Retinal detachment contributes to nearly 500 new blind registrations in the United Kingdom each year. In contrast to other retinal blinding disorders, blindness from retinal detachment is potentially avoidable with a better understanding of the mechanisms defining sub-groups at risk of the event. The majority of retinal detachments are rhegmatogenous, resulting from retinal tears that occur during the process of posterior vitreous detachment. Posterior vitreous detachment is generally considered to be a common, age-related synchitic and syneretic degeneration of the vitreous gel. However, this current understanding fails to explain the significant number of elderly individuals who never undergo posterior vitreous detachment, or the number of young patients with co-existing intraocular pathology who do. Furthermore, the factors distinguishing the majority of patients who undergo ‘physiological’ posterior vitreous detachment (with no associated retinal tears or detachment) from the minority of patients who suffer ‘pathological’ posterior vitreous detachment (associated with retinal tears and/or detachment), remain poorly understood. The objectives of this research project were two-fold: Firstly, to investigate the hypothesis that appropriate prophylactic intervention could reduce blindness from retinal detachment if a high-risk sub-group of individuals were defined. This clinical study retrospectively evaluated a group of molecularly confirmed type 1 Stickler syndrome patients, a homogenous cohort who have been identified to carry the greatest risk of inherited retinal detachment at the time of their posterior vitreous detachment. Multiple analyses comparing patients and eyes that received prophylactic intervention with appropriate controls, consistently demonstrated that the Cambridge Prophylactic Cryotherapy protocol is safe and markedly reduces the risk of retinal detachment in type 1 Stickler syndrome. Secondly, to investigate the anatomical and cellular mechanisms of posterior vitreous detachment in the wider population. This laboratory study sought to isolate and immunohistochemically phenotype posterior hyaloid membranes and associated laminocytes from donor human globes that had undergone ‘physiological’ posterior vitreous detachment. The isolated posterior hyaloid membranes were demonsatrated to be distinct basement membranes composed of type IV collagen and laminin, and morphologically correlated with posterior hyaloid membranes observed clinically in patients presenting with posterior vitrous detachment. Furthermore, the laminocyte cell population adherent to the vitreal aspect of the posterior hyaloid membrane, was identified to express macrophage cell markers.

Corneal dystrophies are a group of inherited eye diseases that affect corneal transparency in humans. I am interested in a subgroup of human corneal dystrophies caused by mutations in the gene Transforming growth factor β-induced (TGFBI). In order to explore the possibility of using Xenopus laevis as a model system to study TGFBI-associated corneal dystrophies, I carried out investigations into Xenopus corneal development, the role of Xenopus TGFBI (XTgfbi) during embryogenesis and the expression pattern of XTgfbi in cornea. The thesis first describes the full process of corneal development in Xenopus laevis. Both light and electron microscopy were used to illustrate the overall structure of the cornea, as well as the subcellular details, throughout the course of development. Interesting structures such as a stroma-attracting centre, multilayered double embryonic cornea and intra-stromal epithelium were described. Overall, Xenopus corneal development is highly complex, and its similarities and differences with that in other model organisms will be discussed in depth. In addition, I performed proliferation assays and found cells with proliferative capacity in all three cellular layers in the mature cornea. The second part of the thesis focuses on the functional analysis of extracellular matrix protein XTgfbi during embryogenesis. After confirming previous data of gene expression and knock-down phenotypes, I showed that XTgfbi specifically regulates the canonical Wnt signalling pathway using semi-quantitative reverse transcription – polymerase chain reaction (RT-PCR), confirmed by luciferase reporter assays. A final set of experiments in this section suggests integrin-linked kinase might mediate XTgfbi’s function in Wnt signalling regulation. The third part of the thesis describes the expression pattern of XTgfbi during Xenopus corneal development. It was found that XTgfbi was present in different types of corneal cells during the course of development. The dynamic nature of its expression pattern implicates its complex yet important roles in this process.

