The retinal pigment epithelium (RPE) is a monolayer of highly polarised cells that lies between the photoreceptors and choriocapillaris and performs a crucial role in the maintenance of visual function. The RPE phagocytoses and digests shed photoreceptor outer segments, transports nutrients, ions and water and secretes various essential growth factors that support surrounding cells. Despite the importance of membrane traffic pathways in the RPE little is known about endocytosis in these cells. Furthermore, although the early stages of phagocytosis of photoreceptor outer segments have been well characterised, the processing of the phagosome after engulfment is poorly understood. The first aim of this PhD project was to characterise the organisation of the endocytic pathway in the RPE and identify potential endocytic cargos that could be used to monitor endocytosis in RPE cells in culture. The second aim was to identify methods of characterising sequential stages of phagosome maturation in the RPE in situ and determine whether the same stages could be reproduced in cultured RPE cells. The final aim was to identify mechanisms underlying phagosome maturation, focusing in particular on the role of interactions with the endocytic pathway. Endocytic compartment markers and potential endocytic cargos were localised in situ on retinal sections and in vitro using primary porcine RPE cells. In situ and in vitro studies showed that Rab11a, a marker for apical recycling endosomes, is found distributed throughout the cell and not restricted to an apical compartment as seen in other epithelial cells. Transferrin receptors are expressed on both apical and basal plasma membranes in the RPE and both fluid phase probes and transferrin endocytosed from apical and basal surfaces meet in a common endocytic compartment. Phagosome maturation was investigated by immuno-electron microscopy using antibodies to two different rhodopsin epitopes. Loss of a C-terminal cytoplasmically exposed epitope was an early step in phagosome maturation, occurring before phagosome:lysosome fusion, which allowed the distinction of early and late phagosomes. Here it is demonstrated that the same stages of phagosome maturation occurred, albeit more slowly, in cultured porcine RPE challenged with isolated porcine outer segments. Treatment with the protease inhibitor, leupeptin, inhibited loss of the C-terminal rhodopsin epitope, suggesting that limited proteolysis can occur within the maturing phagosome. Finally, by loading the endocytic pathway from the basal surface and the phagocytic pathway from the apical surface it was possible to demonstrate acquisition of endocytic content by the maturing phagosome, the timing of which suggests that interaction with the endocytic pathway is likely to be a factor in the limited proteolysis of rhodopsin that occurs in the maturing phagosome. This work opens the possibility to investigate defects in endocytic and phagocytic pathways in retinal disease and determine how these defects could lead to phenotypes associated with aging and retinal degeneration.

The functional integrity of the thalamic reticular nucleus (TRN) is thought to be key in the control of selective attention (Crick, 1984; Pinault, 2004). This GABAergic structure (Houser et al., 1980) is responsible for ensuring synchronous activity within the appropriate thalamocortical circuits required for either sensory perception or for the preparation and execution of distinct motor and/or cognitive tasks. It is therefore imperative to ascertain the exact nature of how inhibitory innervation from the TRN to thalamic nuclei is controlled in order to understand how neurophysiological disease states associated with TRN malfunction (Huguenard, 1999; Rub et al., 2003; Barbas and Zikopoulos, 2007; Pinault, 2011) precipitate. I t has been previously demonstrated that the Group II metabotropic glutamate receptors (mGlu2/3) can modulate physiologically-evoked responses in the VB (Salt and Turner, 1998) by reducing inhibition from the TR N (Turner and Salt, 2003). However, it is not yet known what the relative contributions are of the two subtypes to this modulation, nor to what extent these receptors may be activated under physiological conditions during this process. Using single-neurone recording in the rat ventrobasal thalamus (VB) in vivo with local iontophoretic application of selective Group II mGlu receptor compounds, my findings were threefold. Firstly, I found that both mGlu2 and mGlu3 receptors contribute a component to the overall Group II mGlu receptor effect on sensory responses in the VB. Secondly, I was able to demonstrate that both Group II mGlu receptor subtypes are likely activated by endogenous ‘glutamate spillover’ from the synapses formed between excitatory sensory afferents and VB neurones following physiological sensory stimulation, and that this can lead to a reduction in sensory-evoked inhibition arising from the TRN. I propose that this potential Group II mGlu receptor modulation of inhibition could play an important role in discerning relevant information from background activity upon physiological sensory stimulation: a novel mechanism that could be of importance in attention and cognitive processes, whose malfunction could result in maladaption of sensory perception, such as that which can occur in psychiatric disease. Thirdly, I was able to provide evidence that mGlu2 receptors are likely located on astrocytic processes surrounding the VB- TR N synapse, and are able to mediate a n a st rocyti c mechanism of action that reduces inhibitory synaptic transmission from the TRN to the VB. To the best of my knowledge this provides the first evidence that mGlu2 receptors are able to activate astrocytes, and of the involvement of astrocytes in the modulation of heterosynaptic transmission. As VB astrocytes are able to respond to synaptic stimulation (Parri et al., 2010), maladaption of mGlu2 receptor-mediated endogenous astrocytic activation may therefore have functional implications for the processing of somatosensory information and for the preparation and execution of distinct motor and/or cognitive tasks. Finally, I also investigated the action of the putative endogenous selective Group II mGlu receptor agonist Xanthurenic Acid (XA), which is a metabolite of the kynurenine pathway. Changes in both of these systems have been implicated in the pathophysiology of schizophrenia and other psychiatric disorders, however little is known regarding the mechanism of action of XA. I therefore investigated the effects of XA in modulating inhibition in the VB from the TRN using the same in vivo electrophysiology preparation as described above, and also evaluated the ability of XA to bind to and activate mGlu2 receptors using in vitro molecular pharmacological methods. Selective Group II mGlu receptor compounds that exploit this novel mechanism of endogenous activation may therefore be of importance in the modulation of sensory, attentional and cognitive processes for therapeutic strategies.

