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Ageing is a major risk factor for developing retinal diseases. Ageing is characterised by a decline in repair and disruption in homeostasis, which is accompanied by changes in transcriptomic and protein expression. The contribution of ageing to retinal diseases remains largely unspecified. We investigated age-related transcriptional changes at the single cell resolution using single-cell transcriptomics. We established a single-cell atlas of the ageing mouse neuroretina. We noticed age-associated cell-type proportion changes characterised by rod loss. We identified age-related, cell-type specific transcriptional changes. Ageing rods have shown metabolic reprogramming, while ageing macroglia have shown a reactive gliosis phenotype. Genomic instability and increased proteostasis burden was found in ageing bipolar cells. Meanwhile, the bidirectional communication between microglia and other retinal cells was also found to be altered in the ageing retina that favours a low-grade chronic inflammation (para-inflammation). To understand how inflammation is initiated in the ageing retina, we focused on retinal DNA sensing pathways. Intracellular DNA sensors initiate the host immune response by recognising cytosolic DNA derived from pathogen replication or endogenous nucleic acid released by damaged nuclei or mitochondria. The retinal DNA sensing system requires further investigation. We established an expression profile of retinal intracellular DNA sensors. We further confirmed the expression of the adaptor protein of DNA sensing pathway, STING, in the retina. We demonstrated that STING is critically involved in dsDNA-induced type I IFN response in microglia and RPE cells, but not in photoreceptors and Müller glia. An increased enrichment of DNA sensors was detected in microglia in the ageing retina. Significant increase of Sting expression was found in the ageing mouse neuroretina. Studies have suggested the DNA sensing pathways connects genomic instability and inflammation during ageing. These results suggest that DNA sensors activation may be partially responsible for para-inflammation in the ageing retina.

A subset of retinal ganglion cells expresses the photopigment melanopsin and is thus photosensitive. These intrinsically photosensitive retinal ganglion cells (ipRGCs) integrate inputs from rods and cones as well as from melanopsin. Recent research has shown the variety of visual and non-image forming (NIF) responses that ipRGCs contribute to. However, there is uncertainty around the degree to which these responses arise from melanopsin compared to rods and cones, and how the diversity in ipRGCs supports these functions. This is, in part, due to a limitation in tools to assess melanopsin contribution in humans. By developing our understanding of melanopsin in vision and behaviour, we can better understand the impact of our lighting environment on our physiology and make improvements to advance human health and wellbeing. IpRGCs are a heterogeneous population, with six subtypes in mice varying in morphology and electrophysiology. Research suggests all subtypes are involved in vision, via projections to the dorsal lateral geniculate nucleus (dLGN). However, little is known of the relative contributions of the different ipRGC subtypes to the dLGN projection. We assessed this using the multilabelling technique of brainbow in dLGN-projecting ipRGCs. We find five subtypes involved in these projections and restricted topography of labelled cells in the retina. These results suggest that the diversity in ipRGCs is harnessed to support the function of vision. Assessing melanopsin contribution to responses in humans relies on visual display units (VDUs). The technique of silent substitution allows for the targeted stimulation of melanopsin, whilst excitation for other receptors remains constant. This is achieved through the presentation of metameric stimuli, often incorporating a cyan primary due to melanopsin's peak sensitivity. We discover a visual anomaly of Maxwell's spot in stimuli when the cyan primary is employed. We characterise the stimuli that elicit Maxwell's spot and identify a constraint during stimuli generation that minimises its appearance. This can help create visually uniform metameric stimuli for use in melanopsin research. Many existing VDUs for assessing melanopsin in humans have limitations in contrast, spatial resolution, practicality, and relevance in the modern screen-based world. We developed and categorised a new tool for use in vision research. Our multiprimary VDU can present metameric stimuli that vary substantially in melanopsin excitation. We identify some limitations and offer suggestions for the future development of VDUs for use in vision research. Ambient lighting has an impact on human cognition. However, most studies have been conducted in the lab and have limited application to the real world. We developed a new research tool: an app, able to present performance-measuring tasks whilst recording illuminance. We implement this app to determine the effects of illuminance and time of day on performance in the field. We identify several significant relationships that confirm laboratory findings, as well as new results for the effects of lighting, time of day and subjective sleepiness on visual search. With this new tool, there is a vast avenue of research questions that can be addressed in the context of human cognition in the real world.

