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Current clinical testing of autoregulatory failure involves the patient undergoing a head-up tilt test. This is an experiment to investigate the sequence of physiological events that lead to a syncopal episode by tilting the patient from supine to 70% for up to an hour, or until the patient loses consciousness. This thesis argues that this test is no longer a suitable way of investigating these types of failures. An experiment was carried out to compare the effects of the tilt-test with a battery of other tests that have been used in the assessment of cerebrovascular autoregulation, namely carotid sinus massage, the transient hyperaemic response test, the Valsalva manoeuvre, and lower body negative pressure (LBNP). The first three tests were short, dynamic tests of autoregulation. The final test, and the tilt test, are longer, static tests. It was shown that lower body negative pressure produced more consistent results compared to the tilt-test, principally due to the stimulation of fewer systemic parameters by the test. The thesis also presents an experiment demonstrating for the first time measurement of blood flow to and from the brain during mild LBNP, using quantitative flow analysis conducted using magnetic resonance imaging. The results show a drop in cardiac output, which is reflected in a fall in blood flow in the carotid and vertebral arteries. The near-infrared spectroscopic data suggest that whist there was a drop in cerebral blood volume, sufficient perfusion was maintained through the test. This work provides a basis for future experiments, and the possibility of developing a more specific test to measure changes in blood concentrations locally in the cortex during LBNP.

Myosin light chain₂₀ (MLC₂₀) phosphorylation is thought to be responsible for the initiation of platelet shape change. The aim of this study was to investigate the regulation of MLC₂₀ phosphorylation in platelet shape change. Previous studies have suggested that MLC₂₀ phosphorylation following platelet stimulation is dependent on intracellular calcium release. This study was designed to investigate the role of calcium sensitization in human platelet shape change and the possible intracellular mechanisms involved. An α-toxin permeabilized platelet model was developed using mock intracellular buffers to enable complete control of the intracellular environment. Permeabilized platelets were examined to ensure functional integrity by electron microscopy and were found to be capable of producing platelet shape change. Permeabilized platelets were stimulated by under calcium-clamp conditions and the degree of phosphorylation was determined following separation by two-dimensional gel electrophoresis. Calcium sensitization of platelet shape change following stimulation with both thrombin and GTPγS was produced and this corresponded with an increase in MLC₂₀ phosphorylation. A relatively slow time course was observed and this may represent a second phase of shape change as a result of secreted mediators stimulating further activation. The signalling pathway leading to calcium sensitization was investigated for the possible involvement of RhoA and Rho-kinase activation. Thrombin and GTPγS were shown to directly activate RhoA. The involvement of Rho-kinase was demonstrated using a specific inhibitor Y27632, which inhibited calcium sensitization produced as a result of thrombin and GTPγS stimulation. The calcium sensitivity of MLC₂₀ phosphorylation in platelet shape change is increased by thrombin stimulation and this is likely to involve RhoA and Rho-kinase activation. This is not calcium-independent regulation but does have a requirement for submaximal intracellular calcium concentration to produce an increase in MLC₂₀phosphorylation. This alternative pathway which is not dependent on intracellular calcium release may provide an alternative target in the treatment of disease states associated with haemostasis.

In an attempt to define the role of intravascular coagulation in the pathogenesis of pre-eclampsia, a prospective series of normal primigravidae was studied through pregnancy and the puerperium. A battery of tests of coagulation and fibrinolysis was applied to maternal and cord venous blood, normal pregnancy being oompared with pregnancy preceding pre-eclampsia. A group of established proteinuric pre-eclamptics was also studied. The results failed to implicate typical major D.I.C. even in established pre-eclampsia, although a subtle low grade D.I.C. may be active in the disease. Evidence has been presented of intravascular coagulation localised to the kidneys in the established condition, and the significance of the changes found in this study are discussed in relation to the syndrome of pre-eclampsia.