Elevation of intracellular Ca2+ ([Ca2+]i) is essential for platelet function. Despite the established role of shear stress in haemostasis and thrombosis, the possible contribution of mechanosensitive (MS) Ca2+-permeable ion channels to platelet activation remains unknown. One well-established Ca2+-permeable ion channel which enhances platelet responses at high shear is the ATP-gated P2X1 channel. The relative contribution of this channel to platelet function was studied following the activation of the FcγRIIa immune receptor. qRT-PCR and Western blotting revealed that human platelets and a megakaryocytic cell line, Meg-01, express the MS cation channel Piezo1. To investigate Ca2+-permeable MS channel activity, single platelets and Meg-01 cells were loaded with the Ca2+ indicator Fluo-3, and exposed to arterial shear in flow chambers which induced increases in [Ca2+]i in both cell types in physiological salines. The MS channel blocker GsMTx-4 inhibited these responses and reduced thrombus formation over collagen, whereas Piezo1 channel agonist Yoda1 potentiated platelet shear-induced Ca2+ transients. In Fura-2-loaded platelet suspensions, the GsMTx-4-sensitive shear-evoked responses were shown to be independent of P2X1, Orai1 and TRPC6. FcγRIIa-mediated [Ca2+]i elevations and aggregation were monitored using ratiometric [Ca2+]i measurements and light transmission, respectively. The contribution of P2X1 channels was assessed following inhibition by NF449, and inactivation by pre-addition of α,β-meATP or apyrase exclusion. These treatments significantly reduced antibody- or bacteria-induced FcγRIIa-mediated responses, indicating a significant P2X1 channel contribution. Phosphorylation assays indicated that P2X1 amplifies FcγRIIa-mediated responses via direct Ca2+ influx, rather than via a feedforward effect on early tyrosine phosphorylation. In conclusion, this thesis provides evidence that platelets express functional MS Piezo1 channels which can provide a direct route for Ca2+ entry under normal and pathological arterial shear, and contribute to thrombus formation. Additionally, P2X1 channels were shown to amplify FcγRIIa-mediated platelet Ca2+ signalling and aggregation, which can contribute to platelet activation under shear in infective endocarditis.

Ammonia is produced in the body during the metabolism of amino acids. In the liver, it is converted to urea via the urea cycle and excreted by the kidneys as urine. Normal levels are between 11 to 50 µM, whereas a blood ammonia level of approximately 100 µM indicates pathology. Elevated blood ammonia is associated with a number of pathological conditions including liver and kidney dysfunction. Conditions such as these can affect brain function and can be fatal. Current blood ammonia analysis requires a laboratory blood test. Few, if any of the techniques used are suitable for point of care (POC) testing. The development of a reliable and simple method for blood ammonia determination is essential for clinical diagnosis and management of patient progress in order to prevent further debilitating illnesses developing, and extending life. This is particularly critical in many disorders such as hyperammonaemia of the newborn, inborn errors of metabolism including urea cycle defects, organic acidaemias, hyperinsulinism/hyperammonaemia, liver disease and other cause of hyperammonaemic encephalopathy. This thesis investigates the development of an electrochemical sensor for the measurement of ammonia in blood. Polyaniline has a known affinity for ammonia which operates on the deprotonation of the polyaniline backbone forming an ammonium ion. In this work, polyaniline nanoparticles were fabricated and inkjet-printed onto silver screen printed electrodes. The sensors were then incorporated into devices containing a gas-permeable membrane, which facilitated the measurement of gaseous ammonia from a liquid sample (blood) using electrochemical impedance spectroscopy. The combination of impedance spectroscopy with a gas-permeable membrane allowed the measurement of gaseous ammonia from solution. The ammonia device developed possessed refinements to enhance its sensitivity and included careful optimisation of other aspects of the measurement. For example, an air purge through the device gas chamber was employed to remove matrix interferences from the sensor and improve the specificity to ammonia. The pH of the sample to be analysed was modified in order to increase the mass of ammonia in solution, thus lowering the limit of detection (LOD) of the device. Finally, assay timings were optimised in order to increase the impedimetric response of ammonia. These optimisations resulted in the effective detection of ammonia in a liquid sample down to the lowest clinically relevant levels found in blood. The devices displayed an impedimetric baseline intra- and inter-variability of 25 and 6.9%, respectively for n = 15 over a period of 160 s. A calculated limit of LOD of 12 µM was achieved for human serum measurements. A coefficient of determination of 0.9984, slope of 0.0046 and an intercept of 1.1534 was obtained in human serum across the linear range of 25 to 200 μM ammonia (n = 3). The device was validated against a commercial spectrophotometric assay which resulted in excellent correlation (0.9699, p < 0.0001) with a slope of 1.4472 and an intercept of 0.5631 between both methods (n = 3). The devices could be stored in desiccant for up to five months and displayed minimal variation (0.64%) over time (n = 12).

