Uveitis is a generic term for a wide variety of different types of intraocular inflammation with different clinical phenotypes and visual outcomes. The explanation for why some patients develop chronic anterior disease whereas others do not is unknown. It seems likely that host factors such as the cytokine milieu of the aqueous humor may be an important factor in determining outcome. In turn, their secretion is genetically determined and cytokine gene polymorphisms have been associated with high or low level production whatever the stimulus. Purpose: The aim of this study was to identify key cytokine and chemokine polymorphisms associated with disease susceptibility, clinical phenotype, and development of visually significant complications in patients with anterior uveitis. Methods: PCR amplification was used to genotype a number of biallelic SNPs in several cytokine genes. This genetic data was then compared between patients and healthy controls, and within the patient group itself for association with clinical disease outcomes. Results: Our results show that a significant difference in the frequency of TNF-857T allele in patients with idiopathic anterior uveitis. We found a significant association between TNF-308 allele G and patients with anterior uveitis who were HLA-B27 positive. Patients with HLA-B27 associated anterior uveitis who developed visually threatening complications were more likely to carry the TNFRSF1A-201T or TNFRSF1A-1135T alleles. In addition, the frequency of IL- 1ra allele T was found to be significantly associated with chronicity of the disease. The frequency of MCP-1 (-2076T) allele was found to be significantly higher in healthy individuals when compared to patients with acute idiopathic anterior uveitis. Conclusions: These results suggest that genetic variations in proinflammatory mediators may influence the susceptibility and severity of the inflammatory response in eyes of patients with anterior uveitis. This knowledge may be useful in identifying prognosis and responsiveness to anti-TNF blockade in patients with anterior uveitis.

Usher syndrome (USH) is the name given to a group of recessively inherited disorders characterised by hearing loss, progressive visual loss due to a retinal degeneration termed retinitis pigmentosa (RP) and in some cases vestibular dysfunction. It is the most common form of syndromic RP and is clinically and genetically heterogeneous. There are three clinical subtypes termed USH1, USH2 and USH3, which are defined by the severity of hearing loss and vestibular dysfunction with visual loss due to RP being common to each subtype. To date, mutations in nine genes have been associated with the three clinical subtypes of Usher syndrome as well as non-syndromic hearing loss and RP. This wide spectrum of clinical and genetic variability provides challenges to clinicians in making a diagnosis of Usher syndrome and delivering prognostic information to affected individuals, whilst the genetic heterogeneity presents problems to geneticists attempting to achieve a molecular diagnosis. This study aims to address these issues by determining the distribution of clinical and molecular subtypes of USH in the United Kingdom (UK). This study represents an original contribution to the knowledge of Usher syndrome, as it is the first prospective clinical study to sequence the coding regions of each of the nine genes associated with this disorder in 187 affected families regardless of their clinical subtype. Detailed ophthalmic phenotyping was performed in 219 individuals. This comprehensive strategy of molecular analysis afforded the opportunity to interrogate for the possibility of digenic effects for which no evidence was found. This strategy enabled the discovery of an atypical and novel phenotype associated with the USH1C gene. A molecular diagnosis was achieved in 80% of families with Usher syndrome and the ophthalmic phenotype of a large cohort of affected individuals with Usher syndrome has been further delineated. This study has resulted in a large cohort of UK patients with a confirmed molecular diagnosis and detailed ophthalmic phenotyping, which will provide a framework for subsequent longitudinal studies enabling the characterisation of how visual function progresses over time. Understanding the natural history of this disorder in genotyped individuals will help pave the way for subsequent gene-directed therapy studies in the future.