Background: In ocular diseases such as Age-related Macular Degeneration (AMD) and diabetic retinopathy, dysregulated inflammation in the retina can lead to the formation of a fibrovascular membrane, which can cause severe structural damage to the retina thus leading to severe visual defects and blindness. The process of pathological neovascularisation into the formation of a fibrovascular membrane is not completely understood. Recent research has implicated the Hedgehog pathway transcription factor, Gli1, in the development of fibrosis in numerous organs. This study aims to characterise Gli1 expression in ocular tissues and to elucidate the role of Gli1 in the development of fibrovascular membranes in the retina utilising transgenic mice, and specific small molecule inhibitor of Gli1, GANT61. Methods: Gli1CreERT2 mice were crossed with Ai9(RCL-tdT) (Tomato) mice to produce Gli1-Cre x Tomato crossbreeds. 6-7-week-old Heterozygous Gli1-Cre x Tomato mice received 100 mg/kg Tamoxifen daily (I.P.) for 5 days. Mice were monitored for 80 days to determine the duration Gli1-Tomato expression. Eyes, and other organs, were collected for immunofluorescent staining. For the one laser choroidal neovascularisation (CNV) model, Gli1-Cre x Tomato mice received 3-4 laser burns per eye to induce choroidal neovascularisation and were monitored over 21 days to assess the expression of Gli1 in the injured retina using fundus imaging and immunofluorescent staining. For the two-stage CNV model, Gli1-Cre x Tomato mice received 3-4 laser burns per eye to induce choroidal neovascularisation followed by a second laser directed towards CNV lesions 7 days later. Mice were then monitored over a period of 30 days. Two-stage laser mice received either GANT61 or diphtheria toxin (for Gli1-cre x Rosa mice) to determine the effects of Gli1 inhibition/ablation on fibrovascular lesion development. Cell survival assays of retinal pigment epithelial and microvascular endothelial cells in the presence of TGFβ2 together with/without Gli1 inhibitor GANT61 were utilised to interrogate the role of Gli1 in mesenchymal transition. Results: Expression of Gli1-Tomato was detected as far 90 days in the eye, predominantly in the choroid, sclera, cornea, and ciliary body, although the RPE layer appeared to be Gli1-tomato negative. Immunofluorescent staining displayed co-expression of Gli1-tomato with CD31+, NG2+, and Collagen 4+ retinal microvasculature. Fundus imaging of CNV lesions in one laser model displayed expression of Gli1-tomato in and around the lesion. Immunofluorescent imaging of cryosections and flatmounts showed co-expression of Gli1-tomato with endothelial markers in choroidal neovascular lesions, which persisted up to 21 days after endothelial components diminish. Two-stage laser model CNV lesions displayed co-expression of Gli1-tomato with collagen-1+ lesions up to 30 days post-laser. Knockdown or inhibition of Gli1 did not appear to significantly affect fibrovascular lesion development, however a trend towards decreases in lesion size and collagen-1+ intensity was observed. GANT61 mediated inhibition of Gli1 in vitro demonstrated that epithelial cells undergoing epithelial-to-mesenchymal transition (EMT) were more sensitised than in homeostatic conditions, in contrast endothelial cells undergoing endothelial-to-mesenchymal transition (EndMT) were less sensitised than in homeostatic conditions. Conclusions: Gli1+ cells are expressed throughout the eye, most notably within the uveal tract and retinal microvasculature. In the context of injury, Gli1+ cells are closely associated with endothelial and perivascular components of the CNV lesion. In contrast to published data, results suggest that Gli1+ cells do not drive the development of fibrovascular lesions in a two-stage laser CNV model. Epithelial cells induced to undergo EMT became particularly sensitised to Hh signalling inhibition, suggesting a potential therapeutic avenue for ocular fibrosis. This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 722717.

The aim of this thesis was to evaluate the efficacy and safety of retinal gene therapy in a pre-clinical model of CDHR1-associated retinal degeneration - a hitherto untreatable, blinding disorder. Deep phenotyping of the Cdhr1-/- murine model demonstrated severe early deficits in cone and rod photoreceptor function followed by progressive photoreceptor cell death - recapitulating CDHR1 cone-rod dystrophy. Two AAV8 vectors expressing the full-length human CDHR1 coding sequence were designed, manufactured, validated and titrated in vivo. Sub-retinal injection of AAV8.GRK1.CDHR1.pA (1.5 x 108 vg) in Cdhr1-/- mice at 3-4 weeks of age resulted in functional rescue of cone and rod photoreceptors with improved response amplitudes and decreased implicit times to 12-months post-injection; a slowing of photoreceptor cell death with regeneration of full-length photoreceptor outer segments confirmed on ultrastructural analysis to 21-months post-injection; and a behavioural rescue effect on photopic and scotopic optomotor testing, sustained to 21-months post-injection. AAV8.GRK1.CDHR1.pA at 1.5 x 108 vg appeared safe in C57BL/6J mice by the same outcome measures to 22-months post-injection. Genetic prevalence estimates presented herein suggest more than 200,000 affected individuals worldwide. We characterised 146 individuals with biallelic variants in CDHR1, describing the retinal phenotype, natural history, genotype-phenotype associations, with 25 novel pathological sequence variants. This is the first therapy shown to improve retinal structure and function in a pre-clinical model of CDHR1-associated retinal degeneration. A patent to protect the vectors described herein has been filed on behalf of the University of Oxford, with commercial agreements obtained in preparation for an onward phase 1 clinical trial.

Diabetic macular oedema (DMO) is an important cause of vision loss worldwide. It is characterized by hyperpermeability of retinal vessels due to breakdown of the inner blood-retinal barrier. The goal of this thesis was to better understand the mechanisms through which bradykinin (BK) and thrombin (THR) trigger vasopermeability responses in retinal microvascular endothelial cells (RMECs), with particular emphasis on the role of intracellular Ca2+ and the Ca2+/calmodulin-dependent protein kinase, CaMKII. Ca2+ imaging studies revealed that BK and THR elicit biphasic intracellular Ca2+ responses in cultured bovine RMECs. The initial peak phase resulted from endoplasmic reticulum Ca2+ release via the Gαq-PLC-IP3 signalling pathway, while the sustained phase derived from Ca2+ influx via store-operated Ca2+ channels. Receptor pharmacology studies demonstrated that BK acts primarily through the bradykinin B2 receptor, whereas THR signals through protease-activated receptor 1 (PAR1). Experiments to determine CAMKII phosphorylation upon BK and THR exposure were undertaken in bovine and human RMECs. Western blotting studies identified a time-dependent increase in CaMKII activity upon agonist treatment persisting for up to 24 hours. Hyperglycaemic conditions for 21 days did not have a significant effect on basal or agonist induced CaMKII phosphorylation. Increased FITC-dextran leakage was observed in bovine RMEC monolayers challenged with BK and THR and these effects could be largely abolished in the presence of the CaMKII inhibitor, KN-93, but not its inactive analogue, KN-92. To investigate the relevance of these findings to DMO, the effects of systemic administration of KN-93 and KN-92 on retinal vascular leakage in streptozotocin-induced diabetic mice were investigated. These experiments confirmed an important role for CaMKII in mediating retinal vasopermeability responses in the diabetic retina. Taken together, the work in this thesis suggests that CaMKII could represent a novel therapeutic target for DMO treatment, although further studies will be required to confirm this.

