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Childhood asthma is an extensive problem and is particularly pronounced in the UK. Asthma can restrict activities, cause school absence and can be the source of considerable stress in both children and their parents. Mortality is rare and preventable, although poor perception of symptoms and delay in seeking medical attention are strong risk factors for a fatal asthma attack. Self- management actively involves the child in their own healthcare and entails monitoring symptoms and responding accordingly and has been linked to better outcomes. This can be facilitated by health education. The aims of this project were to develop an educational computer program to promote self-management skills in children and young people with asthma, to evaluate its effectiveness in a clinical sample and to validate measures of asthma knowledge and locus of control. The Asthma Files uses a 'secret agent' theme to encourage users to investigate information about asthma. The program was piloted with 28 children aged 7-16 over a one year period and revised in accordance with both qualitative and quantitative data obtained. To evaluate the computer program, 101 children aged between 7 and 14 years were recruited from three hospital asthma out-patient clinics to participate in a randomised, controlled trial. They were interviewed using asthma knowledge and asthma-specific locus of control measures developed and validated for the purposes of the study. All children were given an information booklet one month later and, in addition, 50 children used the computer program. Baseline knowledge levels were low. At one-month follow-up (n=99), children in the computer group had significantly greater increases than those in the control group (p<0.001), along with an rise in internal locus of control(p<0.01). There was no evidence of changes in objective lung function measures, hospitalisations or oral steroid use between the groups at this time. However, at six months follow-up (n=90), children in the computer group were significantly less likely to have required oral steroids or school absence than the control group (p<0.05). The program was popular with the children across the age range and received positive feedback on both content and mode of delivery. Responding to comments provided by the children in the RCT, some minor amendments were made to the program, which is now available for public use. The Asthma Files computer program was successful in increasing knowledge and promoting internal locus of control. More research is needed to evaluated how this might translate into longer term improvements in self-management. NB. This ethesis has been created by scanning the typescript original and may contain inaccuracies. In case of difficulty, please refer to the original text.

Asthma is an inflammatory disease of the airways characterised by airway remodelling and hyperresponsiveness. New treatments are needed for patients with severe asthma whose disease is not controlled with currently available therapies. Asthma pathophysiology is complex, however, accumulating evidence suggests multiple inflammatory pathways in asthma converge onto a relatively small number of downstream targets that may be of therapeutic interest. These include mitogen activated protein kinases (MAPKs), the pro-inflammatory transcription factors nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) and transcriptional regulators such as histone acetyl transferases (HATs) and histone deacetylases (HDACs). Chemokines are molecules secreted at sties of inflammation, attracting inflammatory cells and perpetuating the inflammatory response. Here we studied the mechanisms by which the pro-inflammatory mediator endothelin-1 (ET-1) and the cytokine tumour necrosis factor-alpha (TNF-alpha) promoted expression by primary human airway smooth muscle cells (HASMC) of two important chemokines, monocyte chemotactic protein-1 (MCP-1) and eotaxin. Further, we studied the mechanisms by which existing asthma therapies (long acting beta agonists (LABA) and glucocorticoids) modulated TNF-alpha-stimulated eotaxin expression. Endothelin-1 stimulated MCP-1 release through a transcriptional mechanism involving NF-kappaB and AP-1; the upstream signalling pathway involved p38 and p44/p42 MAPKs. Previously, this lab showed that TNF-alpha-induced eotaxin release is also NF-kappaB-dependent, involving histone H4 acetylation at the eotaxin promoter. Here we found that TNF-alpha-induced eotaxin release does not involve histone H3 acetylation, and that TNF-alpha-dependent histone H4 acetylation does not occur through alterations in total histone activity or levels of the key HDACs -1 and -2. Similarly, modulation of TNF-alpha effects on eotaxin expression by glucocorticoids and LABA is independent of total HDAC activity and HDAC-1 and -2 levels. These studies support the body of evidence suggesting that multiple inflammatory pathways in asthma converge onto a small number of downstream targets, and are relevant to the understanding and treatment of asthma.

Asthma is a chronic inflammatory disease of the airways characterised by airway hyper-responsiveness (AHR), inflammation of the airways and reversible airway obstruction. Airway remodelling is a feature of asthma, especially in cases of severe and fatal asthma, and includes structural changes such as increased airway smooth muscle (ASM) mass, mucous gland hyperplasia, subepithelial fibrosis and angiogenesis. TGF-β is a pleiotropic cytokine that has been implicated in the development of many of these changes. However, TGF-β is released from cells in a latent complex, associated with its pro-peptide the latency associated peptide (LAP). Extracellular activation of latent TGF-β is the rate limiting step in TGF-β bioavailability. Although TGF-β activation has been investigated in airway epithelial cells, to date, no studies have investigated TGF-β activation by airway smooth muscle cells. The hypothesis of this thesis is therefore that human airway smooth muscle cells can activate TGF-β in vitro. The hypothesis of this thesis has been tested by investigating effects of the serine protease mast cell tryptase, mechanical wounding of cell mono layers and the phospholipid lysophosphatidic acid (LPA) on TGF-β activation by primary airway smooth muscle cells in vitro. We have utilised transformed mink lung epithelial cells, a reporter cell that express a TGF-β responsive promoter driving a luciferase gene, and quantitative PCR for the TGF-β-inducible gene plasminogen activator inhibitor-1 (PAI1) to investigate TGF-β activation. Moreover, we show for the first time that TGF-β activation can be assessed in vitro by detecting the translocation of Smad 2 and 3 from the cytoplasm to the nucleus by western blotting. The results presented in this thesis provide evidence that airway smooth muscle cells are capable of activating TGF-β in vitro. These data show that the serine protease tryptase, released from activated mast cells, can proteolytically activate TGF-β via a mechanism that is independent of the tryptase receptor protease activated receptor-2 (PAR2). This effect is not accompanied by increased expression of the latent TGF-β complex. Furthermore, these data provide evidence that airway smooth muscle cells can activate TGF-β via the integrin αβV5 in response to LPA stimulation. We have found that cells from asthmatic patients activate more TGF-β in response to LPA than cells from non-asthmatic individuals and this is not due to a difference in cell surface expression levels of the αβV5 integrin. LPA-induced TGF-β activation can be inhibited by the β2 adrenoreceptor agonist formoterol, which is a commonly used asthma therapy, and the muscarinic receptor agonist methacholine, which causes cell contraction, also causes TGF-β activation by airway smooth muscle cells. Furthermore, the data presented here show that the cytoplasmic domain of the integrin β5 subunit interacts with the cytoskeletal protein talin to mediate TGF-β activation. Together, these data highlight two previously unreported, biologically relevant, mechanisms of TGF-β activation employed by airway smooth muscle cells in vitro, both of which could contribute to the development of airway remodelling in asthma in vivo. Data concerning a αβV5 mediated TGF-β activation has led us to hypothesise that contraction of airway smooth muscle leads to TGF-β activation in vivo. If correct, this could be vital to our understanding of how airway remodelling is initiated in asthma, and could lead to the development of new therapies aimed at inhibiting contraction-induced TGF-β activation, for the treatment of asthma.