Neutrophils have been implicated in the pathogenesis of many human diseases, including the adult respiratory distress syndrome (ARDS), pulmonary fibrosis and ischaemia-reperfusion injury. Priming of neutrophils by pre-exposure to bacterial products and inflammatory mediators represents a potent means of augmenting both the bactericidal capacity and injurious potential of these cells; in particular, the generation of reactive oxygen species such as the superoxide anion (O2-) may be enhanced by up to twenty-fold. Greater understanding of the signalling mechanisms underlying neutrophil priming could lead to novel therapeutic strategies aimed at preventing neutrophil-mediated tissue injury in these conditions. Exposure of neutrophils to the priming agents lipopolysaccharide (LPS), tumour necrosis factor-α (TNFα) and platelet-activating factor (PAF) was shown to modulate adhesion molecule expression and function (in particular inducing upregulation of β₂ integrins and shedding of L-selectin) in an agonist-specific fashion, with a time-course which correlated with that required to establish the primed state. Neutrophil chemoattractant receptors are coupled to intracellular signalling pathways by heterotrimeric G-proteins, principally Giα2. I have shown that TNFα, LPS and PAF increase the level of Giα2 expression detectable in neutrophil membranes, the time-course for this effect mirroring that for priming. However, the degree of G-protein upregulation is considerably less than the enhancement of the respiratory burst, suggesting that downstream signalling events play a more critical mechanistic role in priming. Two major signalling pathways implicated in the activation of the neutrophil respiratory burst were studied; the cleavage of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) by phospholipase C to yield inositol 1,4,5-trisphosphate (Ins(l,4,5)P3), and its phosphorylation by phosphatidylinositol 3-hydroxykinase (PI3K) to phosphatidylinositol 3,4,5- trisphosphate (PtdIns(3,4,5)P3). Accumulation of neutrophil Ins(l,4,5)P3 (the signal for intracellular calcium release) in response to the secretagogue N-formyl-methionyl-leucyl-phenylalanine (fMLP) was identical in unprimed and primed neutrophils, suggesting that modulation of phospholipase C activity is not involved in signalling neutrophil priming. A small but significant enhancement of fMLP-stimulated PtdIns(3,4,5)P3 accumulation (about 25%) was seen at 10 s in TNFα-primed versus unprimed cells, but 60 s following fMLP stimulation this enhancement was 620%. This suggests that enhanced and sustained PtdIns(3,4,5)P3 generation may be important in signalling mechanisms leading to the primed respiratory burst.