The transport capacity of the retina plays a major role in maintaining health of the photoreceptors cells. In particular, age-related changes in the Bruch’s membrane(BrM) correspond to a decrease in the transport of nutrients and waste products across the Bruch’s. Matrix metalloproteinase(MMP), an extracellular matrix regulator was found to be sequestrated in aged BrM, with decreased availability for activity. Hence, it was hypothesized that a plausible target to rejuvenate the transportation across BrM and prevent the age-related processes in the macular was to increase the activity of MMP in BrM. Previous studies have shown an increase in MMP release secondary to RPE migration initiated by deliberate injury using lasers. However,conventional lasers causes collateral photoreceptor cell damage via primary thermal denaturation and secondary apoptosis. The nanosecond laser used in the present study with its speckled beam configuration and nanosecond pulse is designed to limit the primary damage profile. The main objectives of this PhD was to determine if this novel laser was a viable option to improve transport across BrM while preserving photoreceptor function, further to ascertain its efficacy and safety for use in human subjects to delay or prevent onset of AMD. Laboratory results from this thesis confirmed the importance of MMPs but demonstrated the inadequcy of the speckled beam configuration at higher energy levels. Corroborative clinical results also found beam profile inadequacy with the suprathreshold nature of the current energy-dose but desmonstrated minmal collateral damage. Clinical evaluation found a short term improvement of visual function. It may be concluded this novel laser has potential to be used as a therapeutic modality to delay AMD, but further work needs to be done to address the current issues of beam configuration and energy dosing for its optimal use in the future.

In this study we aimed to identify gene expression differences between macula and periphery within the human neuroretina. To this end we used qPCR and RNA sequencing to analyse post-mortem tissue. Equal sized punches, one within the macula and 4 extramacular areas were isolated and RNA was extracted from 7 donor eyes. We then tested specific genes for differential expression by qPCR. This was done firstly to establish whether mRNA analysis was feasible on human post-mortem tissue, and secondly to validate the dissection method by focusing on genes with known distributions. Next, specific genes with unknown distribution were tested to interrogate whether various key molecular pathways in the retina were differentially active in the macula versus the periphery. This approach revealed many differences but was ultimately limited as each gene had to be measured individually. We therefore decided to use RNA sequencing (RNAseq) in order to obtain expression levels of all genes active in the retina. This method allowed us to identify and quantify transcripts from around 20,000 different genes. We then developed a method to assess the confidence levels in the quantification of individual genes. We also used genes with known distribution for method validation. Focusing on the largest differences between the macula and the periphery revealed marked differential expression components from retinoic acid signalling pathway. Furthermore, roughly ¾ of the genes with the most reliable quantification was used for global pathway analysis. This showed that lipid metabolism and energy production pathways were also differentially used in the macula. 5 In order to further query the role of lipid metabolism in the retina we studied knockout mice lacking the very low density lipoprotein receptor (VLDLR) by using 3D Scanning Electron Microscopy to map mitochondria size and distribution in photoreceptors. This showed marked changes in the mutant animals suggesting abnormal lipid metabolism can directly affect photoreceptors and may play a role in macular pathologies in humans.

Endothelial injury often causes intimal hyperplasia, a disease characterised by local inflammation and critical narrowing or restenosis of the blood vessel. Endothelial cells (EC) grown on collagen particles are highly effective in inhibiting intimal hyperplasia in various animal models, and this effect appears to be, at least in part, the result of EC-derived soluble factors that suppress local vascular inflammation. To test this hypothesis, we produced EC on collagen particles-conditioned medium (ECPCM), which was expected to contain soluble anti-inflammatory factors. Indeed, EC treated in vitro with ECPCM together with pro-inflammatory cytokines including tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) displayed reduced gene expression of the inflammation-related adhesion molecules E-selectin and VCAM-1. Investigation of the molecular mechanism of action for the anti-inflammatory activity excluded mRNA stability of E-selectin and VCAM-1, activation of signalling cascades via the NF-kB and Stat3 pathways, and nuclear localization of transcription factors. ECPCM did affect the TNFα-induced binding of p65, a subunit of the NF-kB transcription factor, to the E-selectin and VCAM-1 promoters. These results suggest that inhibition of gene transcription is responsible for the ECPCM-mediated suppression of inflammatory responses in EC. The therapeutic effects of ECPCM were supported by in vivo experiments performed on the mutant mouse strain JR5558, which develops spontaneous choroidal neovascularization (CNV) lesions associated with inflammatory cell recruitment and expression of inflammatory adhesion molecules. The CNV lesion area and recruitment of activated macrophages were both decreased in JR5558 mice given intraperitoneal injections of ECPCM. ECPCM might therefore have therapeutic potential in treating inflammatory vascular diseases.