The symptomology associated with allergic diseases are a direct consequence of the release of pro-inflammatory mediators from mast cells following bi- or multivalent antigen cross-linking with the high affinity immunoglobulin (Ig) E receptor, FcεR1. Chemokines, small 8-15 kDa polypeptides, control the activation and recruitment of immune cells during the allergic response. Previous studies have demonstrated that co-stimulation by the chemokine, macrophage inflammatory protein-1α (Mip-1α) and cross-linking by IgE with antigen result in four phenomenon 1) enhanced degranulation in ex vivo conjunctival mast cells and rat basophilic leukemia (RBL-2H3) cell line via its chemokine receptor (CCR) 1, cell line also referred to as RBL-CCR1; 2) arrested Mip-1α-induced chemotaxis of RBL-CCR1 cells; 3) enhanced production of proinflammatory mediators from RBL-CCR1 cells and 4) enhanced gene expression in RBL-CCR1 cells of regulatory molecules downstream of CCR1 and FcεR1 signaling pathways, Regulator of G-protein Signaling (RGS)-1 and Tribbles (TRB)- 3. It has therefore been proposed that co-engagement of CCR1 and FcεR1 affects other mast cell processes such as chemotaxis, and moreover these data indicate cross-talk between CCR1 and and FcεR1 signaling pathways. Chemotaxis of mast cells to sites of inflammation and the subsequent release of pro-inflammatory mediators are key to eliciting allergic response. Although there is a vast amount of information pertaining to the molecular mechanisms of chemotaxis in several cell types, there is very little evidence to understand mast cell chemotaxis at this level. Based on current knowledge, the main objective of this thesis was to investigate 1) the effect of CCR1 and FcεR1 co-engagement on mast cell motility and 2) the role of RGS1 and TRB3 on mast degranulation, mediator release and chemotaxis. The data obtained from this thesis is the first to demonstrate the role of WASP, CCR1 and actin polymerisation as mechanisms underlying Mip-1α induced RBLCCR1 chemotaxis, using real time microscopy. Moreover, CCR1 and FcεR1 engagement inhibits RBL-CCR1 actin cytoskeletal re-organisation and significantly increases other cell motility parameters such as directionality and Euclidean distances which are required for efficient Mip-1α-induced chemotaxis. Also, by using a murine model of allergic conjunctivitis, conjunctival mast cells accumulate in the forniceal area of an inflamed conjunctiva in comparison to non-diseased vi mice. In addition, by using siRNA the present study is also the first to show that RGS1 and TRB3 serve as negative regulators of RBL-CCR1 degranulation, mediator release and chemotaxis upon CCR1 and FcεR1 engagement. In conclusion, the data presented in this thesis could advance our understanding of the mechanisms responsible for mast cell migration and arrest during an allergic response, and hence provide new targets for anti-allergic drugs.

Glaucoma is the term used to describe a group diseases characterised by a specific type of damage to the optic nerve head (ONH) known as cupping and a characteristic type of visual field loss. This loss is associated with progressive atrophy and loss of the retinal ganglion cells. Glaucoma is a leading cause of irreversible blindness in the world. This project was aimed at investigating olfactory ensheathing cells (OEC), a population of radial glia proven to be neuroprotective in central and peripheral nerve injury models, and their potential to protect the retinal ganglion cells in glaucoma. We studied the interactions of RGC and OEC in culture. We show that OEC can straighten, ensheath and bundle RGC neurites as well as support the survival of RGC and their synapses in culture. We also show that OEC endocytose dead RGC in culture. We modified a rat model of glaucoma (where paramagnetic microbeads are injected into the anterior chamber of the rat eyes) and characterised the early and late functional changes in the glaucomatous retina. We showed that RGC function was compromised in the early stages of glaucoma, before histological changes set in. We injected OEC into glaucomatous rat eyes to study the effects of OEC on optic nerve damage. The presence of OEC in the vitreous cavity of the glaucomatous rat eye significantly reduced the optic nerve damage in glaucomatous eyes. In summary, the work presented in this thesis provides an insight into • The functional changes of RGC in the early stages of experimental glaucoma and • Protection of RGC in experimental glaucoma by introduction of OEC into the vitreous.

