In the laboratory, prior research has shown that cognitive appraisals of challenge and threat are predictive of differential hemodynamic response patterns during stressor exposure.  Participants who appraise a stressor as a challenge showed a myocardial (heart-mediated) response.  On the other hand, threat appraisal has been shown to be associated with a vascular hemodynamic response pattern. The first aim of the current dissertation was to test whether these relationships between cognitive appraisal and hemodynamic responding generalise to a real-life stressful situation. Stressor exposure was found to elicit both myocardial and vascular response patterns in association with challenge and threat appraisal, respectively.  Thus, this study showed that these patterns can be observed in real life.  In addition, the threat-related increases in vascular responding that were observed in real life were substantial. Thus, these data suggest that interventions aimed at attenuating threat appraisals or enhancing challenge appraisals may potentially affect cardiovascular health.  Therefore, the next aim was to examine whether cognitive appraisals can be manipulated.  Using a bias induction paradigm, participants were trained to interpret emotionally ambiguous situation descriptions as either a challenge or as a threat, depending on experimental condition. Evidence, was obtained in one out of two studies suggesting a bias in cognitive appraisal had been induced successfully. The aim of the final study was to experimentally manipulate appraisal as well as hemodynamic reactivity.  In this experiment, participants received either challenging or threatening task instructions.  Subsequently, participants performed four consecutive mental arithmetic tasks.  Data were analysed using a within- and between-subjects design.  Cognitive appraisal was significantly affected by the experimental manipulation; however, the hemodynamic reaction patterns were not. Some evidence was found suggesting a role for effort expenditure in relation to task difficulty.

Haemopoietic stem cells are capable of self-replicating and differentiating along eight distinct lineages; these properties allow them to reconstitute the entire blood system of irradiated recipients. How these cells choose whether to self-renew or commit to a specific lineage remains unclear, but transcription factors are likely to play an important role in this decision. To gain insight into the transcriptional programmes of haemopoietic stem cells, the regulation of the gene encoding the murine stem cell antigen Cd34 was analysed. Within haemopoiesis, Cd34 expression is restricted to early progenitor cells, but outside haemopoiesis, Cd34 is expressed in vascular endothelium, fibroblasts, brain and testis. Three key regulatory elements were identified: (a) a3 Kb upstream region which functions as a stem-cell specific, chomatin-dependent enhancer in cell-lines, (b) a6 Kb downstream region which seems to have the ability to buffer against position effects, (c) a 1.2 Kb promoter which does not exhibit any stem-cell specificity. These three regions were linked to a human CD34 cDNA reporter and were able to direct high-level expression of the transgene in a Cd34-expressing progenitor cell-line; this construct was then used to generate transgenic mice. RNA and protein analysis were performed on haemopoietic cells derived from bone marrow, AGM, and fetal liver: transgene RNA was detected in Cd34 expressing cells. However, expression was also seen in non-Cd34-expressing cells, as well as in other tissues. To improve the level and the specificity of the targetting, four more constructs were made using combinations of the regulatory elements described previously with different reporters and heterologous promoters to generate transgenic mice. In all these mice, Cd34-expressing cells of the bone marrow were analysed for transgene expression. To assess the extent to which these constructs were subject to position effect variegation, analysis of gene expression by single cell RT-PCR was conducted in stably-transfected clones. The work presented here on the regulation of the murine stem cell antigen Cd34 gene shows that although the elements identified have the potential to target transcription of a reporter to the stem cell compartment, further regions are probably necessary for specific and high-level expression.

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Uncontrolled activation of the coagulation cascade following lung injury has been implicated in both lung inflammation and fibrosis. In addition to its role in coagulation, thrombin exerts pluripotent cellular effects via the activation of its high-affinity receptor, proteinase activated receptor-1 (PAR_1). PAR_1 is a seven transmembrane domain G protein-coupled receptor that exhibits the ability to couple to multiple G protein family subunits, including G\alpha_{i/o}, G\alpha_q and G\alpha_{12/13} within the same cell type. Activation of PAR_1 on fibroblasts, a key effector cell in lung fibrosis, results in the induction of several mediators, including the potent monocyte and fibrocyte chemoattractant CCL2. In this thesis, the G-protein and downstream signalling pathways involved in PAR_1-mediated CCL2 production and release were examined. Using a novel PAR_1 antagonist which blocks the interaction between PAR_1 and G\alpha_q, this thesis shows for the first time that PAR_1 coupling to G\alpha_q is essential for thrombin (10nM)-induced CCL2 gene expression and protein release in murine lung fibroblasts (MLFs). The work presented here further demonstrates that these effects are mediated via the cooperation between ERK1/2 and Rho kinase signalling pathways: a calcium-independent PKC, c-Raf and ERK1/2 pathway was found to mediate PAR_1-induced CCL2 gene transcription; whereas PLC, calcium, calcium-dependent PKC and Rho kinase pathway influences CCL2 protein release. This thesis represents the first demonstration of the cooperation between two pathways in mediating the stimulatory effects of thrombin, or indeed any other extra cellular stimulus, on the induction and release of the potent chemoattractant, CCL2. This thesis also examined the signalling receptor and downstream effectors involved in thrombin-induced CCL2 production in primary human lung fibroblasts (pHALFs). The results demonstrate that PAR_1 coupling to G\alpha_q is also both necessary and sufficient in mediating thrombin-induced CCL2 production at low concentration of the proteinase, whereas at high concentrations, these effects may be partially PAR-independent. This discrepancy between MLFs and pHALFs may be explained by differences of PAR receptor expression between species. Taken together, this thesis proposes that targeting the interaction between PAR_1 and specific G proteins may allow more selective blockade of PAR_1 pro-inflammatory and pro-fibrotic signalling, whilst preserving the essential role of other PAR_1-mediated cellular responses.

