This thesis is focussed on the shape analysis and tracking of neutrophil cells to facilitate the understanding of their behaviour. Neutrophil, one of the important type of white blood cell, protects humans and animals from infections and inflammations. The underlying mechanism is believed to be that when inflammation happens, a changing chemotaxis field causes neutrophils to move to inflammatory sites. During this migration process, neutrophils also change shapes. For example, pseudopod protrusions are formed on the boundary in response to the local gradient of the chemotactic field. After inflammation resolution, some neutrophils go to "sleep" and some move back but what drives these mechanisms are still not fully understood. If the mechanisms were known, it would be helpful to accelerate or slow down the process of treating some diseases. The thesis attempts to provide a quantitative analysis based on time lapse microscopic images of in vivo zebrafish animal models. The underlying premise governing the analysis is to identify if cell states from their motion and shape. The thesis begins with cell centroid tracking and uses three common kinematic models commonly used in the target tracking literature. The interacting multiple model framework, which is underpinned by multiple Kalman filters, is used to determine probabilities of most likely model to explain the cell motility pattern. These different models are then compared to identify if the motion pattern (motility) can be attributed to different cell behaviours. This is then followed by cell shape tracking to characterise not only the cell shape but also to identify regions of protrusions. It addresses the problem of estimating the chemotactic field that acts to recruit neutrophil cells to the inflammation sites based only on observed cell tracks, without any direct measurement associated with the external cell environment. By assuming that the cell velocity is proportional to the local chemotactic gradient, a least squares method in combination with the Kalman filter, was used to estimate this field. Results on a set of real data show the estimated field. Cell shapes were modelled as B-spline parametric active contours. By casting the parametric active contour model in state space form, Kalman filter was employed to track the shapes. Shape tracking required solving the problem of cell boundary association between two time frames which required identification of correspondence points at cell boundaries. This required improvement to a nearest neighbour filter method to give continuity of the cell shapes of the same neutrophils across the different frames. Characterisation of cell shape was carried out by employing Fourier descriptors from which two features, magnitude of the highest descriptor and the magnitude of the lowest frequency, were chosen as proxy for associating cell shapes with cell states. These features for instance are associated with cell roundedness. Both tracking methods, cell centroid and shape tracking, are employed on the real data and results shown to demonstrate the effectiveness of the methods. This thesis makes the following novel contributions. Firstly, a new framework was established to solve the cell centroid and shape tracking towards characterising cell shape behaviour. Secondly, the nearest neighbourhood filter employed to solve the association problem was improved to solve the problem of neutrophil cells disappearing and reappearing in image frames. Thirdly, the low frequency Fourier descriptor, combined with other methodologies, was successfully implemented in detecting the modes of neutrophil behaviour. In addition, the chemotactic field was estimated by using the centroid velocities. Furthermore, the multiple model filter was used for behavioural mode detection of cells.

This thesis presents novel research for real time heart sound detection, heart rate extraction and acoustic respiratory rate extraction algorithms. This was done based on signals obtained with a novel in-house developed wireless acoustic breathing and heart rate monitor. The core aim of this work is to enable additional features with respect to which physiological parameters can be measured by acoustic means with the above- mentioned sensor in the hospital bed environment. The performance evaluation was done with data collected from clinical trials carried out at Queens Square hospital - UCL Institute of Neurology - in London, UK. The respiratory rate extraction algorithm presented achieved a value difference bias of 0.07 and standard deviation of 1.55 breaths per minute with respect to the counted respiratory oscillations on the polysomnography device signals of the flow sensor as well as the abdominal and thoracic band evaluated on more than 21 hours of data from 13 different subjects during sleep. Similarly the novel heart rate extraction algorithm for processing the acoustic signals achieves a performance of 90.20% and 90.26% agreement with respect to heart rate value provided by the Konica-Minolta and SomnoMedics devices respec- tively evaluated on more than 57 hours of data acquired from 13 different subjects during sleep in the clinical trials. This is the largest dataset for acoustic heart sound classification and heart rate extraction in the literature to date. Overall, these results represent: a clear proof of concept for heart rate monitoring with the in-house developed wireless acoustic monitoring system; the addition of two very important monitoring capabilities to the wireless acoustic monitoring system; as well as significant contributions to the field of signal processing for both acoustic respiratory rate and heart rate monitoring.