It was suggested that in addition to its value as a research tool, there may be practical applications of the divided visual field technique. If it is to be used as a method of investigating individual oases in order to assess hemispheric lateralization of function, it must be fully understood. The relative merits of the three major theories of visual field asymmetries were reviewed. A series of ten experiments was performed in which stimuli were presented tachistoscopically to the right and left visual fields. Both verbal stimuli in the form of words and single letters and nonverbal stimuli in the form of shapes, drawings and faces were employed. A variety of problems were considered. The relationship of the serial-parallel processing dichotomy to the left and right hemispheres was considered and not believed to be useful. The distinction between the hemispheres was in terms of visual-verbal processing, although this separation of functioning was not as clearcut as has been thought. It was concluded that the direct access theory was the most adequate explanation of the data, although the results may be partially influenced by scanning and attentional phenomena. Load-sharing between the hemispheres was discussed.

The fovea is a specialised retinal area responsible for high spatial vision. Development of the fovea involves centrifugal migration of inner retinal layers (IRLs) away from the fovea and centripetal displacement of the cone photoreceptors into the fovea and is thought to be complete by 5 years of age. Current understanding of human foveal development is limited to studies of few histological specimens. The recent development of hand-held spectral domain optical coherence tomography (HHSDOCT), can overcome this limitation by facilitating large scale in vivo imaging of the infant human retina, both in controls and in conditions such as achromatopsia and albinism, where foveal development is disrupted. In this thesis, we optimise image acquisition and analysis with HH-SDOCT in young children with nystagmus. We show that HH-SDOCT is reliable in children with and without nystagmus, with an intraclass correlation coefficient of 0.96 for central macular thickness measurements. The non-linear developmental trajectories of each retinal layer modelled in a large cohort of 256 controls suggest that development continues until 12 years of age. A paradigm for the etiological diagnosis of nystagmus using OCT is presented. We describe multiple abnormalities of retinal development in young children with achromatopsia and albinism, including delayed regression of the IRLs from the fovea, diminished elongation of the photoreceptor layers with age and a reduction in perifoveal retinal thickness. This results in significantly increased IRL and decreased photoreceptor thicknesses at the fovea in both conditions (p < 0.05). In contrast, the IRL thickness is significantly decreased at the perifovea in both achromatopsia and albinism (p < 0.01). There is evidence of postnatal development in the achromat and albino retina. In achromatopsia, disruption of photoreceptors is progressive with age. With gene therapy imminent, potentially earlier treatment of these conditions may normalise retinal development and optimise vision.

Inflammation contributes to major causes of visual loss. Both intraocular inflammatory disease (uveitis) and immune-mediated retinal degenerative disorders such as agerelated macular degeneration account for the majority of visual impairment in the adult population in developed countries. Non-infectious uveitis is considered an autoimmune disease initiated by a loss of immune tolerance to retinal proteins, mediated and characterized by infiltration of leukocytes, including T cells and tissuedamaging macrophages. Glucocorticoids remain the first-line treatment of choice, however, despite up to a third of patients failing to achieve disease control at tolerable systemic doses in addition to adverse side-effects. As a preclinical model of human uveitis, experimental autoimmune uveoretinitis (EAU) provides a platform for dissection of the mechanisms responsible for immune-mediated tissue damage, as well as permitting assessment of immunotherapeutic efficacy. The purpose of this thesis was to investigate whether targeted approaches to modulate macrophage responses in EAU could impact disease severity and offer future therapeutic potential. The data presented demonstrates that harnessing CD200 receptor (CD200R) signaling; a homeostatic regulatory mechanism that contributes to the immune health of the normal retina is a viable approach which can suppress macrophage-mediated tissue damage. Specifically, when a CD200R agonist monoclonal antibody (mAb), DX109 is administered either systemically or locally (via intravitreal injection) inflammatory responses and disease severity are suppressed. Triggering of the receptor via DX109 binding delivers a negative signal, and thus mimicking the normal effect of the cognate ligand CD200 to deactivate and tonically suppress macrophage responses. Inappropriate complement activation resulting either from dysregulated para-inflammatory responses or autoimmune inflammation is implicated in ocular tissue damage. By targeting the complement component C5 and preventing generation of the potent pro-inflammatory anaphylatoxin C5a, macrophage responses and tissue damage in the retina can also be suppressed. The results demonstrate that selective blockade with an anti-C5 mAb via both systemic and local routes of administration can be exploited therapeutically. In the final approach, I utilised an S1 P receptor-1 agonist (Fingolimod) to prevent the influx of antigenspecific T cell infiltrate and the subsequent recruitment and activation of macrophages cells, which results in suppression of overt inflammatory responses, and ultimately protects the tissue. Furthermore, employing clinically relevant doses of Fingolimod can acutely suppress active intraocular inflammation, maintain disease remission and support the vascular barrier integrity of the eye.

