EDHF is the endothelium-dependent but nitric oxide (NO) and prostacyclin (PGI2) -independent vasodilatation pathway that is the dominant relaxation mechanism in the microcirculation, and has been shown to require IKCa and SKCa channel activation and also functional myoendothelial gap junctions. Reactive oxygen species (ROS) have been implicated in the response, but their precise role remains uncertain. To investigate this, the cremaster muscle circulation of freshly killed rats was perfused with a Krebs buffer solution containing albumin (1 g.l-1) and either FITC-albumin (2 g.l-1) to measure changes in diameter, or Fura-PE3AM (10 μM) for 60 minutes to selectively load the endothelium, allowing endothelial [Ca2+]i to be recorded. The preparation was placed on the stage of an intravital microscope to measure vessel diameter, and endothelial [Ca2+]i was estimated from the 360/380 nm excitation ratio, emission at > 510 nm. The preparation was superfused with phenylephrine (30 μM), L-NAME (300 μM) and indomethacin (3 μM) to evoke arteriolar constriction while preventing the synthesis of NO and PGI2. Carbachol (10 μM) caused a 74.5 ± 2.3% (n = 65) relaxation and the 360/380 nm ratio increased by 25.6 ± 1.6% (n = 69). Both responses were substantially reduced by a ROS scavenging combination of superoxide dismutase and catalase (100 U.ml-1 each). The possibility that ROS are important for Ca2+ release from stores was examined by applying carbachol in Ca2+-free solution containing EGTA. Carbachol application then resulted in a transient [Ca2+]i increase that was reduced by SOD and catalase. The CYP 2C9 inhibitor sulfaphenazole (10 μM) reduced relaxation and the endothelial [Ca2+]i increase, as did the phospholipase C blocker U73122. These and other data to be presented suggest that ROS produced by arachidonic acid metabolism via CYP 2C9 promote EDHF mediated relaxation mainly by enhancing the inositol trisphosphate-mediated release of Ca2+ from endothelial stores.

The aim of this thesis is to further define the roles of a4 integrin and b1 integrin in the definitive haemopoietic system, more specifically in the initial stages of its establishment. We show that b1 integrin and a4 integrin are expressed on definitive haemopoietic stem cells from the embryonic day 11 aorta, gonads, mesenephorous region, day 13 foetal liver, day 13 peripheral blood and adult bone marrow, which represent the major developmental stages of the definitive haemopoietic system. In the day 13 foetal liver and adult bone marrow some a4 integrin negative definitive haemopopietic stem cells stem cells are also detected. Therefore, the expression of a4 integrin may be modulated as definitive haemopoietic stem cells mature in the later haemopoietic organs. By studying the generation of definitive haemopoietic stem cells in a4 integrin knockout embryos, a role for a4 integrin in the correct localisation and/or development of definitive haemopoietic stem cells in the foetal liver was revealed. In addition, these studies showed that the increasing defects observed in a4 integrin knockout mice are likely due to progressive defects in definitive haemopoietic stem cells as they develop in different haemopoietic microenvironments. Attempts to generate a reversible b1 integrin knockout which would enable identification of the time point at which b1 integrin is essential for definitive haemopoiesis is also discussed. In conclusion the data presented here supports previous studies on the role of b1 and a4 integrin in adult and embryonic definitive haemopoiesis and gives further information as to the time points at which these molecules are essential for the establishment of the definitive haemopoietic system and during its maintenance.

Serial transplantation of bone marrow through irradiated syngeneic hosts resulted in a decline in the capacity of haematopoietic stem cells to (a) self-renew, and (b) regenerate the haematopoietic system of lethally irradiated mice. Repeated regeneration of the stem cell pool in situ following serial injections of hydroxyurea, however, did not result in any decline in stem cell function. A decreased immune responsiveness was observed in mice repopulated with serially transplanted bone marrow. An attempt was made to detect protein-synthetic errors in reticulocytes descended from serially transferred stem cells by measuring the heat-labile fraction of glucose-6-phosphate dehydrogenase. No increase in altered enzyme was observed.