Adeno-associated virus (AAV) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the eye. Whilst some consider the eye to be an immuneprivileged organ, recent reports have begun to suggest that injection of AAV may elicit local and systemic immune activation. The objective of this thesis was to further our understanding of the nature of the immune response to intraocularly-delivered AAV vectors, and to develop strategies to achieve repeated gene transfer (i.e. successful follow-on injections) to the inner retina. A literature review was conducted into the role that pre-existing and induced neutralising antibody responses may play in the context of ocular gene therapy. This concluded that a multifaceted/comprehensive approach may be required to circumvent anti-capsid humoral immunity and enable repeated gene transfer in humans. As such, two possible components of this strategy were tested in this thesis. First, a capsid mutagenesis strategy was tested in which certain capsid residues, termed phosphodegrons, were mutated to attenuate cytosolic degradation of AAV. This was to enable robust transduction of the murine retina at low vector dose and thereby circumvent the generation of neutralising antibodies that is associated with injection of high doses of AAV. In fact, it was found that phosphodegron mutant AAV serotype 2 (AAV2) resulted in higher levels of immune activation across all tested parameters vs. wild-type AAV2, including neutralising and total AAV binding antibody levels, microglia activation, Muller glia activation, CD4+ and CD8+ T-cell infiltration into the retina, splenic germinal center B-cell activation and class-switching, and activation of conventional dendritic cells. Mechanistically, it was shown that the capsids demonstrated reduced binding affinity to the AAV2 primary receptor, heparan sulphate proteoglycan, and as such it was suggested that reduced sequestration of virions in the heparan-rich inner limiting membrane may increase cellular infection levels in the retina, thereby increasing overall immune activation. The phosphodegron mutant capsids also demonstrated that a slight escape from anti-AAV2 neutralising serum, suggesting they may be more refractory to pre-existing neutralising antibodies in seropositive patients. Second, an assessment was made as to whether perioperative administration of immunosuppressive glucocorticoid steroids would circumvent immune responses to AAV. It was found that prednisolone was effective at reducing anti-AAV neutralising and total AAV binding antibody titres. This effect may have occurred via the inhibition of splenic germinal center B-cell reactions and class-switching of immunoglobulin antibody isotypes, and reducing follicular helper T-cell levels. Despite the reduction in NAb and TAb titres, it was found that perioperative prednisolone administration was insufficient to enable repeated gene transfer following a second eye injection of AAV2. Interestingly, repeated bilateral AAV2 injections may have resulted in a slight reduction in electrophysiological activity in the retina compared to mice who received two control PBS injections. In conclusion, this thesis highlighted novel aspects of phosphodegron mutant AAV2 immunobiology and identified a possible utility for the administration of prednisolone as a tool for enabling repeated gene transfer of AAV gene therapy vectors.

Diabetic retinopathy (DR) is the most frequently occurring microvascular complication of diabetes mellitus, where the later stages of the disease are often is chaemia driven which can result in severe vision loss. A key component cell of the retinal neurovascular unit is the vascular endothelium, which is significantly impacted by the diabetic milieu. It has now been established that the endothelium relies on glycolytic metabolism predominantly for ATP generation, however the degree of flux through this pathway in some non-retinal endothelial cell types has been shown to vary in disease conditions. This PhD project has assessed the nature of glycolysis and related plasticity in human retinal microvascular endothelial cells (HRMECs) exposed to in vitro conditions relevant to DR.HRMECs were obtained from human donors and were exposed to either normal glucose conditions of 5 m M D-glucose (NG), 20 m ML-Glucose + 5 m M D-Glucose (HLG), or 25 m M D-Glucose (HDG) for four weeks, along with a hypoxic exposure (1% O2, 5% CO2 )for 24 hours before experiments were performed, mimicking diabetic-like conditions. Assessments of in vitro metabolism were assessed using the Seahorse XF system and qPCR to highlight potential changes in metabolic pathways. Functional assays including 3D-Matrigelexperiments, Clonogenics, proliferation, migration, and barrier integrity were also implemented to ascertain what changes to functionality occur after HDG exposure. Furthermore, the use of pharmacological compounds such as the glycolytic inhibitor 3PO ((2E)-3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one),and Proxison (7-decyl-3-hydroxy-2-(3, 4, 5-trihdroxyphenyl)-4-chromenone) a synthetic flavonoid were also implemented in this study to determine if pharmacological interventions/ modifications to metabolism could be effective in reducing HDG exposure affects. In an attempt to understand angiogenic pathways in more detail, single cell sequencing was also performed on cells recovered from 3D environment. This project together helps to understand the nature of this complex disease and identify potential new targets of therapeutic interventions.