Maintenance of epithelial tissue integrity requires correct organization of the polarity plane. This is promoted by assembly of cell-cell junctions, defining distinctive apical and basolateral domains. The establishment of polarity is a key point in tissue organization since it controls the plane in which cells divide. When a cell replicates, it duplicates DNA and aligns them before partitioning them evenly between the two daughter cells. Although results from different laboratories indicate a role of cell-cell junctions in the orientation of the mitotic spindle, it is poorly understood if there is a cross-talk between the mitotic checkpoint and the attachment status of the microtubules at the cell cortex. Previous works suggest that the small Rho GTPase Cdc42 regulates spindle orientation and chromosome capture. Cdc42 is also a crucial regulator of cell-cell junction assembly and dynamics. Cell junctions need to remodel during cell division to adapt to the changing cell shape, and allow the condensed chromosomes to be properly aligned. Therefore, we hypothesized that specific regulators of Cdc42 guide the interplay between cell junctions and the mitotic machinery. Using a siRNA approach, we identified a GAP for Cdc42, DLC2, that associates with cell-cell contacts and mitotic spindles, and regulates junctional integrity and chromosome attachment during mitosis. Upon depletion of DLC2 in epithelial cells, the normally continuous immunofluorescence staining of junctional markers was disrupted in mitotic cells, and chromosomes are misaligned. Following DLC2 depleted cells on live, it results clear that these cells are arrested in metaphase for longer timing, with some chromosomes unattached. The orientation of mitotic spindles relative to the substrate was not affected, suggesting that the defect was not due to a loss of polarity. Depletion of DLC2 led to increased levels of GTP-bound Cdc42, indicating that deregulation of Cdc42 contributed to the observed phenotypes. Indeed, partial depletion of Cdc42 by RNA interference rescued the phenotype induced by depletion of DLC2. We also found that DLC2 associates with the plus-end motor Kif1B, a kinesin-3 family member, and that depletion of Kif1B resulted in a similar phenotype as depletion of DLC2. Based on our observations, we speculate that DLC2 regulates Cdc42 to guide association of microtubules with the cell cortex and the kinetochores in metaphase and to regulate forces they exert at these sites.

It has been shown that adults and children with amblyopia can experience metamorphopsia (perceptual visual distortions). This body of work chronicles the piloting of a novel dichoptic technique to quantitatively map perceptual visual distortions in adults with amblyopia. It was demonstrated that perceptual visual distortions as measured with this method were more severe in strabismic amblyopes, were not homogenous across the visual field, and were highly individual to each amblyope, in common with the findings of other studies using alternative distortion mapping methods. It was established that perceptual visual distortions in adult amblyopes remained stable geotopically and in magnitude over time, and were closely associated with the angle of strabismic deviation and strength of binocular single vision - unique findings not documented in the literature previously. The dichoptic paradigm was then used to measure perceptual visual distortions in children with amblyopia at different stages of amblyopia treatment, as no study before had attempted to relate severity of perceptual visual distortions to amblyopia treatment outcomes, or establish how prevalent they were amongst amblyopic children. For the first time, a large sample of amblyopic children was tested (n = 82) and compared to agematched visually normal controls (n = 140). It was established that 56.1% of the sample had perceptual visual distortions, and importantly, that the severity of these was independent of amblyopia treatment outcomes. Instead, as in the adult amblyopes, distortion severity was found to be primarily dictated by strength of binocular function and the size of the angle of deviation. Overall, the key message of this work is the importance of evaluating all aspects of the disruption to visual function in amblyopia, and attempting a unified, binocular treatment approach that addresses these aspects, in the hope of producing better amblyopia treatment outcomes for children in the future.