Background - Sustained activation of spliced x-box binding protein-1 (XBP1), an endoplasmic reticulum stress response transcription factor, results in the development of atherosclerosis in apoE -/- mice. Histone deacetylases (HDACs) play a crucial role in transcriptional regulation through modulation of chromatin structure. In particular, HDAC3 is involved in maintaining endothelial integrity. HDAC3 and XBP1 are similarly expressed in the bifurcation regions of aorta. Unspliced XBP1 (XBPlu) is expressed in a similar manner in endothelial cells. In the present study we investigated the role of XBPlu and XBPls in the maintenance of endothelial integrity and endothelial cell migration. Furthermore, the crosstalk between HDACS and XBP1 signalling pathways was examined. Methods and Results - Our study demonstrated that disturbed flow upregulated HDACS and XBPlu protein production through the VEGF receptor 2 / PI-3-kinase pathway. Knockdown of XBP1 by shRNA lentiviral transfection ablated disturbed flow-induced HDAC3 upregulation. Similarly to HDACS, overexpression of XBPlu by adenoviral gene transfer increased Akt phosphorylation at serine 473 and haem oxygenase 1 gene transcription, which showed a protective role in hydrogen peroxide-induced apoptosis of endothelial cells. Co-immunoprecipitation assays demonstrated that HDACS physically associates with XBPlu and Akt. The use of truncated HDACS constructs demonstrated that XBP1 binds to the central section of HDACS, which is predicted to contain a nuclear export signal. Furthermore, we identified a role for XBP1 in mediating endothelial cell migration. Overexpression of both XBPlu and XBPls enhanced the ability of endothelial cells to migrate. Ablation of XBP1 expression and XBP1 splicing, through knockdown of IRE la expression, reduced endothelial cell migration; however this was without a corresponding decrease in eNOS expression and NO production. Conclusions - These results suggest that XBPlu protects endothelial cells from oxidative stress, including that produced by disturbed flow. The interaction with HDAC3 could be crucial for this effect. The ratio between XBPlu and XBPls is also crucial for correct endothelial cell migration. Modulation of this balance and the interaction with HDAC3 may provide novel therapeutic strategies in vascular disease whilst maintaining endothelial integrity.

Anti-neutrophil cytoplasmic autoantibodies (ANCA), targeting the neutrophil protein granules myeloperoxidase (MPO) and proteinase 3 (PR3), activate neutrophils in vitro, and are associated with a systemic autoimmune vasculitis, in which a pauci-immune crescentic glomerulonephritis is common. The in vivo study of ANCA pathogenesis is severely limited, both by the lack of a robust anti-PR3 IgG induced disease model, and by the differences found between human and mouse biology. Therefore the development of a disease model based on humanised mice, defined as mice possessing human immune cells, has the potential to overcome these limitations, while also allowing the direct study of human ANCA in vivo. The aims of this thesis were 1) to establish in vitro assays of ANCA induced neutrophil activation, and thus allow the study of ANCA:neutrophil interactions and 2) to establish a humanised mouse model of ANCA vasculitis. ANCA were purified from patient plasma, and two human neutrophil respiratory burst assays were developed. Subsequently, it was shown that Granulocyte Colony Stimulating Factor (GCSF) primes neutrophils for an anti-MPO induced respiratory burst, and the inhibition of phosphoinositol 3-kinase p/5 abrogates the ANCA induced release of superoxide. Humanised mice were produced by the engraftment of human haematopoietic stem cells into irradiated, adult NOD-scid \\-2rf1’ mice. At least 8 weeks later a population of human neutrophils were identified by flow cytometry, and this population was expanded by treatment with GCSF. It was shown that these cells could respond to inflammatory stimuli in vivo, by modulating their expression of activation markers, and in vitro, by undergoing respiratory burst and degranulation. Patient derived ANCA was passively transferred into humanised mice that had been primed with GCSF and lipopolysaccharide. Mice were culled 7 days later, but there was no histological or biochemical evidence of disease. Thus, this work has identified GCSF and phosphoinositol-3-kinase p/8 as molecules of potential importance in ANCA vasculitis. Furthermore, the passive transfer of patient ANCA into humanised mice did not prove pathogenic in this study, and possible reasons for this are discussed. The demonstration of functional responses in human neutrophils does, however, suggest that this humanised mouse model has the potential be useful for the study of human neutrophils in vivo.

The continuous production of fetal haemoglobin (HbF, a2Ya) into adulthood is an ameliorating factor in sickle cell disease and B-thalassemia. We have previously mapped two quantitative trait loci (QTLs) controlling HbF levels, one in intron 2 of BCL11A gene, and the other, an intergenic region on chromosome 6 between the genes HBS1L and MYB, known as HMIP. Histone modification and RNA polymerase II binding at these loci and at the globin genes themselves were analysed using microarray-based chromatin immunoprecipitation studies of primary human erythroid progenitor cells. In addition, we analysed binding of GATA-1 and KLF1, two major erythroid-specific transcription factors. Strong GATA-1 binding at the HMIP region coinciding with strong histone acetylation and RNA polymerase II activity was seen, indicative of the presence of regulatory elements in the intergenic region. Moreover, differential GATA-1 binding was observed between individuals with low HbF and raised HbF levels at a site within the HMIP region most strongly associated with HbF variance. BCL11A intron 2 also showed strong GATA-1 binding and histone H3 acetylation, and may therefore be responsible for the overall regulation of BCL11A. I have carried out extensive sequence analysis of individuals from the upper and lower extremes of BCLllA-associated HbF levels to fully characterize DNA variants. The nature of this sequence as a regulator of BCL11A expression remains to be determined and functional tests are on-going. The role of BCL11A, in conjunction with KLF1, as a transcriptional regulator of the a and P globin loci, as well as the QTLs themselves, was also investigated. These results show that BCL11A, often alongside GATA-1, is intimately involved in the transcriptional regulation of the globin genes and that KLF1 also regulates BCL11A. The importance of different protein isoforms of BCL11A is also explored.