Angiogenesis is the sprouting of new capillaries from pre-existing vessels and is driven by hypoxia. While physiological hypoxia in the retina is paramount to regulating physiological angiogenesis, pathological ischemia is a cause of pathological neovascularisation and a large proportion of blindness worldwide. The aim of this thesis is to explore ways of redirecting neovascularisation towards healthy revascularisation. I do this by looking at ways of modulating angiogenesis at an early stage in Oxygen-induced retinopathy (OIR) in mice. This model is based on vessel depletion by exposure to hyperoxia, which results in acute retinal hypoxia upon return to room air. This hypoxia then triggers neovascularisation in the remaining vessels after 5 days. Vascular endothelial growth factor (Vegf) plays a critical role in development and disease of the retinal vasculature. Genetic ablation of astrocyte-derived Vegf surprisingly showed minor impacts on retinal vasculature development. However, it had a vessel stabilizing role during the hyperoxic phase and also promoted vessel regeneration in the hypoxic phase. In other experiments, it was shown that C3H/HeJ mice, which contain the retinal degeneration 1 (Rd1) mutation (Pde6bRd1) and have abnormally thin retinas, do not become ischemic despite vaso-obliteration and do not develop neovascularisation. This demonstrates that maintaining a balance between oxygen demand and supply at the onset of ischemia critically influences the angiogenesis outcome. Using this model, vascular tortuosity was established as an early phenotype of OIR possibly predictive of neovascularisation. Finally, in other experiments, it was shown that inflammation can modulate hypoxia. Subcutaneous injection of lipopolysaccharide (LPS) in mice with hypoxic retinas, led to bilateral reduction of hypoxia, reduction of VEGF and healthy revascularization. From a conceptual point of view, this is a paradigm changer because it shows for the first time that it is possible to change hypoxia in the retina without changing the oxygen (i.e. vascular) supply.

Artists and scientists have long had an interest in the relationship between music and visual art. Today, many occupy themselves with correlated animation and music, called 'visual music'. Established tools and paradigms for performing live visual music however, have several limitations: Virtually no user interface exists, with an expressivity comparable to live musical performance. Mappings between music and visuals are typically reduced to the music‘s beat and amplitude being statically associated to the visuals, disallowing close audiovisual congruence, tension and release, and suspended expectation in narratives. Collaborative performance, common in other live art, is mostly absent due to technical limitations. Preparing or improvising performances is complicated, often requiring software development. This thesis addresses these, through a transdisciplinary integration of findings from several research areas, detailing the resulting ideas, and their implementation in a novel system: Musical instruments are used as the primary control data source, accurately encoding all musical gestures of each performer. The advanced embodied knowledge musicians have of their instruments, allows increased expressivity, the full control data bandwidth allows high mapping complexity, while musicians‘ collaborative performance familiarity may translate to visual music performance. The conduct of Mutable Mapping, gradually creating, destroying and altering mappings, may allow for a narrative in mapping during performance. The art form of Soma, in which correlated auditory, visual and proprioceptive stimulus form a combined narrative, builds on knowledge that performers and audiences are more engaged in performance requiring advanced motor knowledge, and when congruent percepts across modalities coincide. Preparing and improvising is simplified, through re-adapting the Processing programming language for artists to behave as a plug-in API, thus encapsulating complexity in modules, which may be dynamically layered during performance. Design research methodology is employed during development and evaluation, while introducing the additional viewpoint of ethnography during evaluation, engaging musicians, audience and visuals performers.

