It has been recognised for many years that phospholipids are essential for the coagulation of plasma, and the evidence which has been obtained so far suggests that they are involved in the formation in the plasma of a substance which will convert prothrombin. The activity of the various phospholipids has been assessed by their ability either to shorten the clotting of recalcified plasma, or to stimulate the conversion of prothrombin. Using this technique the most active phospholipid has been found to be phosphatidyl ethanolamine, but apart from an investigation of the physical and chemical properties of this phosphatide, no information has been gained as to its mode of action. Since plasma already contains phospholipids it was thought worthwhile to investigate whether any reaction took place during the coagulation of plasma, in which they were involved. For this investigation the plasma was clotted either in the presence of platelets, or Russell's viper venom. It was found that when the plasma clotted it was accompanied by the release of a small amount of glycerylphosphorylcholine. This substance was formed however the plasma was made to clot. The results which have been obtained suggest that this compound was formed by the degradation of between 2-3% of the plasma lecithin. Glycerylphosphorylcholine is formed from lecithin by the action of two enzymes, namely, a lecithinase and a lysolecithinase. It has been shown that while Russell's viper venom has lecithinase activity, it is unable to hydrolyse lysolecithin. It was found that plasma had a latent lysolecithinase activity, which could be activated by exposing the plasma to a roughened glass surface. In addition the activated lysolecithinase enzyme would in the presence of a tripalmitin emulsion hydrolyse lecithin to give glycerylphosphorylcholine. It is suggested that the probable role of phosphatidyl ethanolamine is to activate the plasma lecithin so that it can be hydrolysed by the plasma enzyme. The results which have been obtained suggest that the degradation of the lecithin was associated with the appearance in the plasma of a substance which will convert prothrombin, and the conclusion has been reached that the development of both lecithinase and lysolecithinase activity is a necessary prerequisite for the coagulation of the plasma. Born (1958) showed that during the coagulation of platelet-rich plasma there was a loss of adenosine triphosphate from the platelets. Experiments have been carried out to determine whether this loss of adenosine triphosphate was concerned in the clotting of the plasma It has been concluded that the release of adenosine triphosphate from the platelets was only incidental to the clotting of the plasma.

The thesis reports an investigation of the mechanisms whereby the femoral artery supplying blood to a temporarily vasodilated skeletal muscle bed also dilates. Arterial diameter was continuously measured by an ultrasonic transit time technique, calibrated against distance by separate determination of the velocity of ultrasound in blood. An instrument capable of sampling up to four distances at repetition frequencies above 1 kHz was developed, and new methods of transmitter pulse triggering and received pulse amplification and detection are described. Design of optimal transducers for arterial, cardiac, venous and transcutaneous use is discussed in terms of testing by beam visualisation and other methods. The system runs for several hours without temperature drift and will resolve distance changes smaller than one micron. Trials by measurement of dynamic distension of a long water-filled rubber tube and comparison with manometrically measured phase velocity showed that the technique yields results within the range of predicted values from the various theories of pulsatile flow in rubber tubes. Blood pressure and flow and vessel diameter were measured from the exposed femoral arteries of anaesthetised greyhounds. Transient hyperaemia was induced by distal injection of vasodilator substances, nerve stimulation or occlusion of the femoral artery, and sustained high flowrate by establishing an arteriovenous shunt. Dilatation of up to 3% was observed in less than half of the experiments. The effects on the response of altering blood pressure, arterial constriction, and various degrees of arterial exposure and handling were observed. The data on exposure illuminate the controversy over the applicability to unexposed vessels of data obtained invasively. Locally originating response mechanisms were tested by perfusion of excised arteries at varying flowrates, pressures and pharmacologically induced tonic constriction levels. Conduction of constriction and relaxation was tested in vivo and in vitro by procedures to show the distant effects of locally applied pharmacological agents. Pressure and distension data were digitised and analysed by computer to determine wave velocities, elastic moduli and the importance of elastic non-linearity. Methods of finding phase, group and foot velocities were compared. The femoral dilatation response occurred after distal arterial section and when an arteriovenous shunt was established. Therefore the mechanism is not thought to involve conduction of dilatation commands from peripheral vessels. Temperature changes caused an opposite effect to high flow dilatation: the artery dilated slightly on cooling during occlusion and vice versa. The dilatation response is considered to depend on local sensing of increased arterial blood flowrate. Two such possible mechanisms are discussed.

