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In Alzheimer’s disease, the beta-amyloid (Aβ) protein accumulates within neurons prior to amyloid plaque development. Recent evidence suggests that intracellular Aβ accumulation, rather than extracellular plaques, correlates best with disease progression. Aβ is generated by proteolytic processing of the amyloid precursor protein (APP) but the intracellular site of Aβ production is unclear. Aβ has been localised to multivesicular endosomes/bodies (MVBs), which have many cellular functions, including delivery of contents to the lysosome and release of intralumenal vesicles (ILVs) into the extracellular space. Several MVB and ILV populations exist that are formed by distinct mechanisms and have different fates. This study aimed to determine to which MVB population APP traffics, the role of MVBs in Aβ production and the functional consequences of Aβ production and accumulation within MVBs. Immunofluorescence and immuno-EM showed that APP trafficks to the ILVs of an EGF receptor-containing subpopulation of MVBs in a neuroglioma cell line overexpressing APP. To determine the mechanism of APP ILV targeting, we first characterized the effects of inhibition of ESCRT-dependent and ESCRT-independent mechanisms of ILV formation in HeLa cells. We found that EGF stimulation, which promotes ESCRT-dependent ILV formation, causes the generation of enlarged ILVs, whilst depletion of the ESCRT-0 component, Hrs, leads to the reduction of ILV number and size. In neuroblastoma cells overexpressing APP, APP is ubiquitinated and depletion of Hrs inhibits sorting of APP onto ILVs and Aβ secretion, implicating the ESCRT machinery in traffic of APP and Aβ generation. To analyse the effects of Aβ accumulation in the endocytic pathway, we determined the effects of exogenous Aβ endocytosed by HeLa cells and primary neurons, and demonstrated that Aβ can cause disruption of endosome-lysosome morphology and membrane impermeability. These results indicate that APP traffic to MVBs and generation of Aβ within them could contribute to early AD pathology.

Purpose: Retinitis pigmentosa is a form of inherited retinal degeneration, whereby the rod photoreceptors are gradually lost. Patients usually manifest symptoms in the third decade of life, beginning with night blindness and loss of peripheral vision gradually progressing to tunnel vision and eventually complete blindness, as cone photoreceptors die as a secondary effect. Mutations causing non-syndromic, autosomal dominant retinitis pigmentosa (adRP) were recently identified in TOPORS (RP31 locus, chromosome 9p21.1). TOPORS is a ubiquitously expressed gene, encoding a protein showing multi-functional character. It is currently the only known protein to demonstrate E3 ligase activity for both ubiquitin and SUMO-1 (small ubiquitin-like modifier). It also shows tumor-suppressor activity. The purpose of this study is to characterize the role of TOPORS in the retina, in particular in the photoreceptor cells, which may explain the retinal degeneration seen with mutations in this gene. Methods: To identify the functional consequences of the mutations TOPORS was cloned from human retinal cDNA into FLAG-tagged vector pCATCH, identified mutations were introduced by site-directed mutagenesis and transfected into MDCK cells. Over-expressed TOPORS was detected using an antibody directed against the FLAG-tag. Functional analyses of WT lymphoblastoid cell lines and specifically generated patient cell lines was also conducted using the TOPORS antibody. A commercially available antibody was obtained and used for immunofluorescent staining of endogenous topors in a number of cell lines, and in mouse, pig and human retinal sections. Tissue sections were costained for TOPORS with several proteins of known localisation in the retina (for example, RP1 and γ-tubulin), to ascertain a more detailed localisation of TOPORS and to indicate potential interactants. Cellular localisation experiments were carried out in cell lines on a single cell level to determine the exact sub-cellular localisation. To identify functional partners of TOPORS, immuno-precipitation experiments were carried out to substantiate data obtained from localisation studies in retinal sections. Results: Both mutations in TOPORS result in frameshift, prematurely terminating the protein; however this appeared to make no difference to the nuclear localization of the over-expressed WT and mutants proteins. Work on the patient lymphoblastoid cell lines showed that the mutant protein is not expressed although the mutant mRNA is detectable by RT-PCR. Immunoblot analysis confirmed the expression of TOPORS as a 150kDa band in cell lines and retinal tissue extracts. Localisation studies showed a novel localisation for TOPORS in the retina and in confluent, non-diving cells. TOPORS localised to the connecting cilium of the photoreceptors in retinal sections, and to the primary cilia of cells grown at confluence. Further analyses showed that it co-localizes with γ-tubulin to the base of the cilium, to the basal body. Co-immunoprecipitation experiments showed that TOPORS does not interact with many known connecting cilium proteins although it has a similar localization pattern. It did not show interaction with proteins involved in anterograde transport. However, TOPORS does show interaction with some of the protein subunits involved in retrograde transport. Conclusions: Topors is a multi-functional, ubiquitously expressed gene however mutations in it only cause retinal degeneration (adRP). Results suggest that the mutant proteins are not expressed in patients, hence haplo-insuffiency is likely to be the cause of retinal degeneration. TOPORS is expressed in many cell and tissue types, and is primarily a nuclear protein. However, it shows a unique localisation in the retina - to the base of the connecting cilium in the photoreceptors. Similarly, in ciliated cells, topors localised to the base of the primary cilium which was confirmed by co-localisation with γ-tubulin. The co-IP experiments confirmed its interaction with γ-tubulin and also showed that topors associates with protein involved in retrograde transport. This together suggests it plays a novel role in the retina, possibly related to signal transduction and/or trafficking, that when compromised, results in specific photoreceptor degeneration.

