No description available.

No description available.

Bone cell differentiation and remodelling are controlled by hormones, growth factors and specific transcriptional regulators. This thesis aims to investigate the role of two transcription factors, c-Fos/AP-1 and Msx-1/2 in osteoclast and osteoblast differentiation using in vivo and in vitro approaches. c-Fos has been shown previously to be essential for osteoclast differentiation and is overexpressed in osteoclasts of Paget's disease. To investigate the role of c-Fos in osteoclasts, transgenic mice were generated where c-fos was overexpressed in osteoclasts using the TRAP promoter. Several TRAP-c-fosLTR transgenic founders were generated which developed severe bone remodelling lesions with eventual tumour formation. Histological and in situ expression studies showed abundant osteoclasts within these lesions which expressed c-Fos, in addition to the anti-apoptotic gene Bcl-2. These features are reminiscent of Pagetic osteoclasts and suggest that these mice are useful for studying bone remodelling disorders. The effects of c-Fos on osteoblasts was next investigated in vitro using a well defined inducible expression system. Stable MC3T3-E1 osteoblastic subclones expressing a tetracycline-regulatable c-fos gene demonstrated that exogenous c-Fos appeared to augment the proliferation induced by BMP-2, and inhibited BMP-2-induced alkaline phosphatase activity during differentiation. Moreover, ectopic c-Fos expression in these cells stimulated apoptosis induced by serum withdrawal and Etoposide. This apoptosis was not effectively blocked by the caspase inhibitors Z-VAD-fmk and DEVD-CHO, but was blocked by the cell cycle dependent kinase (CDK) inhibitor, Roscovitine, and ectopic Bcl-2 or p2l^{WAF1,CIP1,SD11} expression. These results may provide a novel link between growth control and apoptosis in osteoblasts, such that under environmental stress, c-Fos may drive cell cycle progression and render the cell susceptible to apoptosis. Finally, the regulation of Msx2 by osteotropic factors using two reporter gene constructs was analysed in osteoblastic cells. PTH showed no regulation of Msx2 expression, however, small increases were observed with BMPs. The expression of Msx1 during bone development was also analysed in tissues from Msx1-lacZ transgenic mice. LacZ expression was detected in mineralising tissues of the foetus and neonate, but no LacZ expression was observed after birth. Taken together, these data further delineate the functional roles of these transcription factors in bone development and bone disease. Importantly, these studies provide a role for c-Fos in osteoblast apoptosis and osteoclast function, and may serve as a model for c-Fos overexpression in Paget's disease.

No description available.

No description available.

No description available.

No description available.

Asthma is a common chronic inflammatory condition which affects over 300 million people worldwide. Thickening of the subepithelial layer is a key feature of asthmatic airways and the extent of thickening has been correlated with severity of asthma and increased exacerbations. Recent epidemiological studies have shown a link between fungal sensitisation primarily with Aspergillus fumigatus (A. fumigatus) and exacerbations of asthma leading to increased morbidity and mortality. The airway epithelium acts as an initial defence barrier to inhaled allergens such as A. fumigatus and emerging evidence suggests that as well as orchestrating an allergic immune response, it initiates aspects of airway wall remodelling including subepithelial thickening. However, induction of a profibrogenic response by the airway epithelium following exposure to inhaled fungi associated with severe asthma has not been well documented. The epithelial expression and production of the profibrotic growth factors, TGF-β1, TGF-β2, IL-6, endothelin-1 and periostin, selected as implicated in the aetiology of asthma and their profibrotic activity, were investigated in response to both A. fumigatus spores and culture filtrate in vitro. Furthermore, in vivo chronic inhalation models using either live spores or culture filtrate from two different strains of A. fumigatus (AF293 and CEA10) were used to determine the ability of the fungi to induce murine airway wall remodelling. In vitro, spores from both strains were able to induce the expression and production of IL-6 and endothelin-1 from human bronchial epithelial cells but none of the other profibrotic growth factors. In vivo, despite spores from both strains inducing expression and production of IL-6 and endothelin-1, only CEA10 spores caused significant subepithelial collagen deposition however, both strains induced α-SMA, a myofibroblast and smooth muscle marker around the airways. As a secreted factor was suspected of driving airway wall remodelling, subsequent studies used culture filtrate produced by the two strains, AF293, a low and CEA10, a high protease producer in basal medium. Only AF293 culture filtrate induced IL-6 and endothelin-1 from human bronchial epithelial cells in vitro. However, in vivo, culture filtrate from both strains was able to induce IL-6 and endothelin-1 expression, with AF293 causing a more profound subepithelial collagen deposition and significantly increased α-SMA abundance. It was hypothesised that epithelial-derived endothelin-1 drives airway wall remodelling and hence Endothelin receptor A was inhibited in the in vivo culture filtrate inhalation model. A significant reduction in subepithelial collagen deposition and α-SMA localisation around the airways was demonstrated in mice receiving an Endothelin receptor A antagonist compared with culture filtrate alone. This thesis indicates that A. fumigatus exposure can drive features of airway wall remodelling such as subepithelial fibrosis possibly through the epithelial production of profibrotic growth factor, endothelin-1.

