No description available.

During developmental growth, tissue repair and disease the vasculature is remodelled and expanded through the process of angiogenesis to enhance oxygen and nutrient supply. Sprouting angiogenesis is the principal mechanism by which this occurs and takes place following the activation of endothelial cells (ECs) and surrounding pericytes by pro-angiogenic signals. Subsequent degradation of the basement membrane and invasion of ECs into the surrounding extracellular matrix leads to the formation of an angiogenic sprout that forms contacts with neighbouring vessels and undergoes lumen formation and stabilisation to produce a functional vessel through which blood can flow. This process requires dramatic changes in EC polarity and shape that is orchestrated by a highly dynamic cytoskeleton. Previous work from our lab identified the formin FMNL3 as an important regulator of sprouting angiogenesis. The mechanistic understanding of FMNL3s function in this process is however poorly understood. Through the use of in vitro assays of endothelial migration and polarity FMNL3 was found to be important for the reorientation of cell polarity during endothelial migration. Following the formation of an angiogenic sprout ECs need to become less invasive and reorient their polarity along their apical-basolateral axis to allow lumen formation. The function of FMNL3 in endothelial polarisation during migration led to the investigation of a role for FMNL3 in apical-basolateral polarisation. FMNL3 was found to be required for the localisation of the protein Podocalyxin-like 1 (PODXL) to the apical surface of ECs through the assembly and remodelling of endothelial apical actin filaments. This function of FMNL3 was found to occur downstream of RhoJ. PODXL associates with apical actin filaments and is transported along it through Myosin Vb and Rab25. PODXL is important for specification of the apical surface prior to lumen formation and this study identifies FMNL3 as an important protein in the localisation of PODXL to the apical membrane through regulation of the endothelial cytoskeleton.

Autophagy is a lysosomal degradation pathway conserved from yeast to man, in which a double membrane-bound vesicle (the autophagosome) is assembled to engulf and sequester cytoplasmic material. Autophagy serves a multitude of protective roles in the cell including removal of dysfunctional or redundant macromolecules and organelles, engulfment of pathogens and provision of molecular building blocks upon nutrient deprivation. Autophagy is highly regulated, with more than 30 proteins having been identified in the process. This thesis explores the molecular regulation of amino acid and growth factor starvation-induced autophagosome biogenesis. First, I explored the regulation of autophagosome biogenesis dependency on Atg4 dependent LC3B delipidation. Through a siRNA approach I found that the LC3B puncta formation response upon starvation was surprisingly robust i.e. siRNA depletion of Atg4B has no discernable effect on LC3B puncta numbers. However when using LC3B-mutants impaired in Atg4 binding, I found that LC3B delipidation appears to be involved in regulating autophagosome size and p62 turn-over during starvation. With the aim of identifying a novel autophagy protein involved in the completion of the autophagosome, I conducted a small-scale, targeted siRNA screen for ER and mitochondria resident proteins. From this screen I identified SigmaR1 and Climp-63 as important for the completion of the nascent autophagosome. SigmaR1, which caused the greatest accumulation of incomplete autophagosomes, is a protein that has been implicated in a wide variety of processes, including calcium regulation, ER stress response and lipid transfer. Climp-63 is an ER sheet organising protein. Thus, overall this thesis extends the knowledge of Atg4 dependent delipidation during autophagosome biogenesis and the role of ER and mitochondria- resident proteins during autophagosome formation.