Vascular networks expand in a two-step process that begins with vessel sprouting followed by vessel anastomosis. Sprouting is induced by chemotactic gradients of the vascular endothelial growth factor (VEGF), which stimulates tip cell protrusion. Yet, it is not known which factors promote the fusion of neighbouring tip cells to add new circuits to the vessel network. In this thesis, the role of macrophages in physiological angiogenesis was examined. By combining the analysis of mouse mutants defective in macrophage development or VEGF signalling with live-imaging in zebrafish, I demonstrated that macrophages promote tip cell fusion downstream of VEGF-mediated tip cell induction. Macrophages therefore play a hitherto unidentified role as vascular fusion cells. Moreover, I showed that there are striking molecular similarities between the pro-angiogenic tissue macrophages essential for vascular development and those that promote the angiogenic switch in cancer, including the expression of the cell-surface proteins TIE2 and NRP1. These findings suggest that tissue macrophages are a target for anti-angiogenic therapies, but that they could equally well be exploited to stimulate tissue vascularisation in ischemic disease. The VEGF non-tyrosine kinase receptor neuropilin 1 (NRP1) is essential for vascular development. NRP1 has been previously hypothetised to transduce signals through its cytoplasmic domain to promote angiogenesis independently of the VEGF tyrosine kinase receptors. However, by studying a new mouse mutant for NRP1 I found that the NRP1 cytoplasmic domain did not play any obvious role in cardiovascular development or neo-angiogenesis in a model of CNV. In contrast, I found that loss of the NRP1 cytoplasmic domain confers resistance to VEGF-induced vascular permeability. These findings implicate the NRP1 cytoplasmic domain as a key molecular switch between VEGF-induced angiogenesis and vascular hyperpermeability, making it an ideal pharmacological target in ischemic diseases where oedema caused by leaky vessels exacerbates disease progression.

Reconstruction of the conjunctiva is an essential part of ocular surface regeneration, especially if an extensive area or the whole ocular surface is affected, such as in patients with ocular cicatricial pemphigoid, Stevens- Johnson syndrome or chemical/thermal burns. However, there is a lack of suitable donor tissue for conjunctival replacement, especially when large grafts are required and it is important that new materials and methods are developed for conjunctival reconstruction. The aims of this thesis were; to characterise the conjunctival epithelial cell population and to improve the maintenance of the epithelial progenitor cells during in vitro expansion in order to produce conjunctival epithelial cells suitable for therapeutic use. The final aim was to transfer these cells to compressed collagen matrices and amniotic membrane and test the properties of these cell-matrix constructs. Experiments showed that cryopreservation does not to alter the proliferative potential of conjunctival epithelial progenitor cells. It was also demonstrated that the maintenance of conjunctival epithelial progenitor cells during cell expansion can be improved by mimicking an environment in vitro, which is more similar to the stem cell niche in vivo and that this is accompanied by downregulation of key genes in the wnt signaling pathway. The final experimental series showed that after in vitro expansion, conjunctival epithelial cells can be successfully transferred and cultured on amniotic membrane and compressed collagen gels. In conclusion these studies highlighted the complexity of tissue engineering ocular surface substitutes and provided further clues for the goal to obtain a stable conjunctival substitute, suitable for transplantation.