The scarring response that follows glaucoma filtration surgery (GFS), an incisional surgery aimed at reducing glaucoma-related pressure increases within the eye, often prevents a successful outcome leading to further disease progression. Whilst a number of drugs have been shown to regulate scarring, a number of side effects are also induced largely related to the delivery method. Following on from previous work initiated in the lab whereby drugs (including the cytotoxic drugs, 5-fluorouracil (5-FU) and mitomycin-C (MMC), the anti-vascular endothelial growth factor (VEGF) monoclonal antibody, bevacizumab, and the matrix metalloproteinase inhibitor, ilomastat) had been fabricated into solid, slow release, implantable tablets, a number of questions remained pertaining to their effectiveness within a biological context. Using 5-FU, it was shown that concentrations at which the drug is released from the tablet can inhibit fibroblast activity (such as proliferation) as effectively as when using conventional concentrations for up to a 30 day period. Furthermore, it has been shown, for the first time, how gene expression within healing tissue (in-vivo) is altered upon the application of 5-FU at currently used clinical concentrations. Results from this study indicated that genes involved in apoptosis such as TP53, RelA, Bax, MYC and TXN were downregulated indicative of a response by the cells to limit the effects of apoptosis on tissue exposed to 5-FU. Secondly this thesis focuses on angiogenesis where it was shown that solid-dosage bevacizumab tablets functioned as effectively as bevacizumab solution in-vitro, with excipients of this drug such as trehalose shown to have implications in modulating wound healing. Furthermore, VEGF was shown for the first time to be a positiveregulator of fibroblast activity, such as in its ability to promote matrix metalloproteinase (MMP) production, presenting mechanisms by which anti-VEGF drugs may function to inhibit the wound healing process. Finally this thesis focuses on the effects of exposing cells and tissue to ilomastat, whereby microarray screening was utilised to uncover novel changes in gene expression that both regulate the direct activity of ilomastat, as well as highlight secondary effects of the drug. For example, a dampening of the immune response (such as in the downregulation of IL-1a, IL-6, IL-8, Bip and XBP1) was observed following ilomastat injection. Importantly however, these experiments also highlighted a previously uncharacterised foreign body response that was observed upon implantation of the ilomastat tablet in-vivo, with the widespread upregulation of genes involved in this process observed including TNF, FN1, IL-1β and IL-6. As such, whilst these experiments exposed novel mechanisms through which these drugs function, care will be required in moving forward with the use of such agents and delivery methods to ensure thatmaximum biocompatibility is achieved.

Visual information is sent from the retina to central visual targets through the optic nerve which is formed of retinal ganglion cells’ (RGCs) axons. In rodents, the superior colliculus (SC) is the major site of termination of retinal axons and the lateral geniculate nucleus (LGN) is another target of retinal axons. Glaucoma is a progressive optic neuropathy, characterized by RGC death. Dendrites are fine neuronal processes which support postsynaptic contact elements and are responsible for receiving synaptic signals. Accordingly, the morphology of dendrites has a profound impact on integrating neuronal input to the central nervous system from peripheral targets. Previous studies have documented dendritic changes in neuronal degenerative processes including those occurring in ageing and diseases. However, the morphological changes of dendrites in the visual pathway in glaucoma have not been well characterized. This thesis characterizes morphological changes of dendrites in the retina and the central visual targets in an experimental rat model of glaucoma for the first time. Dendritic labelling was achieved using the fluorescent dye DiI with in vivo and in vitro techniques. Dendrites of neurons in the SC and LGN were labelled in vitro using 0.1% DiI solution, and those of the RGCs were labelled using the biolistic technique. Confocal microscopy was next performed to image neurons, and dendrites were traced and quantified using Image J. Dendritic parameters including the mean dendritic length and the number of dendrites per neuron were analyzed in baseline, glaucoma animals and age-matched controls. Dendritic morphologies were studied in five types of neurons in the SC (including horizontal (H); piriform (P); narrow field vertical (V); wide field vertical (W) and stellate (S) cells), two types of neurons in the LGN (including the relay neuron type I (LG1) and type II (LG2)) and three types of RGCs (including type I (RI), type II (RII) and type III (RIII)). In this thesis, both age-related and glaucoma-related dendritic changes were demonstrated in the RGCs, SC cells and LGN cells in our rat model of experimental glaucoma. Firstly, the mean dendritic length and dendritic number of RGCs, SC cells and LGN cells were significantly reduced during ageing in normal animals, and more pronounced changes were observed in glaucoma animals. Secondly, significant dendritic shrinkage and losses were also shown in glaucoma animals compared with age-matched controls. Thirdly, the RGC was the first site to show dendritic changes following elevated IOP, but the most prominent changes were visible in the SC. The results in this study implicated that both the RGC and SC are potential sites for an early diagnosis strategy. Additionally, the glaucoma-related dendritic degeneration was demonstrated not only in the RGCs, but also in the SC and LGN, indicating that both the retina and the brain should be targeted when considering therapies for glaucoma. In conclusion, this thesis characterizes dendritic changes in the visual pathway in rats with chronic glaucoma, demonstrating that both ageing and elevated intraocular pressure (IOP) can affect dendritic morphology in the RGCs, SC cells and LGN cells. The findings in this study have contributed to the understanding of retinal and central neuronal degeneration in glaucoma, providing new insights into potential diagnosis and therapeutic strategies.