Selective adhesion may be important for the preferential accumulation of eosinophils in asthma and allergic diseases. The Stamper-Woodruff frozen section assay (FSA) was used to define the adhesion of human peripheral blood eosinophils and neutrophils to endothelium in a model of chronic airway inflammation, the nasal polyp. Eosinophil and neutrophil adhesion to nasal polyp endothelium (NPE) was (32 integrin-dependent. Eosinophil adhesion was inhibited to a lesser extent by mAbs against pi integrins and VCAM-1. Adhesion of both eosinophils and neutrophils to NPE was activation-dependent. as shown by inhibition with azide. Neutrophil adhesion was mediated by PAF and IL-8 signalling through pertussis toxin (PTX)-scnsitive G-protein coupled receptors (GPCR). In contrast, eosinophil adhesion was PTX-insensitive. and the chemokine receptor CCR3 was not involved. The eosinophil activation step was further explored under shear flow conditions. Neutrophils firmly arrested on TNF-a-stimulated human umbilical vein endothelial cells (HUVEC) under flow. In contrast, eosinophils rolled unless exogenous chemoattractants such as PAF, eotaxin, and RANTES were added. Priming eosinophils with IL-5 before addition to HUVEC caused arrest of the entire population. The flow assay was also used to investigate the receptors involved in leukocyte adhesion to IL-13- stimulated HUVEC. Eosinophils but not neutrophils showed enhanced binding to IL-13- stimulated HUVEC compared to medium-cultured cells. This adhesion was mediated by P-selectin/ PSGL-1. and to a lesser extent VLA-4/ VCAM-1. These findings were consistent with the pattern of adhesion receptor expression in nasal mucosa, with weak expression of VCAM-1 compared with P-selectin. In summary, I have demonstrated a difference in integrin usage and the mechanism of integrin activation between eosinophils and neutrophils. An additional priming step in the adhesion cascade appears to be required for eosinophils, but not neutrophils, to respond to an activating stimulus and arrest on endothelium. P-selectin/ PSGL-1 interactions were pivotal to eosinophil arrest on Th2- cytokine-stimulated endothelium and are potential targets for inhibition of eosinophil migration.

The leading cause of death in Western countries is cardiovascular disease with over 1 million people dying each year as a result in the United States alone. One condition identified as a risk factor for cardiovascular disease is an increased blood pressure or "hypertension" which has been shown to result in morphological changes in blood vessels at different sites around the body, including narrowing of pre-capillary "resistance" vessels. This thesis has sought to investigate whether or not this narrowing of resistance vessels might result from the increased physical forces of hypertension exerted upon the vascular smooth muscle cells of the vessel wall and to investigate the intracellular signalling mechanisms initiating this cellular response. Results indicate that cultured human vascular smooth muscle cells undergo cellular proliferation in response to chronic cyclical mechanical strain but only in the presence of suitable concentrations of soluble growth factors. Furthermore, these growth factors do not originate from the cells in response to the mechanical strain. Therefore, the proliferation is a direct response proportional to the strain applied but dependent upon the concentration of growth factors in the overlying media. In addition the magnitude of human vascular smooth muscle cell proliferation in response to mechanical strain is dependent upon interactions between the cells and specific extracellular matrix proteins and involves activation of the mitogen-activating protein kinase intracellular signalling cascade. In conclusion, these results suggest that the narrowing of resistance vessels observed in hypertension subjects may be a direct result of the increased physical forces exerted upon the vascular smooth muscle cells in conjunction with circulating growth factors. This biological response is mediated via specific cell/matrix interactions and involves specific intracellular signalling pathways, which may provide new targets for the effective treatment and/or management of these structural alterations observed in hypertension individuals.