Standard automated perimetry (SAP) serves as the cornerstone in the diagnosis and monitoring of functional deficits associated with glaucoma. Achromatic contrast thresholds are typically measured at pre-defined locations in the visual field for circular stimuli of constant area and duration. However, the selection of SAP stimulus parameters was made with little or no regard to the ability of the visual system to sum light energy over both space and time, and how such aspects of visual function might vary across the visual field and in glaucoma. In recent years there has been renewed interest in the spatial summation of perimetric stimuli, it being suggested that the sensitivity of SAP, and thus the relationship of contrast thresholds to underlying retinal ganglion cell density, might be improved through a simple scaling of stimulus area. In this thesis we re-examined temporal summation to provide an evidence base from which the selection of appropriate stimulus parameters for SAP might be made. Specifically, we investigated the various factors that can influence the upper limit of complete temporal summation (critical duration) including stimulus area, rate of high-frequency flicker, age and glaucoma. We observed that the critical duration is intrinsically linked to the degree of spatial summation exhibited at any point in the visual field, with the result that temporal summation varies across the visual field when examined using a stimulus scaled to the localised area of complete spatial summation (Ricco's area). We also found the critical duration to be significantly increased in glaucoma when examined using both a standard Goldmann III stimulus and a stimulus scaled to the localised Ricco's area under the conditions of SAP. Based on the results of this thesis we propose the use of stimuli modulating in area, duration and luminance to improve the sensitivity and specificity of perimetry in the detection of glaucoma.

Age-related macular degeneration (AMD), which results in the breakdown of the retinal layers and the progressive loss of the retinal pigment epithelium (RPEL is the leading cause of bilateral blindness in elderly individuals worldwide. To date, the success of treatments using retinal cell suspensions injected directly into the subretinal space has been limited due to the aged or damaged Bruch's membrane. Therefore, a biomaterial membrane designed to replicate key aspects of Bruch's membrane that can both support the in -vitro culture and in-vivo delivery of RPE cells could provide a promising alternative treatment for AMD. In this work, electrospinning was used to create poly (L-Iactide-co-E-caprolactone) (PLCL) co-polymer biomaterial membranes which were modified using an atmospheric pressure plasma. This created fibre surfaces with chemical and physical characteristics that mimic the native Bruch's membrane, as determined by SEM, contact angle, AFM, FTIR, XPS and tensile strength analysis. Additionally, atmospheric pressure plasma treatment was also investigated as a way to augment the conformation of a collagen IV layer adsorbed from solution onto the PLCL electrospun membranes. In order to determine the biological performance of the various PLCL electrospun membranes to promote and support the development of a functional monolayer of relevant cells, both adult retinal pigment epithelium (ARPE-19) cells and human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cells were assessed utilising a comprehensive range of biological techniques including SEM, gene expression analysis and immunocytochemistry. The work reported here establishes for the first time that surface treated electrospun PLCL membranes are not only capable of enhancing the cellular response and monolayer formation of ARPE-19 cells, they also provide a carrier system that enhances the attachment, proliferation and importantly, the maturation of clinically relevant hESC-RPE cells in serum-free culture conditions.