Multiple aspects of mammalian physiology display circadian rhythms, which allow the organism to better adapt to the external environment. Several circadian rhythms, such as the sleep-wake cycle and the retinal function, are primarily regulated by light detection in the retina. The molecular properties of melanopsin, a blue light sensitive photopigment expressed in a subset of retinal ganglion cells, make it ideal for encoding environmental light levels. Indeed, melanopsin has been shown to regulate several aspects of the retinal circadian function. However, the molecular mechanisms underpinning melanopsin's role in retinal circadian rhythms remain unknown. By analysing the circadian transcriptome of wild-type and melanopsin-null retinas, I discovered that the absence of melanopsin leads to dampened rhythmicity in the expression of core clock genes. Furthermore, while the melanopsin-null retinas retained the rhythmic expression of dopamine- related and gap junction genes, the circadian rhythmicity of sphingolipid and phototransduction pathways were aberrant. Alongside its role in the retinal circadian clock, melanopsin is also modulat- ing functional aspects of retinal light adaption, and it has been suggested that melanopsin may mediate retinoprotection in response to bright light exposure. However, using a mouse model of light-induced retinal damage, I did not detect an increased susceptibility to light damage in animals lacking melanopsin. I further studied how well the mouse model of retinal light damage mirrors the human retinal degenerations. I conducted a meta-analysis of all available datasets probing retinal transcriptome changes in this model and discovered that the genes affected by light damage were enriched for genes implicated in human retinal degenerations, indicating that the model indeed mimics the molecular aspects of human diseases. Finally, melanopsin signalling also regulates the central light responses in the suprachiasmatic nucleus (SCN), but several reports on its role in retinal light sensitivity are conflicting. Therefore, I further researched how retinal and SCN responses to light are regulated. I found that the retinal light responses of animals lacking melanopsin were slightly attenuated, similarly to what is known about their SCN light responses. However, in a mouse model showing increased SCN light sensitivity, the retinal responses were not enhanced, suggesting differential regulation of the retinal and SCN light sensitivity. These results provide valuable insights into the molecular and functional aspects of retinal light responses in both local and central circadian rhythms, and dissect their implications for retinal degenerative diseases, together providing a wealth of avenues for future research.

Dose-dependent inflammatory responses in adeno-associated virus (AAV)-mediated retinal gene therapy trials have restricted the use of high vector doses and impeded the ability to enhance therapeutic efficacy. Characterising the nature of this response, and devising methods to overcome it, are thus becoming increasingly important in the clinical application of gene therapies. In the experiments herein, I investigated the composition, timing, and magnitude of the cellular immune response to retinal gene therapy in mice using quantitative flow cytometric analysis. I observed a significant increase in retinal leukocytes from 14 days after subretinal injection of an AAV8 vector expressing green fluorescent protein (GFP). These consisted of a constant population of resident microglia and infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells. An identical vector at an equivalent dose encoding a human therapeutic transgene elicited a diminished response compared to paired injections with a GFP vector. However, the proportions of the leukocyte populations remained similar, suggesting a common mechanism of immunity. In addition to the induction of a cell-mediated immune response, innate detection of AAV vectors may directly restrict transgene expression. Preliminary evidence suggested in vitro AAV transduction may be limited by the anti-viral nucleic acid sensors Toll-like receptor 9 and cyclic GMP AMP synthase, suggesting possible detection of transgenic AAV genomes. I also showed that the immunomodulatory drug hydroxychloroquine can significantly enhance AAV transduction in vitro, ex vivo, and in vivo, with indirect indication of the partial antagonism of TLR9. The lack of structural change in the retina following subretinal hydroxychloroquine delivery indicates that it could be a safe adjunct for improving retinal gene therapy efficacy.

Microglia are a tissue-resident immune cell of the central nervous system known to possess functions involved in immune surveillance and tissue homeostasis. Transcriptomics has characterised microglia and enabled discovery of microglial heterogeneity in addition to core microglial transcriptional programmes. However, investigation of microglia during severe inflammatory contexts has been challenging because no markers reliably discriminate them from the monocyte populations that ingress during inflammation. Nonetheless, candidate markers have been identified; these show promise in specific microglial identification yet remain to be widely validated. Within the literature, there are conflicting reports on how microglia regulate or promote inflammation depending on the tissue insult. However, it is well-recognised that the homeostatic state of microglia is altered yet it remains unknown if this state is restored post-resolution. Furthermore, understanding the plasticity of microglial responses to both acute and persistent inflammation within the eye will help to determine the extent to which different pathways are perturbed. The purpose of this thesis was to investigate the transcriptional changes that occur in retinal microglia in response to inflammation and whether the homeostatic threshold remains perturbed after acute and/or chronic inflammation. The data presented herein demonstrates how an ultra-low input mRNA-Seq approach was optimised and validated to permit transcriptomic assessment of low numbers of cells isolated from individual retinas. The Cx3cr1CreER:R26-tdTomato mouse line was then validated as microglial-specific during inflammation. mRNA-Seq was utilised to profile the temporal kinetics of the microglial transcriptome in the acute endotoxin-induced uveitis (EIU) model. Restoration of the microglial homeostatic state was confirmed, and key marker changes were orthogonally validated. Furthermore, C5AR1 was validated as a marker for differentiating microglial subsets during inflammation. The next steps have begun to examine microglial behaviour in experimental autoimmune uveitis (EAU), a model of chronic inflammation, and new approaches are being optimised to better understand tissue heterogeneity.

