Cross-linked Actin Networks (CLANs) were first identified in bovine trabecular meshwork (BTM) cells in response to steroid treatment. Since then, work within our group has identified CLANs in human TM (HTM) cells and human tissue ex vivo. An alteration in the cytoskeleton of TM cells has been associated with changes in outflow facility and is a promising therapeutic target in glaucoma research. CLANs which are believed to make TM cells more rigid, were found in increase numbers in tissue from glaucomatous donors, but were also present in tissue from non-glaucomatous donors. This would indicate that CLAN formation is not merely a steroid response but may be triggered by changes within the outflow system. The current work set out to identify CLAN inducing agents present within the normal outflow system and questioned whether age-related changes within the anterior chamber could influence CLAN number in a similar manner to glaucoma. Investigating the influence of aqueous humor (AH) and some of its constituent growth factors on CLAN formation in both BTM and HTM cells revealed that AH itself was capable of inducing CLANs and that the main CLAN inducing agent present was TGF-β2. Conversely mitogenic growth factors (FGF and HGF) did not increase CLAN incidence and implicated that CLAN incidence was linked to cell shape change linked and therefore to function. In vitro analysis of CLANs in reference to donor age revealed that TGF-β2 could induce a higher percentage of CLANs in HTM cells from older donors compared to HTM cells derived from younger donors. Ex vivo analysis run in parallel demonstrated that CLAN incidence increased with increasing donor age. In order to identify how age-related stress within the TM environment influenced CLAN incidence cells were treated with hydrogen peroxide. In BTM cells pre-treatment for 1 hour was sufficient to significantly increase CLAN incidence. In further experiments it would seem that CLANs are not associated with apoptotic cells nor with senescent cells. The findings of this study indicate that CLANs can form when no glaucomatous pathology is detected and that changes in the extracellular environment can influence their formation. This is especial evident in older tissue.

Over expression of lens epithelium derived growth factor (LEDGF) is described in a number of different tumours. In addition, self-antibodies against this protein are detectable in patients with lymphomas and chronic lymphocytic leukemia (CLL). Alternative splicing of LEDGF results in a number of different isoforms, and thus the initial aim of this thesis was to determine the variability in LEDGF isoform expression in a cohort of CLL cases. Utilising RT-PCR, it is shown that CLL cells variably express four different LEDGF isoforms. The study was then extended to quantitate the expression levels of these four isoforms in individual CLL cases, and to compare them to normal B cells. RT-qPCR analysis showed that the longest of these isoforms, p75, is significantly over expressed, whilst the shortest form, p52bΔE6 is significantly under expressed in CLL, compared to normal B cells. All four isoforms of LEDGF are generally over expressed in CD38 positive CLL cases compared to those that are deficient for surface expression of this molecule. Alternative splicing occurs as a result of the inclusion or exclusion of different exons within the mRNA. This process is governed by the splicing machinery, of which Splicing Factor 3B, subunit 1 (SF3B1) is a critical component. Recently, it has been shown that mutations within SF3B1 are recurrent in CLL. Such mutations impact on the splicing of a significant array of genes within the cell. One of the genes predicted to be affected is LEDGF. Therefore, the mutational status of SF3B1 was examined in the same cohort of CLL cases and correlated with the expression of the LEDGF isoforms. As well as the commonly reported mutations in SF3B1, a novel nonsense mutation was identified. Although not statistically significant, cases with unmutated SF3B1 have higher levels of all isoforms of LEDGF and a possible explanation for this is discussed. That two of the major isoforms of LEDGF (p75 and p52) have differing functions within the cell is well documented and can be explained by virtue of their participation in distinct protein complexes. In order to identify components of the complexes generated by the four LEDGF isoforms identified in this study, stable cell lines exogenously expressing these isoforms were generated. Each of the isoforms was ‘double-tagged’, and a purification protocol optimised that would allow successful characterisation of the complex associated with each isoform. The results provide useful insights and provoke thought for the role of alternative splicing in the altered phenotype of cancer cells and its contribution as an additional epigenetic mechanism in normal and malignant gene regulation.