The retinal pigment epithelium (RPE) is a monolayer of cells that are vital for visual function and play a key role in the maintenance of the photoreceptors. Within RPE cells are melanin granules which absorb stray light and minimise scatter within the eye, therefore, protecting the RPE from damage. Albinos lack this protective capacity of melanin as a result of mutations of the tyrosinase gene. The first half of this thesis investigates heterogeneity within the RPE in both pigmentation phenotypes. Furthermore, the effect of ageing on the RPE is examined. Immunohistochemistry highlighted the molecular heterogeneity of the RPE and how this varies with pigmentation phenotype. In aged animals, SEM revealed abnormalities occurring within the photoreceptor outer segments of albinos, while ERGs and QRT-PCR illustrated that albinos show the signs of an age-related decline in visual function much sooner than pigmented animals. These studies revealed that in the outer retina, albinism is a progressive disease and not simply a congenital abnormality. The second half of this thesis investigates migration and proliferation of RPE cells in healthy pigmented animals. Using BrdU and DiI it was established that individual RPE cells have the ability to migrate. In addition, the effect of inducing lesions in different retinal locations was studied to determine whether this affects the response of RPE cells to the damaged area. The results conveyed that a single unilateral lesion in the RPE caused an upregulation of proliferating cells not only in the lasered eye, but also the contralateral unlasered eye in a quadrant specific manner. An additional experiment investigated the effect of treating rats with Glatiramer Acetate and found that it elevates levels of cell proliferation in the RPE of treated animals. Together, the data presented in this thesis demonstrate that the mature RPE is a dynamic heterogeneous epithelial cell layer.

Vascular remodelling and angiogenesis are commonly associated with sight threatening diseases such as age related macular degeneration (AMD) and diabetic retinopathy. Current treatments for retinal vascular pathology are limited to inhibitors of potent growth factors such as vascular endothelial growth factor (VEGF), which despite preventing any further vascular growth cannot mend the damage already done. Vascular abnormalities are associated with late stages of disease, meaning we are treating patients too late. We must identify new, earlier therapeutic tagets to preserve vision. Gene expression profiling was performed on retinal vessels isolated from three mutant mouse strains (Curlytail, VLDLR-/- and RD1) that display retinal vascular abnormalities. Sixty-two genes were found to change in all three models. One gene which was significantly up-regulated was LBP (lipopolysaccharide binding protein). This is an acute-phase response glycoprotein, which through its binding to LPS and activation of TLR4 is involved in inflammatory signalling. We have tested the hypothesis that LBP has a novel function separate from its characterised LPS recognition. qPCR analysis of VLDLR-/- mice has shown LBP to be expressed in the neuroretina and isolated vessels. qPCR data has shown LBP to be up-regulated in VLDLR-/- mice just prior to an increase in VEGF expression and the vascular abnormalities being observed. Testing LBP affects in Matrigel, Aortic ring and Metatarsal assays revealed an increase in vessel sprouts and branching. Western blot analysis has suggested LBP can induce phosphotyrosine and phophoERK responses in a variety of cell lines and immnocytochemistry data provides evidence that this is not occuring through the well-established LBP-LPS NfkB pathway. PCR analysis of older passage HUVEC which are unresponsive to LBP has given us a candidate receptor. In conclusion, we have revealed a potential role for LBP in contributing to angiogenesis.

