No description available.

No description available.

The holy grail of human embryonic stem cells (HESC) is the generation of a spectrum of differentiated cell types that may be used for clinical and therapeutic use in the treatment and cure of infinite diseases. Over recent years, research into the process of generation of specific cell types from HESC have advanced very rapidly and these advances appear only to be moving with greater speed as scientists sought to understand the specific mechanisms in the generation of these differentiated cells types. This study focused on furthering this understanding through the discovery of novel haematopoietic genes that may contribute to haematopoietic development by screening the entire human genome. This comparative microarray experiment relied heavily on the isolation and utilisation of haematopoietic-enriched cells acquired through the haematopoietic differentiation of HESC lines H9 and H1. The haematopoietic-enriched populations were generated using an approach that adapted published haematopoietic differentiation protocols, which was then optimised within this experiment for the generation of significantly greater quantities of haematopoietic progenitors compared with the standard foetal bovine serum differentiation approach. These differentiated cells were sorted for a haematopoietic-specific population by fluorescence-activate cell sorting (FACS) for KDR+ CD31+ (markers of early haematopoiesis). Microarray analysis revealed 3,162 transcripts that may play important roles in haematopoiesis, with pathway analysis supporting these findings revealing an overwhelming preference for vasculogenesis, angiogenesis, and erythropoiesis process networks within this gene list. A select number of transcripts were flagged for further investigation, and of these SOST, FLI1, EPO, and SCL were of particular interest and hypothesised to enhance in vitro haematopoietic differentiation from HESCs. The effect of SCL on haematopoietic differentiation was assessed using SCL over-expressing HESC lines, and it was shown to significantly enhance the efficiency of haematopoietic differentiation. The critical role of SCL in erythropoiesis has been well described in literature and within this study, it was discovered that SCL over-expression dramatically enhanced erythropoiesis of HESCs by over 25 fold compared to the control. The differentiation of SCL over-expressing HESCs within erythropoietic-optimised media may be the missing link in the efficient generation of therapeutic functional erythrocytes.

The vascular endothelium regulates vasomotor tone and blood fluidity through the release of paracrine factors, such as nitric oxide (NO). Acutely activated neutrophils have been shown to consume vasodilatory NO in vitro through the NAD(P)H oxidase-dependent generation of superoxide. As such, neutrophils may contribute to the endothelial dysfunction and increased vascular tone commonly observed in inflammatory vascular diseases. Herein, the experiments in this thesis set out to examine a role for neutrophils in the regulation of vascular tone. Initial experiments aimed to assess the capacity of inflammatory-primed neutrophils to consume NO in vitro. Inflammatory priming of human neutrophils resulted in significantly accelerated rates of NO disappearance from aerobic buffer in vitro in an NAD(P)H oxidase-dependent manner. Thus, neutrophil priming may have a detrimental effect on vascular homeostasis in vivo. With this in mind, subsequent chapters aimed to elucidate a role for neutrophils in regulating vascular tone in vivo using immunodepletion techniques. Following neutrophil depletion, mice exhibited a gradual fall in systolic blood pressure over 3 days, and phenylephrine-induced vasoconstriction was significantly attenuated in thoracic aortae isolated from neutropenic animals compared to controls. There effects were due, at least in-part, to the interferon-y-dependent upregulation of inducible nitric oxide synthase (iNOS) bioactivity in the thoracic aortae of neutropenic mice. Thus, under physiological conditions, neutrophils may modulate vascular tone by negatively regulating the bioactivity of pro-inflammatory enzymes, such as iNOS. Finally, the effect of acute neutropenia on angiotensin (Ang) II-induced hypertension in vivo was examined. Neutrophil depletion prevented the onset of Ang II-induced hypertension, indicating that neutrophils may be directly involved in the development of hypertension associated with this octapeptide in vivo. In conclusion, the data described in this thesis highlight the diversity of neutrophil activities in vivo and in vitro and how changes in neutrophil phenotype may impact upon vascular physiology.