The human retina is the tissue at the back of the eye responsible for converting light stimulus into neuronal signal that can be interpreted by the brain. To perform this integral role within the central nervous system, the retina has a complex and layered structure, with each layer performing a vital step in the signal transformation process. Changes in the morphology of this structure are often a consequence of disease, which can affect the function of the eye. Better understanding of the genetics influencing this structure may teach us about the biological processes underlying these diseases as well as general eye development. The retina is imaged routinely in the clinic using optical coherence tomography (OCT), providing a non-invasive imaging technique that produces high-resolution, three-dimensional representations of the retina from which measures describing retinal morphology can be extracted. This thesis summarises my research into the genetic variation underlying retinal morphology. Firstly, I explored the morphology of the inner retina, whose thickness is used as a biomarker of glaucoma, using quantitative phenotypes extracted from OCT. I conducted genome-wide association studies (GWAS) of the thickness of the retinal nerve fibre layer and the ganglion cell inner plexiform layer to understand the genetic variation driving inner retinal morphology. I further explored the causal relationship between the inner retina and glaucoma using Mendelian randomisation analysis. I next performed GWAS of the thickness of the outer retinal layers, including both the component photoreceptor cell layers (the outer nuclear layer, inner segment, and outer segment), and the retinal pigment epithelium layer. I explored how genetic variation was affecting the outer retinal morphology at a higher dimension by looking for genetic variants that were differentially affecting the outer retinal thickness at the central macula compared to the peripheral macula. To further explore the rich dimensionality of OCT data, I developed several image analysis techniques to gain more granular information about the morphological variation being affected by the discovered genetic variants. In doing so I established a novel population level trait and examined its effect on visual acuity. In summary, this thesis provides a well-rounded and detailed look into the genetic variation underlying morphological variation of the retinal layers. As the largest study of retinal layer genetics of its kind, it offers insight into clinical ophthalmology and retinal development, and furthermore opens new avenues for clinical research.

Cellular senescence is the permanent state of growth arrest with significant phenotypic changes including the senescence-associated secretory phenotype (SASP) and triggered by mechanisms such as telomere shortening and DNA damage. Diabetes is associated with endothelial dysfunction, which is characterised by increased vascular permeability, deficit in blood perfusion, vessel loss and increased inflammation. Cellular senescence has been implicated in the pathogenesis of diabetes; however, a role for cellular senescence in diabetic retinopathy is unclear. Therefore, the aim of this study is to investigate the senescence profile of human retinal microvascular endothelial cells (HRMECs), to elucidate how this is affected by the diabetic milieu. HRMECs undergo replicative senescence in vitro and they acquire gene and protein expression profiles that are characteristic of a senescent phenotype. Senescent HRMECs show a SASP signature, changes in organelle profiles, loss of endothelial function and metabolic impairment. Chronic exposure to high glucose (25mM D-glucose) induces premature senescence in HRMECs. The accelerated senescence programme was characterised by enlarged cellular and nuclear size, upregulation of p53 and CCL2 (MCP1), increased mitochondrial mass but, with alterations in mitochondrial cristae ultrastructure and decreased cellular bioenergetics both glycolysis and oxidative respiration. The aged mouse retina shows cellular senescence, accompanied by visual dysfunction and structural changes. The diabetic milieu, both hyperglycaemia and ischaemia, lead to accumulation of senescent cells in the retina, including the retinal vasculature. Senolytic drugs are investigated as a therapeutic approach for elimination of senescent HRMECs. Navitoclax was found to be an effective senolytic for HRMECs in vitro.

Embryonic development is a highly complex procedure, leading from initial, unspecialised cell types - the stem cells - to ever more specialised ones. In part, this process is regulated by genes; but interactions between cells, such as mechanical contacts and the exchange of diffusive signalling molecules, also play pivotal roles for the correct execution of developmental programmes. Therefore, unravelling the rules of cell differentiation requires insights from both biology and physics. In this dissertation, we focus on the process of interkinetic nuclear migration (IKNM). IKNM takes place in cells of so-called pseudostratified epithelia (PSE) during development. In these tissues, the nuclei of cells move in a cell cycle dependent manner and position themselves in a specific region of the cells for each cell division. The correct nuclear positioning has been shown to be crucial for proper development in PSE. And because organs like the brain and the spinal cord develop from pseudostratified epithelial tissues, IKNM appears to be of paramount importance for the entire embryo. The work presented here concerns IKNM in the retina, an experimentally accessible outgrowth of the brain, and relies on experimental data obtained from zebrafish. However, the conclusions are likewise relevant for understanding the development of many other PSE tissues. Based on the experimental data, two previously posed hypotheses on how the majority of nuclear movements might be driven can be tested. The data is consistent with the idea that nuclear movements depend on the build-up of a gradient in nuclear packing density across the retinal tissue. Consequently, we develop the first mathematical model for the distribution of nuclei across the retinal tissue as a function of time. Underlying this model is the notion that individual nuclear trajectories phenomenologically resemble random walks during most of the cell cycle. Therefore, we model the time evolution of the nuclear density using a diffusion equation with an effective diffusion constant to be determined from the data. Furthermore, we specifically account for the fact that nuclear divisions always take place in a defined region of the cells - leading to the aforementioned gradient in nuclear packing density. Finally, we also pay attention to the spherical geometry of the retinal tissue. Although the simplest linear model describes well the data from early in the experiments, it fails to do so for data from later stages in which nuclei approach close-packing. We hypothesise that the reason for this mismatch between model and data might result from the neglect of crowding. Therefore, we present a second, nonlinear model which now takes the volume of nuclei into account by introducing a maximum possible packing density. This enables us to replicate the experimental nuclear distribution across the whole range of experimental time points. We finally employ this second model to make statements about the influence of experimental parameters, specifically incubation temperature, on the dynamics of IKNM. The result also provides some indications for possible microscopic mechanisms underlying the nuclear movements. Having studied the distribution of nuclei across the retinal tissue, we aim to investigate the significance of our obtained results on the level of individual cells. First, we compare the mobility of nuclei during IKNM with the expected mobility in the cases of Brownian motion and membrane-hindered Brownian motion. We find that IKNM appears to be both membrane-hindered and additionally driven throughout the entire cell cycle. Assuming a stochastic driving force and calculating its typical strength we deduce IKNM to be consistent with cytoskeletal transport. We then devise possible Langevin models for individual nuclear movements which are consistent with the model for the distribution of nuclei derived previously. The numerical simulation of each of these Langevin models enables us to distinguish between them; we identify the model which most likely reflects the biological process in each individual cell. This again leads to predictions about the potential microscopic underpinnings of nuclear movements during IKNM. The apparent importance of the cell membrane in restricting nuclear mobility prompts us to examine the shape of PSE cells in closer detail. We numerically solve the Helfrich elastic model for lipid bilayers for increasingly large cell aspect ratios. In the case of long, slender cells and high membrane tension, we recover shapes not unlike those previously reported for membrane tethers. In contrast, shorter cells are almost cylindrical. The results of this systematic investigation into cell shapes might explain the different geometries of cells in various types of PSE. Furthermore, they might also be of relevance for more generally understanding peculiar cell shapes, such as those of neurons.

