased Ca2+ affinity. The observed rate constant of Ca2+ dissociation was unchanged for all mutations except for ∆28+7. In conclusion, we have observed multiple structural and functional consequences from different TnT mutations that occur in different regions of the molecule. Overall the data suggests that it is the functional changes caused by mutations that are critical in developing the disease and not the specific location of the mutation.

Diurnal variation has been reported in many cardiovascular haemodynamics parameters such as heart rate and blood pressure and the cardiac action potential. This variation may result from the diurnal variation in sympathetic activity or in cardiac gene expression. However, it is unknown whether these time-of-day dependent changes impact on excitation-contraction (EC) coupling. There is also a morning peak in the onset of ventricular arrhythmias and associated sudden cardiac death in man, which appear linked to the increase in sympathetic activity. Therefore, the aims of this investigation were to determine whether there was a time-of-day dependent variation in EC-coupling and its modulation by sympathetic stimulation. Left ventricular myocytes were isolated during either the resting period (ZT3) or the active period (ZT15) o f the adult Wistar rat. [Ca[superscript 2+]][subscript i] was determined using Fura-2 and contraction strength was determined using cell-edge detection in response to electrical field stimulation, and gene expression was determined using quantitative real-time RT-PCR. To determine the effects of hypertension-induced hypertrophy, myocytes were isolated from pre- and post-hypertensive spontaneously hypertensive rats (SHR). The basal Ca[superscript 2+] transient, contraction strength and SR Ca[superscript 2+] content were significantly greater in resting period (ZT3) myocytes than active period (ZT15) myocytes. Systolic [Ca[superscript 2+]], amplitude of Ca[superscript 2+] transient and SR Ca[superscript 2+] content in response to isoproterenol (> 3nM) were significantly greater in resting period (ZT3) myocytes. The percentage of myocytes developing arrhythmic activity in response to isoproterenol was greater in resting period (ZT3) myocytes. Nitric oxide synthase (NOS) inhibition using L-NNA significantly increased systolic [Ca[superscript 2+]], amplitude of Ca[superscript 2+] transient, SR Ca[superscript 2+] content and the percentage of myocytes developing arrhythmic activity in active period (ZT15) myocytes thereby depressing time-of-day dependent variation in these parameters. In addition, expression of NOS1 was significantly greater in active period (ZT15) myocytes. Diurnal variation in the Ca[superscript 2+] transient and its responsiveness to isoproterenol were depressed in adult SHR, however, this did not reflect a depression of diurnal cycling in NOS1 expression. This shows for the first time a time-of-day dependent variation in the Ca[superscript 2+]-transient and resulting contraction strength, reflecting levels of SR Ca[superscript 2+]-loading, due to a NOS-signalling pathway. There was also a reduction in sympathetic-induced arrhythmic activity in active period (ZT15) myocytes which was associated with increased NOS activity. Therefore, variation in NOS may be a means of protecting against arrhythmias during severe sympathetic stimulation. Loss of protection through disruption to the circadian clock resulting from cardiomyopathies such as hypertension-induced hypertrophy may result in a decreased threshold for sympathetic-induced arrhythmias, however; this requires further work to elucidate the underlying molecular mechanisms.

Gold nanoparticles effects on neurite outgrowth were assessed by measurement of neurite outgrowth in differentiating mouse neuroblastoma cell line NB2a exposed to 5 nm, 10 nm and 15 nm conventional gold colloids and monolayer protected clusters. Neurite outgrowth enhancement was the predominant effect of most particles types. The thesis also investigated the uptake and intracellular fate of gold nanoparticle in the NB2a cell line in a serum free environment, employing a 2-D profile-based counting method. It was found that the monolayer clusters were taken up in significantly less amounts than their conventional counterparts following 24 hour exposure. No evidence for cellular accumulation was found after seven days as particle clearance was shown to be greater than 90 %. In addition, the thesis describes the development of functionalised monolayer protected gold clusters for application in bilirubin extraction. Bilirubin neurotoxicity was determined by measuring neurite outgrowth in NB2a cells and the application of the gold nanoparticle system in ameliorating this effect was attempted. No significant changes were observed. The presence of virus-like particles in the neuroblastoma cell line was investigated in the line of a potential confounding factor to the observed toxicity results. The thesis therefore also describes the morphology and genomic features of a virus-like particle. The morphological appearance describes intracisternal A-particles (IAP) but the nucleotide sequence revealed evidence of the B-tropism of mouse leukaemia viruses suggesting a rare form of an infectious IAP. The finding of an infectious IAP in mouse NB2a cell has implication for the continued use of this cell line.

The effects of thrombosis greatly contribute to the incidence of mortality in the Western World. Understanding thrombosis is therefore crucial in providing the correct treatment for the underlying pathologies. Numerical methods have previously been used to investigate various factors associated with thrombosis, usually starting from solutions of the Navier-Stokes equations. This thesis presents the development and implementation of models of thrombosis using the lattice Boltzmann method, which is a relatively new technique for simulating fluid dynamics. The advantages and disadvantages of this methodology are critically reviewed and two major pathologies, atherosclerosis and deep vein thrombosis have been chosen to demonstrate principles of the application. The first part of the work concentrates on the simulation of flow and clotting in idealised stenotic occlusions representative of the geometry and flow conditions in a diseased human femoral artery. Simulations of unsteady flow are reported and comparisons are made to previous flow visualisation studies. Stability issues regarding the diffusion algorithm are investigated in detail. In the first instance, clotting is simulated with the use of an aging model with extensions including proximity and shear stress. Comparisons are made with experimental results obtained using milk as a blood analogue. )'he second part of the work focuses on increasing the complexity of the models to incorporate the representations of the actions and distribution of the platelets, proteins and enzymes involved in the coagulation cascade. The models are tested in 2D geometries to demonstrate their functionality. As an example of this work, a model of deep vein thrombosis was developed, based on a hypothesis supported by the clinical literature. The foundations laid in this project allow for future developments, which will incorporate further details of thrombotic processes, in the hope that a valuable predictor of thrombosis can be developed.

NADPH oxidases are major sources of reactive oxygen species (ROS) involved in the pathophysiology of many cardiovascular diseases, acting through the modulation of redox-sensitive signalling pathways as well as the inactivation of nitric oxide and generation of endothelial dysfunction. There are five oxidase isoforms (Nox1 - Nox5), which have different tissue distributions, are subject to stimulus-specific activation and may exert distinct downstream effects. Endothelial cells co-express two of these isoforms, the classical Nox isoform, Nox2, and a novel isoform, Nox4. Recent studies indicate that Nox4 generates predominantly hydrogen peroxide (H2O2) but its role in vivo remains unclear. To address this question, transgenic mice with endothelial-specific overexpression of Nox4 were studied as an approach to investigate the in vivo functional significance of Nox4 in the endothelium. Unexpectedly, Nox4 overexpression was found to significantly enhance acetylcholine-induced vasodilatation compared to wild-type littermates. This resulted from increased H2O2 production and not from altered nitric oxide bioavailability. Transgenic mice also had lower systemic blood pressure compared to wild-type littermates which was normalised by three structurally separate antioxidants. Endothelial Nox4 exerts potentially beneficial effects on vasodilator function and blood pressure that are attributable to H2O2 generation. These effects are quite distinct to those reported for Nox1 and Nox2 which involve superoxide-mediated inactivation of nitric oxide. These results suggest that therapeutic strategies to modulate ROS production in vascular disease may need to separately target individual Nox isoforms.