In this thesis, we describe the development of a new technique for determination of the mechanical properties of the red blood cell membrane from measurement of its thermal fluctuations. Experimentally, the shape fluctuations of the equatorial contours of red blood cells are recorded using fast phase-contrast video microscopy, from which the Fourier fluctuation spectrum is obtained and analysed. The experimentally obtained fluctuation spectra are interpreted using a coarse grained particle dynamics simulation which models the thermal fluctuations of an elastic mesh endowed with bending and shear elasticity, and constant volume and surface area. We demonstrate that the simulation correctly describes the mean shape of the red cell as well as the membrane thermal fluctuations. Comparison between theory and experiment leads to physically sound values for the relevant membrane elastic moduli and helps distinguish between the contributions of the lipid bilayer and the membrane skeleton. We extend this technique to investigate the mechanical response of red blood cells to oxidative stress. We show that it is possible to discriminate between the actions of different oxidising agents from their distinctive effects on the membrane thermal fluctuation spectrum. This allows comparative measures of the membrane material properties to be extracted using an approximate analytical model thus discriminating between the effects of each oxidising agent on different structural components of the membrane. This technique was also applied to investigate the response of the red blood cell to oxidative stress under simulated hyperglycemic conditions characteristic for disease states such as diabetes. We established that the membrane elasticity of glycated cells deteriorate much fasted under administration of hydrogen peroxide, which may be related to the observed microvascular complications in diabetes, characterised by disproportionately high levels of reactive oxidative species. We demonstrate that metformin, one of the most widely prescribed anti-diabetic drugs, has an ameliorative effect on the membrane mechanical properties, which is probably due to its anti-glycating effects. The technique provides a reproducible means to assess the effects of reactive oxidative species on the red blood cell membrane mechanical properties and distinguish between effects on the protein membrane skeleton and the lipid bilayer. This makes the new method of potential value in monitoring the effects of drug induced changes hence assessing progress of treatment in terms of the antioxidant or anti-glycation properties of administered drugs in diabetes or other conditions characterised by high levels of oxidative stress.

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Endoglin is an accessory receptor for the transforming growth factor beta family. Patients carrying mutations in the endoglin gene develop the inherited vascular dysplasia, Hereditary Haemorrhagic Telangiectasia (HHT). However, it is becoming apparent that endoglin may also be important in acquired cardiovascular diseases such as hypertension, atherosclerosis and heart failure. The work in this thesis investigates the role of endoglin in the structure and function of the adult cardiovascular system by utilising a conditional endoglin knockout model and murine cardiac magnetic resonance imaging (CMR). I established and validated murine CMR at Newcastle University allowing accurate quantification of in vivo cardiac structure and function in a number of mouse models of cardiac disease either at single time points or in longitudinal studies. Immunohistochemical analysis confirmed that endoglin is expressed in the endocardium and vasculature of the adult mouse heart. Following myocardial infarction there was increased endoglin expression which co-localised with endothelial cells and myofibroblasts. Cre-lox genetics was then used to ubiquitously knockdown endoglin in the adult mouse. This resulted in significant ventricular remodelling within three weeks, with ventricular dilatation associated with cardiomyocyte hypertrophy. These changes gradually progressed over three months following endoglin knockdown; however, overt heart failure was not seen within this time frame. Invasive measurement of ventricular function suggested an impairment of vasomotor control and reduced contractile reserve following endoglin knockdown. Also, using an endothelial-specific endoglin knockdown mouse I demonstrated that these novel cardiac changes were due to endoglin depletion in endothelial cells. Together these data suggests that the alterations in cardiac structure and function are secondary to alterations in the wider cardiovascular system. This is supported by evidence of eNOS uncoupling following endoglin knockdown. The results reported in this thesis describe a novel phenotype and highlight the importance of endoglin in the maintenance of cardiac structure and function. Further work will clarify the mechanism behind these alterations.

Present real-time echocardiographic visualisation methods and their clinical application have been reviewed. Ultrasonic instruments for visualising the heart were considered to have a number of limitations. Experimental and theoretical considerations of ultrasonic access and interaction with the heart and its surroundings have led to the design and development of a mechanical sector scanner. Rotating transducers within an oil filled cavity facilitates acoustic contact and vibration-free operation. The small dimensions and the 900 field of view optimise visualisation of the heart avoiding bone and lung, provide a point of entry aspect allowing flexible scanning action and permit the application of two scanners simultaneously at adjacent intercostal spaces. These aspects also allow subxiphoid application of the scanner to visualise the heart and conventional T-M echograms to be obtained. A real-time heart scanning system with grey scale display and recording facilities has been developed. This system is versatile allowing two scanners to be driven in several operating modes. The scanner has been evaluated on a representative group of patients suffering from cardio-respiratory disease. Visualisation of cardiac structures and their function has been successful. The possible visualisation extensions and improvements through the use of two scanners simultaneously has been investigated. Real-time compounding and biplane visualisation can be achieved. The brief clinical use of this scanner in pediatrics has indicated the potential applicability of this design philosophy in the diagnosis of congenital heart defects.

In view of the recent work on isolated cardiac muscle which has shown that situations can occur in which the force and velocity of contraction are chanced in different directions, we have questioned the usefulness of the search for a single index of contractility related to the rate of rise of tension, a trend which dominates studies of the heart in vivo. He have attempted to show, in the heart in vivo, that situations do occur in which the duration of contraction can determine the force of contraction or in. which force and velocity of contraction are changed in different directions such that purely a change in the velocity dependant aspects of contraction would not describe the inotropic intervention.

No description available.