This thesis describes a body of work examining associations between retinal biomarkers (retinal microvascular parameters [RVP] and retinal layer thickness measurements) and measures of renal function and damage (estimated glomerular filtration rate and urinary albumin to creatinine ratio). Retinal biomarkers have previously been associated with the most significant causes of chronic kidney disease (CKD), hypertension and diabetes, and the most important effect of CKD, cardiovascular disease. As a result of recent advances in retinal imaging technologies, and the routine collection of fundus photographs and optical coherence tomographs at diabetic eye screening clinics and high-street opticians, retinal biomarker measurement has become a potentially cost-effective source of structural information from the microvasculature and neuronal layers in vivo that can be collected semi-automatically and non-invasively as part of routine eye screening or measured from pre-existing images. This may allow for cost-effective improvements in the early prediction of symptomatic CKD, and/or progression of CKD, helping to target earlier treatment initiation, and facilitating new therapeutic avenues. To assess the association between retinal arteriolar and venular calibre, arterio-venous ratio (AVR), fractal dimension, and tortuosity were measured and their associations with estimated glomerular filtration rate (eGFR) and urinary albumin to creatinine ratio (ACR) were tested in population-based and clinically derived populations. Measurements of retinal thickness were also taken and their association with eGFR tested. Overall, no evidence of an association between retinal microvascular calibres and renal function (eGFR) or damage (ACR) was found independent of known confounding influences. Cross-sectional analyses of UK biobank data showed that sparser retinal microvascular networks (lower arteriolar and venular fractal dimensions) were associated with an increased risk of ACR > 3mg/mmol. In a prospective cohort, RVPs did not predict reductions in renal function in individuals with type 2 diabetes. These findings do not provide evidence for associations between measures of retinal microvascular calibre and CKD or for predictive value of RVP for the progressive loss of renal function in diabetes. Associations between both renal health and RVP and two potential confounding factors (diet and cognitive function) were also assessed and models of the association between RVP and ACR were constructed to adjust for these factors. The results of these analyses did not provide evidence for an association between Alzheimer’s disease and eGFR < 60mL/min/1.73m2 , nor a clinically significant association between cognitive function and ACR > 3 mg/mmol. Consistent with this, models of the association between RVP and ACR were not altered following adjustment for cognitive function. In the Irish Nun Eye Study, adherence to an unhealthy dietary pattern was associated with poorer renal function but no association between the dietary intake of various food groups and ACR was found in the UK biobank population and associations between RVP and ACR were not altered following adjustment for dietary intake. The work presented in this thesis did not, therefore, provide sufficient evidence that either cognitive function or dietary intake warrant inclusion as confounding variables in future studies of RVP in CKD. Finally, novel associations between inner retinal layers and eGFR < 30 mL/min/1.73m2 were found in conjunction with altered fractal dimensions in the retinal microvasculature which supplies the inner retinal layers. Longitudinal analysis of this association may be warranted to assess the predictive potential of retinal thickness measurements for CKD.

Age-related macular degeneration (AMD) is the most common cause of visual loss amongst the elderly in developed countries. AMD is a complex disease with a highly variable phenotype, which makes generating reliable disease models difficult. In addition, lack of knowledge of the underlying pathological mechanisms makes development of an effective treatment difficult. To address the lack of knowledge of the molecular mechanisms affected in AMD a robust cell-based model system is needed. Late-onset retinal degeneration (L-ORD) is a rare autosomal dominant disorder caused by a p.S163R, p.P188T, or p.G216C missense mutation in the C1q and tumor necrosis factor-related protein 5 (C1QTNF5) gene. L-ORD has a very similar phenotype to AMD, including sub-RPE deposit formation, the hallmark of AMD. Studying L-ORD could therefore potentially reveal common molecular pathways affected in macular degenerations. Here, I used CRISPR/Cas9 gene editing to correct the p.S163R mutation in C1QTNF5 in patient-derived induced pluripotent stem cells (iPSCs) and generated C1QTNF5 null iPSCs. The iPSC lines were differentiated into retinal pigment epithelium (RPE), the cell type that highly expresses C1QTNF5 and is severely affected in macular degenerations. RNA sequencing of L-ORD and control iPSC-RPE revealed that the extracellular matrix, complement system, lipid and general cell metabolism, and oxidative stress pathways are affected in L-ORD. C1QTNF5S163R in L-ORD iPSC-RPE was found to form less high molecular weight multimeric structures compared to its isogenic gene-corrected control. In addition, L-ORD iPSC-RPE were also found to phagocytose and possibly adhere differently compared to their isogenic controls. Both oxidative stress and the complement system play an important role in the pathology of macular degenerations. RPE were found to undergo regulated necrosis in response to oxidative stress and L-ORD iPSC-RPE were found to be more sensitive to UV light-induced oxidative stress. In addition, the terminal complement system complex C5b-9 was found to bind more frequently to L-ORD iPSC-RPE and colocalises with APOE in sub-RPE drusen-like deposits after human serum exposure. Together, the results in this thesis reveal that L-ORD iPSC-RPE have a molecular phenotype that could explain part of the clinical presentation of this disease and possibly of AMD. Further study of the affected molecular mechanisms could potentially lead to therapies for both L-ORD and AMD.