Doctor of Philosophy (PhD) / Asthma is an inflammatory disease characterised by tissue remodelling. A prominent feature of this remodelling is an increase in the number and size of the blood vessels- formed from pre-existing capillaries – angiogenesis (Siddiqui et al., 2007; Wilson, 2003). This is triggered by many different endogenous angiogenic stimulators such as vascular endothelial growth factor (VEGF), and inhibited by endogenous angiogenic inhibitors such as tumstatin. Tumstatin is the non-collagenous domain (NC1) of the collagen IV α3 chain which, when cleaved, inhibits endothelial cell proliferation and induces apoptosis. Experiments described in this thesis have for the first time demonstrated the absence of tumstatin in the airways of individuals with asthma and lymphangioleiomyomatosis (LAM) as well as the functional responses to tumstatin as an angiogenic inhibitor, both in vitro and in vivo, in the airway. Although tumstatin was absent from the airways of asthmatic and LAM individuals it was present in the airways of individuals with no airways disease, chronic obstructive pulmonary disease, bronchiectasis and cystic fibrosis. No significant difference was seen in the levels of the Goodpasture Binding Protein (GPBP), a phosphorylating protein responsible for the alternate folding of tumstatin, between asthmatic, LAM and individuals with no airways disease. The αvβ3 integrin, reported to be necessary for the activity of tumstatin, as well as the individual αv and β3 sub-units were shown to be equally expressed in the airways of all patient groups. Co-localisation of tumstatin, VEGF and the αvβ3 integrin was seen in the disease free airways, however, a different pattern of VEGF and the αvβ3 integrin expression was observed in asthmatic and LAM airways with minimal co-localisation. Tumstatin was detected in serum and bronchoalveolar lavage fluid (BAL-f) samples from asthmatics and individuals with no airway disease, however there was no significant difference in the level of expression between the two groups. It was demonstrated that the tumstatin detected in the serum and BAL-f samples from asthmatics and individuals with no airway disease was part of the whole collagen IV α3 chain and not in its free and potentially active form. The ability of recombinant tumstatin to inhibit tube formation and proliferation of primary pulmonary endothelial cells was demonstrated for the first time. Further, the functional response of tumstatin was demonstrated in vivo in a mouse model of allergic airway disease. Tumstatin inhibited angiogenesis in the airway and decreased airway hyperresponsiveness. Whether there is potential for tumstatin, or a derivative thereof, to be of therapeutic value in airways diseases in which angiogenesis is a component should be the subject of future studies.

The family of Nucleoplasmin (NPM) proteins play an important role in a number of chromatin remodelling processes. The first NPM protein discovered in the eggs and oocytes of Xenopus laevis was NPM2, a tissue specific histone chaperone. In Xenopus, NPM2 has been linked to paternal chromatin decondensation following fertilization through the removal of sperm proteins, nucleosome assembly through the storage and addition of H2A-H2B dimers and apoptosis. In mammals, NPM2 correlates strongly with nucleolus-like bodies, and has been suggested by various groups to differ in its roles when compared to the X. laevis homologue. However, the exact roles of NPM2 in mammals remain to be fully elucidated. In this dissertation, attempts are made to determine the physical interaction sites between mouse NPM2 and core histone proteins, H2A, H2B, H3 and H4, as well as physical interactions between mouse NPM2 and protamines (sperm proteins) P1 and P2. Interaction sites between mouse NPM2 and various chromosomal proteins were investigated using a number of different techniques. First, NPM2: chromosomal protein binding assays were attempted to determine the ratio of NPM2 to both core histones and protamines. When visualized through 12% Native gels, NPM2 was determined to interact with histone octamers at a molar ratio of 1-1.5 mol NPM2/mol histone octamer. Mouse sperm protamines were determined to form complexes with mouse NPM2 at a molar ratio of 2.5 mol protamine/mol NPM2 (or mol protamine/0.4 mol NPM2). Analytical Ultracentrifuge (AUC) analysis was conducted on NPM2 and chromosomal proteins separately and in complex formation. Although determining that isolated, full length mouse NPM2 exists in a pentamer form, attempts with AUC were unsuccessful in determining specific NPM2:chromosomal protein binding affinity and complex formation. Specific physical interaction sites between NPM2 and chromosomal proteins were investigated using Cross Linking Mass Spectrometry. Here, a number of new interaction sites as well as sites previously identified by other groups were determined. In combination, our results present likely interaction sites between NPM2 and chromosomal proteins and represent an interesting point of reference for future work. / Graduate