Angiogenesis is a complex process regulated by co-ordination of extracellular matrix (ECM) and growth factor signalling (via integrins and growth factor receptors respectively). Dysregulated angiogenesis contributes to disease pathology and progression, while an adequate blood supply is crucial for successful tissue repair and reconstruction. Thus an understanding of the mechanisms by which ECM controls angiogenesis would contribute to development of therapeutic strategies. Decorin is a small leucine-rich repeat proteoglycan consisting of a core protein and a single glycosaminoglycan. Decorin has been reported to contribute to ECM organisation through interaction with numerous ECM components, including collagen types I, VI, XII and XIV, fibronectin, thrombospondin, tropoelastin and tenascin-X. Further, decorin influences growth factor activity, interacts with a2pl integrin, and can activate growth factor receptors. In the absence of decorin, angiogenesis is dysregulated however it is not known which of these interactions are responsible. This thesis investigates the role of decorin interactions with collagen type I, a2pl integrin (a collagen I receptor), and the IGF-I receptor in modulating endothelial cell behaviour. This thesis demonstrates that both collagen-bound and soluble decorin enhance endothelial cell adhesion and migration, and that the latter may involve activation of the IGF-I receptor, and the small GTPase Rac, by decorin. In accordance with this, decorin induced morphological changes consistent with activation of small GTPases. Further, decorin interacts with a2pl integrin via the GAG moiety, and may influence integrin activity in an allosteric manner, although the intact proteoglycan is required for modulation of endothelial cell behaviour. It was also demonstrated that decorin supports a2pl integrin activation in the presence of the specific inhibitor rhodocetin. Additionally, decorin activates transcription factors associated with long-term cell survival and quiescence. Together, these data implicate decorin as an important regulator of several aspects of angiogenesis pertinent to establishment of mature neo-vessels.

The primary function of the cardiovascular and pulmonary systems is to produce a flow of oxygenated blood in sufficient supply to maintain aerobic metabolism in all organs and tissues. The system is required to be energy efficient but also receptive to changes in cellular metabolic demand. In order to function both at rest and during demand the system must also match oxygen allocation to metabolic requirements at a localised level. Once blood enters an area that requires substrates, oxygen should have the capacity to efficiently move from the blood, across the vessel wall and into the tissue. Global oxygen delivery (DO2, product of cardiac output and arterial oxygen content) under normal, resting conditions is more than adequate to meet metabolic demands/oxygen consumption (VO2) (Figure 1.1). At a tissue level, oxygen delivery is governed by two processes, convective and diffusive oxygen transport. Convective transport can be described as the bulk movement of oxygen in the blood, encompassing changes in cardiac output and the mechanisms that regulate flow in the microcirculation. Diffusive oxygen transport simply refers to the movement of oxygen from the blood into tissue, down the capillary-intracellular oxygen tension (PO 2) gradient and is governed by arterial oxygen tension (PaO2).

It was hypothesised that generation of endogenous glucocorticoids by 11βHSD1 within the vessel wall regulates angiogenesis. <i>In vitro</i> mouse aortic ring cultures established that physiologically-relevant concentrations of glucocorticoids inhibit angiogenesis in a glucocorticoid receptor-dependent manner.  In addition 11βHSD1 was found to modulate glucocorticoid-induced angiostasis, for 11dehydrocorticosterone (a substrate for 11βHSD1) although angiostatic in C57B16 aortae did not inhibit angiogenesis in llβHSD1 deficient animals. <i>In vivo</i> using subcutaneous sponge implants in mice, endogenous glucocorticoids were found to inhibit angiogenesis: sponges in adrenalectomised mice grew more vessels compared to sponges from sham-operated animals. 11βHSD1 regulated the angiostatic effects of glucocorticoids, for cortisone (the human equivalent of 11dehydrocorticosterone), although angiostatic in controls did not inhibit angiogenesis in 11βHSD1 deficient mice. <i>In pathology</i> in cutaneous wounds and infracted myocardium endogenous glucocorticoids were found to inhibit angiogenesis. RU38486, (a glucocorticoids receptor antagonist) in comparison to placebo enhanced angiogenesis in both tissues. In similar studies in C57B16 or llβHSD1 deficient mice, 11βHSD1 was found to tonically repress angiogenesis and impair left ventricular remodelling post infarction. Thus 11βHSD1 deficient mice had increased myocardial revascularisation and preserved left ventricular function. In conclusion, by using <i>in vitro</i>, <i>in vivo</i>, and <i>pathological </i>models, endogenous glucocorticoids were seen to inhibit angiogenesis. In addition, 11βHSD1 regeneration of glucocorticoids tonically repressed angiogenesis and influenced left ventricular remodelling post myocardial infarction. Thus 11βHSD1 appears to be an attractive therapeutic target for the management of tissue revascularisation.