People with Down’s syndrome (DS) are known to experience premature ageing and have a high propensity for clinical diagnosis of dementia due to Alzheimer’s disease (AD). In DS there is a unique and natural model of over-expression of amyloid-beta (Aβ) protein, the accumulation of which is proposed to be the central early event in the pathogenesis of AD. In DS, AD neuropathology is universally seen in the brain from the fourth decade. Identifying biomarkers are essential to the evaluation of future treatment trials. The retina has been shown to experience changes in patients with AD, such as retinal thinning, compared to age-matched controls. As an extension of the brain, the retina can be quickly and non-invasively imaged and may provide a proxy measure of brain changes in AD. Using optical coherence tomography (OCT), cross-sectional retinal examinations were completed in 50 people with DS aged 18 years and over. Comparisons between retinal thickness of the DS and control groups were examined, as well as the effect of age on thickness in both groups. For the DS group, further investigations were made into the relationships between retinal thickness and (i) cognitive performance, (ii) diagnosis of dementia, (iii) cortical thickness and, (iv) presence of Aβ binding in the brain. Contrary to expectations, people with DS had thicker retina compared to age-matched controls. In addition, normal age-related retinal thinning was not seen in the DS group. People with DS have a life-long overproduction of Aβ, deposits of which have been previously imaged in the retina. Aβ may be responsible both directly, through physical mass, and indirectly through inflammation as a response to Aβ, for increased retinal thickness in people with DS. Consequently, retinal thickness in DS may be a proxy measure of Aβ deposition in the retina. As part of a collaborative study, brain Aβ binding was measured using positron emission tomography neuroimaging in a subset of the DS group. Individuals with positive Pittsburgh compound [11C]-PIB (PIB) binding to Aβ displayed a trend towards having thinner retina than those with negative PIB binding. These results indicate that a shift towards thinning retina in people DS may reflect changes in brain pathology. Future studies are discussed which aim to investigate Aβ and Aβ driven pathology in the retina.

The central nervous system is a complex, yet well-organised, often laminated, tissue. This robust organisation is evident in the architecture of the retina: consisting of 5 different neuronal types organised into distinct layers: Retinal Ganglion Cell (RGC), Amacrine Cell (AC), Bipolar Cell (BP), Horizontal Cell (HC) and Photoreceptor cell (PR) layers. This remarkable organisation is evolutionarily conserved in vertebrates, yet little is known about the mechanisms by which these cells form the correct layers. Live imaging has revealed overlapping periods of birth and extensive inter-digitation followed by cells sorting out into their appropriate positions, suggesting cell-cell interactions are important. To investigate possible cellular and molecular mechanisms responsible for the establishment of the tissue architecture I developed an organoid culture system for zebrafish retinal cells. To identify the cells in culture I used a Spectrum of Fates fish line which is a multiply transgenic line in which each retinal cell type can be identified based on expression of a combination of fluorescently tagged cell fate markers. The development of the protocol by which I cultured the cells and observed their cell-cell interactions involved establishing the best methods to dissociate and culture zebrafish retinal cells in a non-adhesive environment, then imaging the resulting reaggregates to examine the position of the different retinal cell types. By doing this I observed their inherent self-organising properties, in the absence of extrinsic cues or scaffolds. These cells appeared to be arranged in an inside-out layering, although all cell types are layered in the same relative order as they are in vivo. To analyse the organization in these aggregates I developed a Matlab script in collaboration with Leila Muresan which analyses the relative positioning of cells in concentric rings from the periphery to the centre of the aggregates according to the cell fate-tagged fluorescent markers. The script then fits this data as an empirical cumulative distribution function for different groups of cells to determine how spatially distinct populations of cells are. This gave me my measure of organisation. I then investigated the cell-cell interactions involved in this self-organisation by genetically or pharmacologically removing individual cell types and assaying the resulting organisation of the reaggregated, cell-type deficient, retinal organoids. I revealed that Müller Glia are important for retinal cell self-organisation. I also investigated the role of Retinal Pigment Epithelial (RPE) cells and Retinal Ganglion Cells and found they had no impact on the ability of the remaining cell types to organize. I began to investigate the role of Amacrine Cells but found that retinas void of ACs were susceptible to disaggregating in our dissection setup, preventing me from collecting the material needed for culture. I also investigated the role of candidate molecules in this system and revealed that R-Cognin is critical for retinal cells to reaggregate. Not only can I remove cells or molecules from the system, but I show how it can also be manipulated to replace molecules of interest such as laminin, by coating beads with the substance of choice and placing it amongst the cells to see if their organisational behaviour is affected. In summary, I have developed a system which provides a simple and easy platform to manipulate in various ways to help us potentially reveal some of the important players in neuronal patterning.