The aim of this project was to confirm and extend previous work on this subject by studying the expression of t-PA, u-PA, PAI-1 and uPAR in the walls of human arteries and veins as well as in rabbit iliac arteries that were normal or undergoing neointima formation. In normal human internal mammary arteries and saphenous veins, immunohistochemistry showed all four proteins to be associated with endothelial cells with little present on smooth muscle cells. Surgical distension of saphenous veins resulted in increased immunostaining to all four proteins probably due to extravasation, as specific mRNA levels were lower in the distended compared to undistended vessels. Human atheroma demonstrated intense immunostaining to all four proteins in keeping with the increased antigens on immunoassay, increased (uPA and PAI-1) mRNA on quantitative RTPCR, and greater signal (for tPA, uPA and PAI-1) with in situ hybridisation compared to normal artery. Although PAI-1 antigen was increased in atheroma, PAI-1 activity was decreased. In human saphenous vein grafts or saphenous vein in organ culture, tPA, uPA and uPAR immunostaining was increased compared to normal undistended saphenous vein in keeping with increased signal for tPA and uPA mRNA on in situ hybridisation. PAI-1 immunostaining and mRNA signal was prominent on the endothelial cells of venous hyperplastic vessels but unlike arterial atheroma, they were largely absent on the neointimal smooth muscle cells. Experimental angioplasty of rabbit iliac arteries resulted in increased immunostaining for all four proteins and an increase in uPA activity. These results are compatible with and extend previous reports implicating the plasminogen activator system in the control of cell migration and matrix remodelling during normal and pathological vessel growth and repair, but also emphasize the complexity of this process.

The use of postmenopausal oestrogen replacement (HRT) has increased more than threefold over the last two decades. Apart from its effect on menopausal symptoms and-consequently-on quality of life, many women embark on HRT because of its presumed benefits on cardiovascular health. However, some of the effects of oestrogen on the cardiovascular system remain controversial and poorly understood. The present investigation aims to examine the effect of oestrogen on aspects of the female cardiovascular system in vivo and in vitro. In the clinical arm of the study, transdermal oestrogen treatment in oophorectomised women was shown to be associated with an overall reduction in ambulatory blood pressure (ABP), whereas with oral therapy ABP remained unchanged. With both delivery systems, ABP increased significantly in a proportion of women. The reason for this is unclear, however, further investigation did not reveal a demonstrable association with molecular variants of the angiotensinogen genotype. In a pilot study looking at carotid disease in postmenopausal women, oral oestrogen treatment was associated with significant plaque regression, as assessed by ultrasonography. Confirmation in a definitive trial is awaited. In the in vitro arm of the study, a model for the induction of intimal hyperplasia in human ovarian veins was introduced. Intimal thickness increased significantly in culture, and was attenuated by the addition of oestrogen. The underlying mechanisms may be linked to increased VEGF expression in vitro in response to oestrogen, which was also demonstrated in this experiment. When tested in vivo, however, serum VEGF were reduced following exposure to transdermal oestrogen, possibly by reciprocal regulation of VEGF and nitric oxide. In conclusion, postmenopausal oestrogen replacement therapy is associated with demonstrable benefits on blood pressure and vascular disease, and is capable of vascular protection in vitro. Further work is needed to fully investigate underlying mechanisms.

The SA gene encodes a 578 amino acid protein of unknown function. SA was first identified as a candidate gene for hypertension and blood pressure regulation due to its increased expression in the kidneys of genetically hypertensive compared with normotensive rats. The aim of the work undertaken in this thesis was to investigate the function of the SA gene by gene targeting in the mouse and to assess any possible involvement of the SA protein in BP homeostasis. We utilised ES cell technology to generate a mouse model carrying a null mutation of the SA gene. Mice lacking the protein product of the SA gene are viable, reproductively normal and have no overt phenotype. Body weight and kidney, liver and heart weights are not affected by the absence of the SA protein. Comparison of basal blood pressures (BP) revealed no differences between SA-null and wildtype littermate controls in either male or female mice. Exposure of male mice to a high salt diet caused an increase in BP in wildtype mice. However in SA-null mice no effect of salt intake on BP was observed. It therefore appears that absence of the SA protein may offer some protection against a sodium induced rise in BP. The mechanism for this remains to be elucidated but may include an involvement of the SA protein in sodium retention. Administration of dihydrotestosterone (DHT) to female wildtype mice caused no increase in BP. However in SA-null mice BP was increased in response to DHT administration implying a protective effect of SA against a DHT induced rise in BP. These findings provide for the first time direct evidence of the involvement of the SA protein in BP regulation under certain conditions.

No description available.

No description available.

No description available.