Adenosine receptors are members of the G protein coupled receptor (GPCR) superfamily. Neurochemical and electrophysiological experiments point to a neuromodulatory role for adenosine. Adenosine inhibits the release of several neurotransmitters in brain by pre- and postsynaptic actions at adenosine A1 receptors. In this thesis the pharmacology and modulation of adenosine A1 receptors in the central nervous system (CNS) of different species was characterised. Rat renal adenosine A1 receptors were also studied. In addition the central penetration by adenosine A1 receptor antagonists following peripheral administration was investigated. An in vitro [3H]DPCPX ligand binding assay utilising a range of receptor agonists and antagonists, were used to compare the pharmacology of rat, mouse, guinea pig and human adenosine A1 receptors. Standard xanthine-based adenosine antagonists had 10-fold lower affinity in human and guinea pig in comparison with rat and mouse. Several novel nonxanthine A1 antagonists from the Fujisawa Pharmaceutical Co. Ltd., retained high affinity for adenosine A1 receptors in all species, including human. Modulation of ligand binding to adenosine A1 and A2a receptors by Gpp(NH)p and magnesium was examined. In common with other GPCRs, agonists bind to high and low affinity states, with the equilibrium modified by GTP analogues and magnesium, whereas effects on antagonist affinity are minimal. Agonist radioligands under the conditions used labelled predominantly the high affinity state of adenosine A1 and A2a receptors. Gpp(NH)p and magnesium have essentially inverse effects on radioligand (agonists and antagonists) binding to both adenosine A1 and A2a receptors. In addition their effects on [3H]agonist and [3H]antagonist binding are generally opposite, which is consistent with effects observed for other GPCRs. To act centrally, compounds must cross the blood brain barrier (BBB). A modified radioreceptor assay, involving a denaturation step to overcome the lipophilic nature of adenosine A1 receptor antagonists was developed. This assay accurately measures the central penetration of adenosine A1 receptor antagonists. Adenosine A1 antagonists administered peripherally at equipotent behavioural doses, were found to be present in brain, at concentrations between 50-500 fold greater than in vitro Ki values for adenosine A1 receptors. In addition, there was an excellent association between antagonist affinity in vitro and antagonist brain concentrations at the equipotent behavioural dose. Adenosine and its receptors are widely distributed throughout the body. It is important to determine if peripheral adenosine A1 receptors are identical to those in the CNS. Renal adenosine A1 receptors were examined using radioligand binding, in vitro autoradiography and in situ hybridisation. Even using this combined approach, identification of adenosine A1 receptors in rat kidney was not possible. Alzheimer's disease involves a myriad of neurochemical changes and neurotransmitter deficits. By antagonising the actions of endogenous adenosine, A1 receptor antagonists could compensate for some of these deficits, by enhancing synaptic facilitation and increasing neurotransmitter release. Analogues of FK453, a non-xanthine adenosine A1 antagonist, which are BBB permeable and potent and selective for human adenosine A1 receptors, could prove to be clinically useful compounds, by enhancing cognition, in conditions such as Alzheimer's disease.

No description available.

No description available.

Thermal dilution as a method for measuring peripheral blood flow has been the basis of at least two major pieces of work in the past, (Clark 1966, Richards 1970). It is questionable whether anybody to date has been able to make a thermal dilution probe which, when inserted into a blood vessel of a living subject, could measure with accuracy the blood flow rate minute by minute over a long period. The possible reasons for the failures in vivo are examined in this thesis. The presence of blood clot on and around the probes is shown to be a major problem caused by the use of a centralising structure on the probe tip. Methods for making the centralising cage less thrombogenic are examined in the light of an enquiry into blood clotting mechanisms. Available techniques for making plastic surfaces less thrombogenic are reviewed, and their suitability for use on the thermal dilution probe examined. An anti-thrombogenic catheter is developed and tested in vivo using radioactive labelling techniques to monitor clot formation. The outcome of subsequent tests result in the abandonment of the centralising cage technique, the ramifications of which have necessitated the redesign of the flow probe and restatement of the problem of blood flow measurement by thermal dilution. A new method (based on that of Fronek and Ganz 1960), is investigated using a single hole for injecting saline. The effects of the injection on flow and pressure are considered both in vivo and in vitro, for various angles of injection, rates of injection (i. e. various kinetic energies of jet), flow rates and vessel diameters. The effect of the distance between the injection site and the measuring site on the accuracy of the method, is investigated. A venous velocity trace and an arterial velocity trace are examined using a mathematical model in order to demonstrate the unpredictability of "distance-distortion". A conflict of interests arises between the need to reduce changes in flow during injection to a minimum, whilst obtaining a suitably homogeneous mixture for measurement to be made in a short enough distance to reduce distance distortion to an acceptable level.