Podocytes are highly specialised epithelial cells, located on the urinary side of the glomerular filtration barrier. Disruptions to podocyte structure and function results in a disruption to the filtration process and proteinuria. Therefore, an understanding of factors that influence podocyte biology is desirable. Podocytes respond to insulin both in vitro and in vivo and the podocyte-specific knock-down of the insulin receptor results in a glomerular pathology with features characteristic of diabetic nephropathy. There is also evidence that podocyte insulin signalling is disrupted in models of insulin resistance and diabetes. The aim of this thesis was to investigate how both positive and negative modulators of insulin signalling specifically influence podocyte responses in vitro. Initial work was undertaken to characterise the responses of a recently developed mouse podocyte cell line. These cells express the insulin and insulin-like growth factor {IGF)-I receptors, and the phosphorylation of these proteins is evident following insulin stimulation. Downstream of receptor activation, increased phosphorylation of insulin receptor substrate (IRS) proteins and activation of phosphoinositide 3-kinase {PI3K)/Akt and MEK/ERK mitogen activated protein kinase (MAPK) cascades is observed . Furthermore, these cells activate glucose transport pathways following insulin stimulation. The effects of the anti-diabetic agent metformin and the selective chemical inhibition of protein tyrosine phosphatase 1B (PTP1B) were next examined, as potential enhancers of podocyte insulin signalling. Data herein demonstrates that these chemicals have direct effects on mouse podocytes in vitro and influence cellular glucose uptake. The modulation of podocyte signalling by diabetic factors, specifically the chronic exposure to high insulin, high glucose and inflammatory cytokines, was also investigated. Data reveals that the exposure of podocytes to a combination of these diabetic factors disrupts the activation of insulinstimulated signalling and glucose uptake. Individually, these factors exert their effects at different points within the signalling network. Quantitative peR arrays were also performed to identify changes at the mRNA level. Interestingly, the mRNA encoding the neurotransmitter Neuropeptide Y (NPY) was down regulated by 7-fold following cytokine exposure. Further investigation demonstrates that this peptide signals to both human and mouse podocytes in vitro stimulating the phosphorylation of Akt and ERK1/2, as well as influencing calcium signalling. Overall, work within this thesis demonstrates both the positive and negative modulation of podocyte insulin signalling in vitro and identifies a novel role for NPY in podocyte biology.

The aim of this thesis was to investigate the effects of cyclical mechanical stimulation on chondrocyte proteoglycan (PG) metabolism and to explore the mechanisms involved in the transduction of the mechanical stimulus into a biochemical response. Chondrocytes were exposed to cyclical pressurisation in an apparatus that functions to produce strain of the base of culture dishes with deformation of attached cells. PG synthesis during pressurisation, was increased significantly compared to controls following 6 h and 3 h cyclical pressure-induced strain (PIS). These experiments provided the first evidence that stretch-activated ion channels are involved in the signal transduction process that leads to accelerated PG synthesis following cyclical deformation of chondrocytes. Two antibodies (3B3 and 7D4) raised against epitopes within the chondroitin sulphate chains of cartilage PG have been shown to be of particular value in detecting structural changes early in the development of OA. Methods were established for the measurement of these epitopes and normal chondrocyte glycosaminoglycan (GAG) epitopes by flow cytometry. 3B3 and 7D4 epitopes were expressed at low levels in both human and bovine chondrocytes from normal tissue. There was no increase in their expression up to 24 h after stimulation of chondroytes for 3 h with cyclical PIS. A new apparatus was developed for the production of cyclical PIS, which could accommodate two strained and two unstrained dishes under conditions of pressurisation. This apparatus proved suitable for further studies of the signal transduction mechanisms involved in responses to PIS. Chondrocytes treated with adhesion-blocking anti-α5 and anti-β1 integrin antibodies had a reduced response to strain, whereas those treated with an adhesion enhancing anti-β1 integrin antibody results in an increased response. These results implicit α5β1 integrin, which is the cell surface receptor for fibronectin, as a mechanotransducer in chondrocytes. The studies demonstrate that chondrocytes respond to cyclical deformation by increasing PG synthesis but that the expression of GAG-epitopes associated with OA was not increased.