The purpose of this project is to identify the causative gene for one type of autosomal recessive retinitis pigmentosa, RP25. Through CGH (comparative genome hybridisation) and mutation screening, independent mutations were identified in arRP affected Spanish families mapping to RP25. These mutations were identified within a cluster of uncharacterised gene transcripts all which have EGF-like repeat domains; Q5T669, Q5T1H1, Q9H557_human, Q5TEL3_human, Q5TEL4_human, Q5VVG4_human, and Q5T3C8. Through 5` and 3` RACE PCR analysis, the full length gene was revealed to incorporate the EGFL11 gene. On assembling all available data we noted that RP25 gene encompasses 30 exons belonging to nine previously predicted genes and 13 newly identified exons, totaling 43 exons and spanning the interval between 64,487,835 and 66,473,839 on chromosome 6q12. The RP25 full length gene transcript is retinal specific. The genomic length covers over 2.0 MB in size and is therefore the largest eye specific gene identified to date. It is also the fifth largest gene in the human genome to date. Homologs of the RP25 gene to Drosophila eys/eys-shut (Spacemaker) were identified, leading to the annotation of the name EYS (SPAM). An apparently intact eys gene is found across the mammalian clade, including monotremes (platypus) and marsupials (opossum). However, despite the mutations and the presumed loss of function associated with human disease, this gene has been dispensed with on at least four separate occasions in the last 100 million years of mammalian evolution including in the armadillo (Dasypus novemcinctus), little brown bat (Myotis lucifugus) and ruminant (cattle and sheep) lineages. EYS has acquired several (<3) reading-frame disruptions in three rodents (mouse, rat and guinea pig) representing two of the three major rodent clades. Through immunohistochemical and electron microscopy analysis, a signal for SPAM was identified in the outer segments of photoreceptor cells.

Genetic defects in AIPL1 cause a heterogeneous set of clinical conditions depending on the severity of the mutant alleles. Diseases can range from Leber Congenital Amaurosis (LCA), the severest form of early-onset retinal degeneration, to milder forms such as retinitis pigmentosa (RP) and cone-rod dystrophy. There is currently no effective treatment for LCA and inherited retinal dystrophies, which are the commonest cause of childhood blindness. AIPL1 is expressed primarily in retinal photoreceptors and is required for the biosynthesis of photoreceptor phosphodiesterase (PDE). This thesis describes a programme of work that examines the potential and efficacy of gene replacement therapy in the treatment of AIPL1- associated retinal diseases. It centres on the use of recombinant adeno-associated virus for the transfer of murine and human AIPL1 cDNA into photoreceptor cells. AAV-mediated gene replacement was assessed in two genetically engineered mouse models carrying null and hypomorphic alleles, Aipl1 -/- and Aipl1 h/h mice, which simulate retinal degenerations similar to human LCA and RP respectively. Three different rates of photoreceptor degeneration were simulated using the mouse models. To treat the different rates of degeneration, two pseudotypes of AAV (serotype 2 and 8) exhibiting different transduction kinetics were used for gene transfer. Substantial and long term rescue of the disease phenotype was seen as a result of Aipl1 transgene expression mediated by AAV2/2 vector in Aipl1 h/h mice and by AAV2/8 in rapid degenerations in light accelerated Aipl1 h/h mice and in Aipl1 -/- mice. Thus, the results presented in this thesis validates the efficacy of AIPL1 gene replacement using AAV vectors in varying rates of degeneration that reflected the clinical spectrum of disease. This is the first study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of rapid photoreceptor degeneration. The development of an efficient therapy depends on the identification of patients and characterisation of disease phenotype. A panel of DNA samples from patients with LCA and early onset severe retinal dystrophy was screened for mutations in the AIPL1 gene. Patients identified with AIPL1- associated disease demonstrated varying severity of disease from LCA to milder form of rod cone dystrophy. Clinical characterisation and imaging of the patients highlighted distinctive features which will direct future identification and molecular screening of patients. Residual retinal integrity and function in young patients and patients with milder phenotype suggests that AIPL1 defects may be amenable to treatment.