This investigation was concerned with extracorporeal oxygenators used to support the pulmonary function of patients undergoing open heart surgery. Of the two problems investigated, the first was a biological study of blood damage. For many years the advantages of membrane oxygenators as against direct contact oxygenators have been postulated. A review of past work shows that many of the physical and chemical factors known to cause changes in the blood are not properly understood. A study of red blood cells using the scanning electron microscope was conducted to obtain greater understanding of the changes that occur during clinical bypass. This investigation was carried out with blood taken during open heart surgery from extracorporeal circuits which losed different types of oxygenator. The results demonstrated changes in red blood cell shape and size under different conditions and in particular, they were found to undergo the echinocyte transformation. The second part of the investigation was aimed at providing an analytical description of the mechanics of mass transfer in a flat plate membrane oxygenator. In order to highlight the general characteristics of such a device, the analysis includes consideration of non-reactive fluids (e.g. water, saline) as well as reactive fluids (e.g. blood). Data obtained from a series of tests on an experimental oxygenator agreed well with the analytic solution derived for both blood and water. This solution illustrated the importance of various parameters and non-dimensional groups in die process of gas transfer in an oxygenator. In particular, the transfer rate was found to be less sensitive to variations in membrane thickness than was at first suspected. Furthermore, it was discovered that water may be used as a satisfactory substitute for blood in order to predict and test the performance of membrane oxygenators.

The lymphatic system returns fluid to the blood stream from the tissues to maintain tissue fluid homeostasis. The collecting lymphatic vessels actively pump fluid against a body scale pressure gradient, i.e., from tissue interstitial space to the venous side of the blood circulatory system. The collecting lymphatic vessels pass the lymphatic fluid to lymph nodes that filter the lymph before it is returned to the circulatory system. This thesis presents work undertaken to create a fluid structure interaction model of a lymph node with afferent and efferent lymphatic vessels. The model is built in COMSOL Multiphysics, a commercial finite element software. Four pieces of novel work are presented in this thesis. Firstly, an optimisation method used to approximate the material properties for the collecting lymphatic vessel from the pressure diameter behaviour. Secondly, model of the collecting lymphatic valve with surrounding wall used to investigate valve closing behaviour. Thirdly, an image based model of a lymph node where the material properties are optimised to experimental data and based on selective plane illumination microscopy images. Finally, an image based model of a lymph node based on computed tomography images that shows how the structure within the node affects the fluid flow pathways.

The aim of this thesis is to investigate the ability of cardiovascular biomarkers calculated from peripheral pulse waveforms to estimate central properties of the cardiovascular system (e.g. aortic stiffness) using nonlinear one-dimensional (1-D) modelling of pulse wave propagation in the arterial network. To test these biomarkers, I have produced novel 1-D models of pulse wave propagation under normal and pathological conditions. In the first part of my thesis, I extended the modelling capabilities of the existing 1-D/0-D code to represent arterial blood ow under diabetes, hypertension, and combined diabetes and hypertension. Cardiac and vascular parameters of the 1-D model were tailored to best match data available in the literature to produce generalised hypertensive, diabetic, and combined diabetic and hypertensive population models. Using these models, I have shown that the pulse waveform at the finger is strongly affected by the aortic flow wave and the muscular artery stiffiness and diameter. Furthermore the peak to peak time measured from the pulse waveform at the finger can identify hypertensive from diabetic patients. In the second part, I developed a new methodology for optimising the number of arterial segments in 1-D modelling required to simulate precisely the blood pressure and flow waveforms at an arbitrary arterial location. This is achieved by systematically lumping peripheral 1-D model branches into 0-D models that preserve the net resistance and total compliance of the original model. The methodology is important to simplify the computational domain while maintaining the precision of the numerical predictions { an important step to translate 1-D modelling to the clinic. This thesis provides novel computational tools of blood flow modelling and waveform analysis for the design, development and testing of pulse wave biomarkers. These tools may help bridge the gap between clinical and computational approaches.