There has been renewed interest in foetal phonocardiography since the recent advent of wide-bandwidth (0.5-250 Hz) phono-transducers which yield higher signal-to-noise ratios than were hitherto obtainable. With this passive and non-passive transducer there is the potential to realize the goal of long-term continuous foetal heart monitoring. In order to achieve this goal, signal processing and analysis methods are required to automatically extract from the phonocardiogram (PhCG) the beat-to-beat temporal parameters of foetal cardiac function. The research detailed in this thesis investigates the temporal and spectral morphology of the princicpal heart sounds, and proposes a method which detects and identifies the first and second heart sounds in the PhCG on a bcat-to-beat basis. Examination of the princicpal heart sounds in 252 PhCGs, each of 4.1 seconds duration, from 19 subjects has found that wide variations exist in the temporal morphology of these sounds. It was also observed that the PhCG is susceptible to persistent contamination by many adventitious sounds. These latter sounds often coalesce with the principal heart sounds and obscure the end points of both. Second heart sounds were noted to be subject to sudden and severe attenuation, but were more morphologically stable than first heart sounds. The frequency analysis of the principal heart sounds was performed using Fourier transformation, a high-resolution parametric technique based on the Burg algorithm. In all the 741 principal heart sounds examined from 12 subjects, it was observed that the majority of the energy in their spectra is concentrated in the previously unmonitored 20-40 Hz band. In this sample set of first and second heart sounds, it was found that each set of values docs not produce a spectral signature with which it may be uniquely associated. Results arc presented to substantiate this observation. A procedural knowledge-based system (KBS) was developed to process and analyse the PhCG with the objective of detecting and identifying individual first and second heart sounds. In this the KBS has been successful. The KBS analyses PhCGs over a wide range of FHRs (80-220 bpm), irrespective of either short-term variabiity in FHR or contamination by adventitious sounds. Results are presented which illustrate the performance of this system over the spectrum of PhCG types.

The thesis describes the measurement of the local mass transfer coefficients on the surface of a small convex disc and on the surface of Petichek's stagnation point blood flow chamber. No correlation was found between the go mass transfer coefficients and the limited blood clot data available for those surfaces. The mass transfer measurements were made using the shrinking film holographic interferometry technique. The thesis details the interpretation of fringe patterns obtained on small, curved surfaces, and a method of determining the fringe order from a single hologram.

Approximately one-third of trauma patients are coagulopathic on arrival to the emergency department. Acute traumatic coagulopathy and systemic inflammatory responses are serious secondary consequences of severe trauma and are linked to increased morbidity and mortality. Early tissue hypoxia is a major component in the aetiology of both complications. New resuscitation strategies are aimed at improving tissue oxygenation in the pre-hospital phase, and may attenuate coagulopathy and inflammatory sequelae. This is of particular importance in military personnel who suffer complex injuries, often from blast exposure, and may have extended evacuation times. This thesis evaluates the effect of a novel hybrid (NH) resuscitation strategy on coagulation and inflammation. Terminally anaesthetised pigs were randomised to one of two injury strands of haemorrhage +/- blast injury; initially resuscitated with 0.9% Saline to a hypotensive systolic blood pressure of 80mmHg for one hour. This was followed by either a return to a normotensive pressure (110mmHg) (NH) or a continuation at the hypotensive level. Over both injury strands NH significantly reduced Prothrombin Time, PT (mean proportion of baseline: 1.40±0.05 vs. 1.80±0.09; p=0.001) and interleukin-6 (IL6) levels (mean 1106±153 vs. 429±79 pg/ml; p=0.001) compared to the hypotensive groups. PT was positively correlated with IL6 (p=0.002) and base deficit (p=0.0004). These findings indicate that improving tissue oxygenation reduces the coagulation derangement and the pro-inflammatory response. No difference in coagulopathy was found between injury strands although blast did cause greater inflammation. Early identification of coagulopathic casualties is essential and a separate feasibility field study was preformed to assess the use of thromboelastometry in a deployed military hospital, evaluating the degree of coagulopathy in battlefield casualties and to monitor the coagulation status during the resuscitation process. In conclusion, NH attenuated the acute traumatic coagulopathy and inflammatory responses and therefore should be considered when an extended casualty evacuation is enforced.

No description available.