This thesis describes the water chemistry of the River Dee and its tributaries, and the potential water chemistry changes that may occur under acid deposition and land use change scenarios. Historic water quality and flow records were collated and supplemented with new water chemistry data. These data were analysed in relation to catchment geography and river flow using both mathematical modelling and novel, GIS based techniques. This analysis established the importance of diffuse inputs and highlighted differences between upland and lowland regions in the catchment. In headwater streams, different geological types create hydrochemical source areas that strongly influence stream chemistry whilst in lowland tributaries, agricultural sources are particularly important. In the upland region most major ions were diluted as flows increased, further emphasizing the influence of deeper geological sources on baseflow chemistry, but showing soilwater controls on stormflow composition. The headwaters, which drain predominantly acid rocks, are presently oligotrophic but threatened by the impact of acid deposition and land use change (re-afforestation). In some of the lowland tributaries, increased NO3-N concentrations have resulted from more intensive land management. The potential impacts of acid deposition and land use change were simulated in both upland and lowland catchments by considering existing and new models within a Functional Unit Network. For upland regions this consisted of developing a new, two component hydrochemical mixing model to simulate the spatial and flow-related variations in streamwater acidity. The mixing model was based on End Member Mixing Analysis (EMMA), and site specific end members (alkalinity and Ca) could be predicted from emergent catchment characteristics (soil and land use) using linear regression.

The ocular microenvironment is immunosuppressive in animal models of antigen presenting cell function. My hypothesis was that in humans the normal ocular microenvironment maintains an immature dendritic cell (DC) phenotype, whereas in intraocular inflammation (uveitis) this regulation fails, permitting full DC maturation leading to the production and recruitment of pathogenic effector T cells to the eye. Using an in vitro model of DC function, I observed that non-inflammatory aqueous humour (AqH) inhibited DC maturation, with reduced MHC and CD86 expression, and reduced capacity to induce proliferation of allogeneic T cells, an effect which was cortisol and TGFβ2 dependent. In contrast, exposure to uveitis AqH generated a distinct DC profile with IFNγ dependent elevation of MHC class I, but reduced MHC class II and CD86 expression and impaired induction of T cell proliferation. Exposure to uveitis AqH from patients on topical glucocorticoid treatment caused additional suppression of CD86. Characterisation of ex vivo myeloid DC from patients with uveitis supported the findings of the in vitro model, with AqH-derived myeloid DC showing elevated MHC, but reduced CD86 expression. In summary human AqH is shown to be a powerful inhibitor of DC maturation, retaining this regulatory role during uveitis.

Thyroid-Associated Ophthalmopathy (TAO) is a debilitating inflammatory condition of the orbit occurring in 30-50% of Graves' Disease (GD) patients. It is not currently possible to predict which GD patients will develop TAO or the severity of their eventual ophthalmic manifestations. The aim of this thesis was to evaluate novel biomarkers for this purpose. I developed two immunoassays to detect serum antibodies to insulin-like growth factor-1 receptor (IGF-lR-Ab) in GD, TAO and healthy controls (HC). Assays were validated to measure commercial monoclonal IGF-lR-Ab but no study group differences, or correlation with clinical activity or severity, were noted with sera. Differential IGF-lR expression on peripheral blood CD4+ and CDS+ T lymphocyte memory subsets was observed, although without variance between groups. However, T cell differentiation was perturbed, with elevated proportions of naïve, and reduced cytokine-producing effector memory T cells, in GD and TAO compared to HC. Nuclear magnetic resonance-based serum metabolomic analysis differentiated GD and TAO subjects, and varying TAO clinical activity, with good uncorrected sensitivity and specificity. Distinguishing metabolites included lactate, isopropanol, methylguanidine and pyruvate. Collectively these data cast doubt on a simple model of IGF-lR-Ab being responsible for orbital inflammation in GD, but highlight the biomarker potential of metabolomics in TAO.

Haemopoietic Stem Cell Transplant (HSCT) is used as a treatment for a number of conditions particularly leukaemias. Following conditioning and HSCT, there is a ‘resetting’ of the immune system, which reconstitutes over a number of months. Graft-versus-Host Disease (GvHD) is a life-threatening complication of HSCT that includes severe, sight-threatening dry eye disease. In GvHD transplanted immune cells mount an immune response against the host. This thesis investigated how the immune cells of the conjunctiva are affected by HSCT and how the ocular surface leukocytes reconstitute. A non-invasive technique, ocular surface impression cytology (OSIC), was used to demonstrate that whilst there was no apparent depletion of innate immune cells in the conjunctiva, there was a marked reduction in the lymphocytes, which gradually reconstituted, returning to normal levels at the 6 months timepoint. Secondly OSIC was used to profile the leukocyte population in a cohort of patients post-HSCT with and without eye disease. In patients with dry eye disease following HSCT, the conjunctiva contained increased CD8+ lymphocytes, macrophages and neutrophils; a pattern that was distinct to that found in patients with dry eye disease following HSCT.