Whether nitric oxide is implicated in the contractile function of isolated cardiac ventricular myocytes forms the major part of the work of this thesis. Contracting guinea-pig cardiac myocytes were studied in isolation <I>in vitro</I> using a videomicroscopy length detection system. Studies are presented which establish that nitric oxide attenuates contractility of cardiac ventricular myocytes, both when it is derived from exogenous sources, and when nitric oxide is released from adjacent endothelium in coculture with cardiac myocytes. The coronary microcirculation is in close proximity to cardiac myocytes within the myocardium, thus endothelium-derived nitric oxide may have an important tonic effect on myocardial contractility. This may be particularly important when the diffusing distance from endothelial cell to myocyte is altered in disease states. Myocardial contractility is impaired in endotoxic shock. The hypothesis that this is caused by production of nitric oxide within cardiac myocytes is examined. A model of endotoxic shock was developed. Contractility of cardiac myocytes was substantially impaired. Much of this impairment was caused by nitric oxide production within the cardiac myocytes themselves. Inhibition of nitric oxide synthesis in these cells restored contractility towards normal. Healthy myocytes did not produce effective amounts of nitric oxide. Induction of nitric oxide synthase activity within cardiac myocytes may account for much of the depressed contractility of endotoxic heart failure. Myocardial contractility is impaired following ischaemia-reperfusion. Experiments examining myocyte behaviour in this situation are discussed, but whether activation of nitric oxide synthase contributes to the impaired contractility of myocytes following ischaemia is not established.

No description available.

No description available.

Background: HDLs are heterogeneous particles, and apoA-I is the major apolipoprotein in human HDL. CVD is a multifactorial condition, and a various lipids and lipoproteins in the plasma are involved in the development of CVD. It has been suggested that HDLs have an inverse association with the risk of CVD. A high sugar intake (especially fructose and sucrose) was found to associate with a low HDL-C level and increased risk of CVD. However, the effect of dietary sugar on HDL kinetics is unclear. An insight of HDL subclass kinetics may provide a better understanding of the whole dynamic of HDL metabolism. Methods: Two studies were undertaken: 1) A controlled, randomized crossover dietary intervention study was carried out in 6 overweight middle-aged men. Subjects underwent two 12-week dietary interventions with high and low non-milk extrinsic sugar diets. Total HDL kinetics was measured using a primed constant intravenous infusion of [1-13C] leucine for 10 hours. 2) A HDL subclass kinetic study was carried out in 6 healthy subjects (3 males and 3 females). An intravenous bolus injection of [1-13C] leucine was applied to measure HDL subclass kinetics. Blood samples were taken during a 10-hour study and the following 2 weeks. Total HDL, HDL2 and HDL3 were separated from the plasma by ultracentrifugation, and αHDL and preβHDL were isolated by agarose gel electrophoresis. ApoA-I in HDL fractions was separated by SDS-PAGE. After purification, hydrolysis and derivatization, the isotopic enrichment of apoA-I in HDL was measured by GC-MS and apoA-I fractional catabolic rate (FCR) and production rate (PR) was calculated for each subclass and total HDL. Results: In the dietary intervention study, the FCR of total HDL apoA-I on the high and low sugar diet (0.20 ± 0.02 and 0.18 ± 0.02 pools/day) was similar, as was the PR (7.33 ± 0.66 and 6.05 ± 0.72 mg/kg/day respectively). In the HDL subclass study, the concentration of αHDL apoA-I (0.97 ± 0.05 g/L) was significantly higher than that of preβHDL apoA-I (0.15 ± 0.03 g/L) (p<0.001). The FCR of αHDL and preβHDL apoA-I was 0.10 ± 0.02 and 0.13 ± 0.04 pools/day, and the PR of αHDL and preβHDL apoA-I was 3.94 ± 0.73 and 0.67 ± 0.12 mg/kg/day respectively. The concentration of HDL3 apoA-I (0.68 ± 0.04 g/L) was significantly higher than that of HDL2 apoA-I (0.23 ± 0.06 g/L) (p=0.002). The FCR of HDL2 and HDL3 apoA-I was 0.15 ± 0.02 pools/day for both, and PR of HDL2 and HDL3 apoA-I was 1.35 ± 0.35 2 and 3.81 ± 0.51 mg/kg/day respectively. A significant difference was observed between αHDL and preβHDL apoA-I PR (p=0.010), and between HDL2 and HDL3 apoA-I PR (p=0.030) in the whole group. The concentration of HDL2 apoA-I was higher in women (0.32 ± 0.08 g/L) than men (0.13 ± 0.02 g/L) though the difference was not significant. HDL2 apoA-I PR was significantly higher in women than men (p=0.017). Conclusion: The high and low sugar diet did not affect HDL metabolism in overweight men. The higher apoA-I concentration of αHDL and HDL3 might be due to the higher apoA-I PR of αHDL and HDL3 compared to preβHDL and HDL2 respectively in healthy subjects. The higher level of HDL2 apoA-I in female than male subjects might be due to the higher PR of HDL2 apoA-I in women.

No description available.