In the first part of the thesis, temporal and spatial localisation of several different proteins has been studied using immunocytochemistry. Novel results regarding localisation of androgen receptor and inhibin subunits are reported which demonstrate differences in their patterns of expression in fetal and adult Leydig cells. Specifically, in contrast to their adult counterparts, fetal cells do not express androgen receptor, suggesting that the negative feedback loop of testosterone on its own production, established in adult testis via the androgen receptor, is not functional in the fetus. Inhibin α and βB subunits were localised to Leydig cells from days 14.5 and 16.5 of gestation respectively. In addition, functional differentiation of fetal Leydig cells has been studied by examining the expression of two steroidogenic enzymes 17α-hydroxylase/17,20-lyase (P450c17) and 3β-hydroxysteroid dehydrogenase (3β-HSD) and two orphan nuclear receptors, steroidogenic factor-1 (SF-1) and DAX-1. As expected 3β-HSD and P450c17 immunolocalised exclusively to fetal Leydig cells. Data on immunoexpression of DAX-1 has not been reported previously; DAX-1 protein was first detectable in the fetal rat testis on day 15.5 in the interstitial cells, simultaneously with the onset of testosterone production. Co-localisation of SF-1 and DAX-1 in the fetal testis demonstrated that both proteins are not exclusively co-localised in the same cell types, a finding at odds with suggestions that SF-1 regulates expression of DAX-1. The second part of the thesis describes experiments designed to elucidate potential mechanisms underlying the influence of oestrogens on the developing fetal testis. A significant decrease in mRNA expression and enzyme activity of P450c17 occurred in fetuses of mothers treated with DES or the environmental oestrogen octylphenol (OP) but this was not mirrored by an obvious reduction in 3β-HSD immunostaining.

No description available.

Oxidative DNA damage is thought to play a role in the aetiology of ageing and a number of diseases including cancer, chronic inflammation, ischemia, degenerative arterial and autoimmune diseases. 8-oxodeoxyguanosine (8-oxodG), an oxidative DNA adduct, has gained much popularity as a biomarker of damage to DNA. In this thesis the reliability of 8-oxodG as a marker for oxidative stress and its involvement in ultraviolet (UV)-mediated DNA damage in cells was investigated. Because of our concern and those expressed in the literature over the possible induction of artefactual 8-oxodG during phenol extraction procedures, a comparative study on DNA extraction methods was undertaken. It was found that phenol isolation of DNA yielded higher levels of 8-oxodG when directly compared to pronase E isolation, irrespective of the model of oxidative stress used to treat DNA or cells. Furthermore results from peripheral blood mononuclear cells from patients under oxidative stress (systemic lupus erythematosus and rheumatoid arthritis) showed a high degree of variability leading to inconclusive results. This lead to investigation of alternative methods of analysis of the 8-oxodG lesion. 8-oxoguanine is measured conventionally as the deoxynucleoside by high performance liquid chromatography with electrochemical detection (HPLC-ECD) or as the free base by gas chromatography combined with mass spectrometry. A 'hybrid' analysis of the 8-oxoG base by HPLC was established. The new procedure combined formic acid hydrolysis of DNA with guanase treatment of the resultant base mixture. Guanase was found to specifically degrade guanine thus allowing reversed-phase HPLC quantitation of 8-oxoG. This procedure avoided problems with enzymes used to degrade DNA to deoxynucleosides which may lack specificity toward oxidatively damaged DNA. A reliable method to measure oxidative DNA damage, including 8-oxodG, at ultra-low levels, without artefactual generation of 8-oxoG which is suggested to be unavoidable when extracting and/or derivatising DNA, is clearly of great importance. Therefore, as a model, a polyclonal antibody to UV-induced DNA damage was developed and utilised in flow cytometric and immunocytochemical techniques to detect UV-mediated DNA damage in cells. Ultraviolet radiation has been reported to produce a number of potentially mutagenic photoproducts and has consequently been implicated in skin tumourigenesis. The polyclonal antibody utilised was successful at detecting UVB-induced DNA damage and UVA-induced single strand breaks. The levels of DNA damage and p53 expression following UVA irradiation of keratinocytes were found to be cell-cycle and dose dependent. UVA was found to cause a dose-dependent increase in 8-oxoG formation also as determined by HPLC. Therefore it is likely that oxidative DNA damage has a role in UVA-induced DNA damage in cells. It is concluded that the use of specific antibodies may represent an accurate and reliable measure of oxidative lesions directly m cellular DNA as an alternative to procedures that require DNA extraction.

No description available.

No description available.