Ocular Mucous Membrane Pemphigoid (OcMMP) is a blinding immunobullous disease, characterised by auto-antibody driven conjunctival inflammation and scarring. My hypothesis was that progressive fibrosis in OcMMP, occurring in the apparent absence of clinical inflammation, was driven by underlying inflammatory processes. I observed that in OcMMP, progressive scarring did occur in the apparent absence of clinically identifiable inflammation and I was able to improve clinical documentation by developing and validating an objective Fornix Depth Measurer (FDM) for assessment of scarring. I optimised non-invasive Ocular Surface Impression Cytology (OSIC) combined with flow cytometry to characterise conjunctival leukocytes. I found that CD8αβ+ effector memory, cytotoxic, mucosal-homing T cells were the dominant population in health. This population was unaltered with age but CD4+ T cells, capable of producing IFN-γ, increased. In OcMMP, the conjunctiva was characterised by decreased CD8+ lymphocytes and an elevation in CD45INTCD11b+CD16+CD14- neutrophils. Although neutrophils correlated with clinical inflammation, they were even present in the absence of identifiable conjunctivitis. This elevation was associated with progression of scarring assessed by FDM, even in the clinically Non-inflamed eye. These findings confirmed my hypothesis and provide a platform for quantifying neutrophils as a biomarker of sub-clinical inflammation and their role in the scarring process.

Cataract is the opacification of the crystalline lens of the eye. Both childhood and later-onset cataracts have been linked with complex genetic factors. Cataracts vary in phenotype and exhibit genetic heterogeneity. They can appear as isolated abnormalities, or as part of a syndrome. During this project, analysis of syndromic and non-syndromic cataract families using genetic linkage studies was undertaken in order to identify the genes involved, using an autozygosity mapping and positional candidate approach. Causative mutations were identified in families with syndromes involving cataracts. The finding of a mutation in CYP27A1 in a family with Cerebrotendinous Xanthomatosis permitted clinical intervention as this is a treatable disorder. A mutation that segregated with disease status in a family with Marinesco Sjogren Syndrome was identified in SIL1. In a family with Knobloch Syndrome, a frameshift mutation in COL18A1 was detected. Analysis of families with non-syndromic autosomal recessive cataracts was also performed, identifying homozygous candidate regions, and sequencing candidate genes within these regions. The identification of a potential putative mutation in one family in CDC25A illustrated the challenges of distinguishing between rare benign variants and pathogenic mutations. Identification of novel genes involved in cataractogenesis will increase understanding of the pathways involved in cataract formation, and benefit affected families through genetic counselling, and, potentially personalised management.

Betaglycan, also known as TGFβRIII, is a component of the TGFβ receptor, being thought to act as a co-factor in signal transduction. It has proven roles in TGFβ-mediated embryogenesis and carcinogenesis, and facilitates signalling by activins and inhibins within the endocrine system. Despite the recognised importance of TGFβ in immunity, little is known about betaglycan in immune regulation. In mice, absolute deficiency of any of the three isoforms of TGFβ, or any of the individual TGFβ receptor components results in embryonic or perinatal lethality, limiting the ability to study peripheral immune responses in these models. We developed a chimeric model in which betaglycan-deficient stem cells were transferred to immunodeficient hosts, allowing study of mature animals with betaglycan deletion restricted to T and B lymphocytes. We were thus able to investigate peripheral immune responses in vivo, and assess TGFβ signalling to T lymphocytes in vitro, in the presence and absence of betaglycan. Betaglycan deficiency resulted in upregulated CD4+ and CD8+ T lymphocyte activation, with increased Th1 polarisation in peripheral lymphocyte compartments in both naïve and antigen-experienced animals. These observations confirm the involvement of betaglycan in T lymphocyte biology, providing the first evidence for its role in regulation of peripheral immune responses.

Introduction A risk factor for POAG is elevated intraocular pressure induced by reduced outflow of aqueous humour through the fibrosed trabecular meshwork, causing retinal ganglion cell death. This study aimed to: 1) create a rodent model of increased intraocular pressure by inducing trabecular meshwork fibrosis; 2) evaluate the ability of the anti-fibrotic agent, Decorin to diminish trabecular meshwork fibrosis and lower intraocular pressure; 3) correlate these findings to RCG survival and 4) investigate alternative translational methods for delivery of Decorin into the eye. Methods Fibrotic agents, kaolin or TGF-β, were intracamerally injected to induce fibrosis and intraocular pressure elevation. Following this human recombinant Decorin was administered intracamerally. Fibrosis was measured by immunohistochemistry and electron microscopy. Retinal ganglion cell counts were performed from retinal whole mounts. Results TGF-β induced fibrosis of the trabecular meshwork leading IOP elevation and retinal ganglion cell death, thus providing a reliable in vivo model of the ocular fibrosis. Decorin reversed the TGF-β induced fibrosis in the trabecular meshwork, lowered intraocular pressure and indirectly prevented retinal ganglion cell death. Decorin was successfully delivered to the anterior chamber in an eye drop formulation. Conclusion Decorin may be an effective treatment to preserving visual function in patients with POAG.