Age-related macular degeneration (AMD) is the major cause of vision loss in the developed world. AMD is a chronic progressive disorder of the outer retina leading to vision loss from atrophy (geographic atrophy) and/or the development of choroidal neovascularisation (CNV). Mounting evidence indicates the importance of innate immunity in its pathogenesis. This thesis describes a programme of work conducted with the aim of further understanding the role of innate immune cells in AMD. Analysis of mouse cell suspensions by flow cytometry demonstrated significantly greater densities of innate immune cell populations in the RPE-choroid than in the neurosensory retina. Dendritic cells accumulated in both tissues with increasing age, and this process was accelerated in mice deficient in a chemokine upregulated in the aged choroid - CCL2. Innate immune cells were recruited to CNV lesions in mouse models of laser-induced and spontaneous CNV. Increasing age was found to correlate with the extent of laser CNV lesion size but not with recruitment of innate immune cells. Impaired recruitment of innate immune cells in CCL2-deficient mice was associated with a smaller laser-CNV lesion size, which nonetheless increased with age. Attenuation of CNV lesion size by targeting key angiogenic pathways using the small molecule pazopanib or by direct targeting of innate immune cells by induction of alternative activation using a CD200R agonist was associated with subtle increases in the recruitment of innate immune cell subpopulations to CNV lesions. Findings did not support the hypothesis that age-related vulnerability to laser CNV is a consequence of age-related changes in innate immune cell populations. However, the results indicated that CCL2 controls dendritic cell migration in the ageing retina and the recruitment of innate immune cells to CNV lesions. Further investigation of these pathways may lead to better treatments in the prevention and management of AMD.

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the UK. In 2005, the first publication of a genome-wide associated study identified a single nucleotide polymorphism in complement factor H (CFH) as a genetic risk factor for AMD. CFH is a secreted regulator of the alternative complement pathway and therefore key to controlling the inflammatory response. Prior to 2005, little was known about the role of CFH in the retina. This study addresses this question in order to understand how this protein could contribute towards AMD pathology. Initial experiments confirmed that retinal pigment epithelial (RPE) cells are capable of secreting detectable levels of CFH, and that RPE cells were able to enhance the secretion of CFH in response to inflammatory stimuli. The main focus of this study was to characterise the effect of loss of CFH on young and aged retina in Cfh-/- mice. Immunohistochemical studies revealed that signs of stress and re-distribution of complement proteins appear at one year of age. Genome-wide microarray analysis of the RPE and choroid or neuroretina, showed that loss of CFH has little effect on gene expression in young mice but that the impact of CFH loss increases with age. The largest group of genes to change were involved in antigen presentation and immunity suggesting that CFH has an important role in immune regulation in the eye. Analysis of visual function using electoretinograms revealed that dysfunction seen at two years was not present at one year, indicating that age-related gene expression changes are likely to be involved in the pathogenic process in these mice. This study reveals the importance of CFH in maintaining retinal health and good visual function with age.

Polarized epithelial cells form selective barriers between tissues and various body compartments that are essential for normal development and organ function. A mature apical junctional complex (AJC), consisting of tight junctions (TJ), adherens junctions (AJ), and desmosomes is crucial for functional polarized epithelia. Rho-GTPases are key regulatory proteins of many cellular processes, including epithelial adhesion and polarization. These small GTPases are in turn controlled spatially and temporally by guanine nucleotide exchange factors (GEFs) that promote GTP-binding, resulting in their activation; and GTPase activating proteins (GAPs) that promote GDP hydrolysis, resulting in their inactivation. In this thesis I studied a novel junction associated GAP protein known as SH3BP1 in a variety of epithelia. SH3BP1 was identified in a functional siRNA screen that was designed to identify actin regulators of epithelial polarisation and differentiation. I will show that SH3BP1 localises to the early AJC when TJ and AJ are not yet properly separated. SH3BP1 regulation is important for tight junction formation and, its depletion affects polarity and junction integrity. I will demonstrate that SH3BP1 is functionally important in epithelial cell lines from different tissues as well as in organotypic three dimensional cultures. A major part of my thesis will focus on the demonstration that SH3BP1 is a crucial EGF receptor signalling effector that guides morphological alterations and actin dynamics. Using the A431 cell line EGF signalling model, I will demonstrate SH3BP1 is required to regulate both Rac1 and Cdc42 signalling, and subsequently its role in the recruitment of junctional proteins first to dorsal ruffles and then to forming tight junctions. I will provide evidence that SH3BP1 forms a heteromeric complex with the scaffold JACOP/paracingulin and the actin capping regulator CD2AP that has a key role in the regulation of actin dynamics.