The last decade has seen significant progress towards the elimination of blinding trachoma as a health problem. However, gaps in our understanding of the epidemiology of trachoma at large scales are increasingly important in the context of programmatic scale up. This thesis therefore aimed to define the current distribution and burden of trachoma, in addition to investigating the spatial heterogeneity of trachoma and underlying risk factors at different scales. A systematic review of trachoma prevalence data was used to generate the Global Atlas of Trachoma, a unique spatially-referenced global database. In addition to highlighting important regional differences in the geographic distribution of trachoma, this database was used to quantify the disease burden in Africa; estimating nearly 153,000 disability-adjusted life years (DALYs) attributed to trachomatous vision loss and 155,500 additional DALYs to trichiasis. Detailed analyses of individual and cluster-level risk factors underlying the distribution of trichiasis in Nigeria and active trachoma in Kenya identified a number of key socio-demographic and environmental factors. Both analyses suggested that spatial dependency (generated by underlying associations with shared risk factors at larger scales) may vary in endemic areas. These findings emphasise the importance of local epidemiology and the need for robust and well-designed survey methodologies to identify areas of high risk. Computerised simulations were used to evaluate the performance of Integrated Threshold Mapping (ITM) in comparison to the accepted gold standard trachoma survey design. The results found that ITM tended to underestimate the prevalence of trachoma across a range of epidemiological contexts where attendance was low and/or the risk of disease was lower in school-going children. This thesis provides the first systematic investigation into the geography of trachoma; highlighting heterogeneities at different scales and their potential programmatic implications. In particular, the findings and methods from this thesis may help to inform future survey design.

Inherited retinal dystrophies constitute one of the most prevalent causes of bilateral visual loss, affecting as many as 1 in 3000 people. Genetic therapies have been developed following the identification of disease-causing mutations, using an understanding of how these mutations cause retinal degeneration and improved viral vectors designed to give safe and efficient gene delivery. Hence a broad range of retinal disorders may be treatable with gene therapy, although many aspects of ocular gene therapy warrant further investigation. The studies presented here aim to improve gene therapy in two well-characterised models of retinitis pigmentosa, the Prph2Rd2/Rd2 mouse and the RCS rat. Gene replacement therapy can be effective in these and other models, mediating both structural and functional improvement. However, in many cases long-term rescue from degeneration is not achieved, as evidenced by a continuing loss of photoreceptor cells. These studies aim to improve adeno-associated virus (AAV)-mediated gene delivery to the murine retina by using novel promoters to investigate the consequences of gene replacement therapy. Additionally an alternative viral vector (AAV2/5) will be used to enhance gene replacement, which gives faster and stronger transgene expression than the AAV2/2 used in previous studies. Cell death can also be prevented by administration of vectors expressing neurotrophic factors, but no study to date has identified such a factor that enhances gene replacement therapy indeed, AAV-mediated CNTF expression has serious deleterious effects on retinal function. Hence here we have investigated these deleterious effects, and determined whether CNTF can be used to prevent photoreceptor death without the reduction in retinal function seen previously. In addition, the ability of AAV-mediated GDNF expression to enhance gene replacement therapy in both the Prph2Kd2fRd2 mouse and the RCS rat has been evaluated in detail. These studies show for the first time that neurotrophic factor delivery, when used in combination with gene replacement therapy, can enhance structural and functional improvement in models of retinitis pigmentosa.

Inherited retinal diseases include a number of disorders which typically affect photoreceptor/retinal pigment epithelial function, and can lead to severe visual impairment. Advances in molecular genetics have allowed the identification of many of the genes responsible for particular conditions, and progress in viral gene transfer technology has enabled the replacement of specific genes into the retina. The first human clinical trial of gene therapy for inherited retinal disease was carried out at Moorfields Eye Hospital and UCL Institute of Ophthalmology, involving 12 patients with RPE65 deficiency - an early-onset retinal dystrophy. The results from this trial are described and provide evidence for the safe administration of viral vectors in the eye, and also demonstrate improvements in retinal function in a number of patients. However, the extent and duration of the response did not match that observed in prior animal studies, suggesting improvements in gene expression level may be required in humans. In addition, consideration for future involvement of the foveal region is highlighted, since retinal thinning was observed in a number of patients following subretinal delivery. Achromatopsia is an inherited disorder of congenitally absent cone photoreceptor function. Gene replacement therapy in animal models of achromatopsia has shown evidence of restored cone function, suggesting that this condition may be an appropriate target for gene therapy in humans. Recent studies have suggested that achromatopsia is a progressive condition with deterioration in cone structure with age, implying that the window of opportunity for therapeutic intervention may be narrow. In this study of retinal structure and function (in preparation for a gene therapy trial) in 40 patients with achromatopsia, an age-associated deterioration in cone structure was not identified, suggesting that the age range for potential intervention is wider than recently suggested, and prospective patients should be assessed on an individual basis, irrespective of age.