Background: Hypoplastic left heart syndrome (HLHS) describes a spectrum of underdevelopment of the left heart, rendering it incapable of supporting the systemic circulation. Improved results from Norwood palliation mean more children are surviving into later childhood. The assessment of right ventricular (RV) function is an important prognostic factor, but is complicated by wide heterogeneity and complex geometry. Novel MRI and echocardiographic techniques are non-invasive and may offer insight into the pathophysiology of the systemic RV. Methods: Current methods for assessing the RV were reviewed. MRI and echocardiography were used and compared prospectively in HLHS patients to investigate RV performance and changes in ventricular volumetry across the palliative stages. The novel approach of pre-Fontan assessment using MRI and central venous pressure (CVP) measurement alone was compared to the current literature. Results: Echocardiographic subjective assessment of RV function in HLHS had little concordance with MRI ejection fraction, showing the limitation of using this method alone. MRI demonstrated significant RV volume unloading after hemi- Fontan, with a shift of the Starling curve suggesting improved contractility. The novel pre-Fontan assessment showed no difference in outcomes from the published literature. Tissue Doppler time intervals were significantly different in HLHS patients compared to normal hearts. Differences were also seen in tissue Doppler indices and speckle tracking derived strain between those with a significant residual left ventricle and those without Conclusions: Novel MRI and echocardiographic techniques give unique and reproducible insights into the morphologic and functional development of the systemic RV across the stages of surgical palliation. Important differences between the morphological subtypes were also noted. Based on this MD thesis, reliable, easy to use, reproducible and non-invasive screening tools have been established, validated and used for longitudinal follow-up. These techniques may also lead to improved follow-up: predicting, or possibly preventing systemic RV failure.

Inflammation is a response of the immune and vascular systems to the stimuli perceived as harmful to the host. Inflammation can be acute or chronic, and the recruitment of leukocytes to the affected tissues is a fundamental process during the course of both these events. Leukocytes are recruited in a process known as the multi-step adhesion cascade, which is a highly co-ordinated series of adhesive events mediated by the cell adhesion molecules (CAMs) on both leukocytes and endothelial cells. L-selectin is a CAM involved in the initial stages of the cascade, i.e. tethering and rolling, although there is a mounting body of evidence that points towards its role at later stages of the cascade, such as chemotaxis beyond transendothelial migration (TEM). The work outlined in this thesis explores the possibility that L-selectin actively contributes to TEM in monocytes. There are two important and measurable properties of L-selectin: (i) its rapid proteolysis (or “shedding”) upon cell activation, and (ii) its transition from being monomeric in the plasma membrane to being clustered, following ligand binding, which is a hallmark of downstream signalling. By expressing C-terminally green fluorescent protein (GFP)-tagged L-selectin in THP-1 monocytes, it was possible for the first time to monitor and analyse the spatio-temporal distribution of L-selectin and its shedding during TEM. In addition, co-expressing L-selectin-GFP with L-selectin tagged to red fluorescent protein (RFP) enabled measurement of L-selectin clustering during TEM via the use of fluorescence lifetime imaging microscopy (FLIM) to monitor Forster resonance energy transfer (FRET) between the GFP and RFP-tags. Interestingly, the majority of L-selectin was found to be clustered exclusively in pseudopods protruding beneath the endothelial monolayer, where L-selectin ligands are known to exist. L-selectin clustering was also found to occur following cross-linking of either CD43 or PECAM-1, suggesting inside-out signalling is an important factor in modulating L-selectin function. Moreover, both the extracellular cleavage domain and two cytoplasmic tail serine residues were involved in fundamentally regulating L-selectin clustering. Finally, the sub-cellular distribution of L-selectin clustering correlated tightly with the dynamics of the pseudopods that protruded beneath the endothelial monolayer during TEM. This thesis aims to understand the contribution that L-selectin has during the later stages of the adhesion cascade, and will re-shape the currently held perspective that this cell adhesion molecule’s role is solely restricted to just tethering and rolling.

Cardiovascular disease remains the primary cause of death worldwide. Medical imaging plays a critical and increasing role in both its diagnosis and characterisation. Recent advances in hyperpolarised 13C Magnetic Resonance (MR) allow, for example, imaging an injected molecule and its downstream metabolites to uncover biochemical changes in the myocardium. On the other hand, radionuclide imaging using Positron Emission Tomography (PET) and Single Photon Emission Computed Tomography (SPECT) has significantly higher sensitivity than MR, but the signals from injected tracers and their metabolites are indistinguishable. All these imaging modalities are able to produce dynamic data containing information of the kinetics of their respective tracers. In order to relate the measured signal or activity to the underlying physiological or biochemical processes mathematical models have to be used. In this thesis, experimental protocols have been developed and semi-quantitative and quantitative methods have been evaluated for the analysis of hyperpolarised 13C dynamic time-series and time-activity curves of PET tracers acquired ex vivo from Langendorff perfused rat hearts. A number of compartmental models were explored to fit in vitro and ex vivo hyperpolarised 13C time-series acquired for pyruvate and its downstream metabolites to derive apparent rates of the enzymatic reactions involved in pyruvate metabolic pathways. Compartmental modelling was also used in combination with Monte Carlo simulations to explore the detection limits of transmural cardiac ischemia in vivo in small rodents using hyperpolarised 13C spectroscopic imaging. The feasibility of using a model free maximum entropy/nonlinear least square method (MEM/NLS) for the kinetic analysis of hyperpolarised 13C dynamic data was explored in this thesis for the first time and validated using Monte Carlo simulations and experimental hyperpolarised 13C in vitro time-series. Finally, the feasibility of extending the analysis methods validated for in vivo PET experiments (spectral-based algorithm, Patlak graphical plot and the semi-quantitative index RATIO) to the kinetic analysis of time-activity curves of PET tracers acquired ex vivo was also assessed in this thesis.