The retinal blood vessels can become occluded due to both inflammation and thromboembolic diseases, and the main aim of this thesis is to examine the features of retinal vein occlusion (RVO) in patients with co-existing ocular inflammation to determine risk factors for the development of RVO, risk factors predictive of a poor visual outcome in uveitis, the prevalence of anti phospholipid antibody-based disease and the role of antiphospholipid antibody (aPL) testing. In this thesis, I also explore the efficacy of new treatments for retinal vein occlusion, particularly the Ozurdex intravitreal dexamethasone implant, which can also be used to treat uveitis and uveitis macular oedema. Demographic and clinical variables were extracted from the medical notes of three separate sample groups of patients attending Moorfields Eye hospital including: 1) patients attending a Uveitis clinic between 2009-2011 with a new or past history of RVO; 2) any patient who had aPL testing performed during 2010; 3) patients recruited onto the initial Ozurdex for uveitis phase III clinical trial. 34 RVO events were recorded during a two year period with an overall clinic prevalence of 1.83%. Presenting ocular features and risk factors for RVO in uveitis patients were explored. aPL testing was commonly performed on patients with RVO in an Ophthalmology setting, and the usefulness of this and its relation to RVO events were examined. Finally, follow up data for uveitis patients treated with a single Ozurdex implant were explored to determine the longer-term outcome of this treatment, and the strategies employed as and when patients relapsed, comparing these outcomes with those of the Ozurdex implant.

The corneal epithelium is continuously renewed by a population of stem cells that reside in the corneo-scleral junction, otherwise known as the limbus. These limbal epithelial stem cells (LESC) are imperative for corneal maintenance, with deficiencies resulting in in-growth of conjunctival cells, neovascularisation of the corneal stroma and eventual corneal opacity and visual loss. One such disease that has traditionally been thought to be due to LESC deficiency is aniridia, a pan-ocular congenital eye disease due to PAX6 heterozygosity. Corneal changes or aniridia related keratopathy (ARK) seen in aniridia are typical of LESC deficiency, however, the pathophysiology behind ARK is still ill defined. Recent studies, utilising heterozygous Pax6 mouse models suggests that ARK is not solely due to LESC deficiency. Current theories suggests it may be caused by a deficiency in the stem cell niche and adjacent corneal stroma leading to abnormal differentiation of epithelial cells, with an altered wound healing response also playing a role (Ramaesh et al., 2005a, Li et al., 2008). The ultimate goal of biological research is to further the understanding of disease mechanisms with aim to develop improved therapies, therefore this thesis examines the pathogenesis of the ARK with this in mind. The difficulties found with the initial assessment of gene replacement therapy in the mouse highlighted the need for further investigation into LESC location and ARK progression. The examination of corneal epithelial label retaining cells indicated an increase in numbers and abnormal location of putative LESC in the heterozygous Pax6 mouse, suggesting these cells may fail to differentiate into progeny cells. Furthermore, analysis of corneal epithelial and fibroblast cells with PAX6/Pax6 down regulation has further established that an abnormal wound healing response may be involved in disease progression. Overall, these studies have highlighted the complexity of the disease and the requirement for further investigation to dissect the mechanisms underlying ARK, providing clues for future directions in therapy development.

The human macula is essential for high acuity vision but its biochemical and cellular properties are poorly understood. A disease that specifically affects the macula, Macular Telangiectasia (MacTel) type2 was investigated by studying postmortem tissue from a single donor. This revealed Müller cell loss specifically in the macula, which might be responsible for the vascular changes and photoreceptor degeneration typical of this disease. To establish whether the disease has subclinical vascular changes outside the macula, the peripheral retina was studied. This showed that contrary to previous reports, abnormal looking capillaries are not disease-specific but a normal ageing phenotype in humans. Proteomics and immunohistochemistry was used to characterise maculae in healthy donors. Comparative proteomics identified differentially expressed proteins and immunohistochemistry confirmed the distribution of selected proteins. This led to the discovery of several Müller cell markers, lactate dehydrogenase (LDHB), glial fibrillary acidic protein (GFAP),αB crystallin (CRYAB) and αA crystallin (CRYAA), which are expressed at higher levels in the macula, demonstrating that Müller cells can take on different differentiation phenotypes depending on retinal area. Furthermore, the spatial expression pattern of LDHB, GFAP and CRYAB was found to correlate with the size/shape of the area that is affected in MacTel type2, providing a possible explanation why the disease affects only the macula. Embryonic development of the macula was investigated in eye tissue obtained from abortions by using immunohistochemistry and gene expression analysis (qPCR and differential gene display). Müller cell markers, CRYAA, CD44 and CRALBP were found to be specifically expressed in the presumptive macula, starting at 7 gestation weeks (CS20), prior to ganglion cell and photoreceptor differentiation. Genomic comparison between this region and peripheral retina revealed further differentially expressed genes and led to the identification of a morphogen, Retinoic acid, which might play a role in macula development in the human retina.