Diabetes mellitus currently affects over 422 million people globally and over 80% of patients with diabetes will develop diabetic retinopathy. Patients with diabetic retinopathy initially develop background retinopathy, which does not cause significant deterioration of visual function; however, background retinopathy may progress and lead to proliferative diabetic retinopathy and diabetic macular oedema, both of which cause severe visual dysfunction if left untreated. Current therapies for diabetic retinopathy include invasive intravitreal injections and laser photocoagulation; however these treatments only attenuate the progression of proliferative diabetic retinopathy and diabetic macular oedema. Aside from prevention by maintaining good blood glucose and blood pressure control, there are currently no treatments to prevent progression to late-stage diabetic retinopathy and new innovations in the field have not significantly progressed. For this reason, we have used untargeted –omics approaches to identify previously unknown pathological pathways in diabetes. In this thesis, I have analysed a range of trace metals in donor retinas and found that total copper was increased in diabetic retinas compared with non-diabetic. This result was replicated in streptozotocin-induced diabetic rat retinas and further evidenced by upregulation of metallothioneins and caeruloplasmin in diabetic rat retinas compared with non-diabetic. Treatment with the copper chelator triethylenetetramine modulated these changes, the downstream effects of which require further study. This is the first description, to our knowledge, of dysregulated copper homeostasis in the diabetic retina. I have also mapped metabolic changes in streptozotocin-induced diabetic rat retinas and found previously undescribed metabolite changes such as diabetes-induced downregulation of scyllo inositol. This coincided with substantial changes to retinal lipids during diabetes and changes to individual lipids were consistent within their respective class. I have also found a pattern whereby regardless of the extent of change to a lipid class in diabetes, lipids containing docosahexaenoic acid (22:6 carbon chain) were consistently downregulated. This is thought to be the first study to describe this range of metabolite changes in the diabetic retina but also the first study to describe this range of metabolite analysis concomitantly within the same tissue sample. The data from this study provides new insights into metallomic and metabolic dysfunction in diabetic retinopathy and shown that these data are reproducible. We suggest that there is plenty of scope for further research to investigate mechanisms behind copper dysregulation, how this affects pathogenesis of diabetic retinopathy along with new insights into dysregulated metabolic pathways.

Retinal imaging with a fundus camera or scanning laser ophthalmoscope (SLO) allows a fast, non-invasive view to the body’s microvasculature. Evidence suggests that features associated with retinal blood vessels (for example, narrowing of arteries and increased vascular tortuosity) measured near to the optic disc are early biomarkers of disease such as diabetes, hypertension (HT) and cardiovascular disease. Ultra-widefield (UWF) ophthalmic imaging allows unique views of peripheral locations as well as the posterior pole, potentially facilitating a more comprehensive study of the state or health of the microvasculature than is afforded by conventional retinal imaging. It is envisaged that this will reveal new candidate biomarkers derivable from the retina which could help identify the presence of disease or improve risk stratification for serious illness. In this thesis, 532 individuals were recruited from a trial evaluating the added benefit of using computed tomography (CT) imaging in the diagnosis of coronary heart disease (CHD) to measure atherosclerotic plaque in the arteries of the heart muscle tissue. The trial participants were deeply phenotyped which allowed access to additional information including: presence and severity of CHD, hypertensive status, presence of diabetes, age, gender, and smoking status (all risk factors commonly associated with CHD). After CT imaging patients were invited for undilated UWF ophthalmic imaging using an Optos P200C SLO. To accurately measure features of blood vessels indicative of microvascular health or disease in these images required the development of a novel semi-automatic computerised technique to segment and analyse the retinal vasculature. This involved implementation of a supervised vessel segmentation algorithm utilising multi-scale matched filters, a neural network classifier and hysteresis thresholding. A true positive rate (TPR) of 0.702 (and standard deviation of 0.059), false positive rate (FPR) of 0.011 (0.006) and accuracy (Acc) of 0.965 (0.006) was achieved by the algorithm. This compared to TPR of 0.674 (0.062), FPR of 0.017 (0.004), and Acc of 0.957 (0.006) for state-of-the-art fundus camera vessel segmentation applied to UWF SLO. After segmentation and prior to the measurements of retinal vessel parameters, the distortions introduced by the instrument mapping the 3D retinal surface onto a 2D image plane were also accounted for utilising an established technique. This is especially important for measuring in the periphery of UWF images and has not previously been reported for biomarker discovery. Measurements from UWF SLO were compared between those participants with CHD (where a reduction in arterial width was hypothesised based on existing research with fundus cameras into cardiovascular disease), and those without to investigate whether a difference between the two groups existed. After appropriate statistical correction for confounding variables (i.e. age, gender, and hypertensive status) the results did not show a statistically significant difference for presence of CHD or for risk stratification. However, the analysis techniques that were developed in this thesis do allow a rapid investigation of retinal vascular parameters in UWF SLO. This has application to a number of other diseases, such as HT, where a more pronounced change to the appearance of vessels is anticipated, and to different areas of the peripheral retina not previously measurable with standard imaging techniques and existing algorithms.

Aims: The electroretinogram (ERG) can be used to independently assess the function of rod and cone photoreceptors within the human retina. The work in this thesis sought to investigate an alternative method of recording the ERG, using the silent substitution paradigm (Estevez and Spekreijse 1982). The aims are separated into two parts, firstly, the isolation and characterisation of the non-dark adapted rod photoreceptor response, and secondly, characterising the ERG response from L-, M- and S-cones. Methods: Rod, L-, M- and S-cone isolating as well as non-isolating sinusoidal flicker and transient square-wave stimuli were generated on a 4 primary LED ganzfeld stimulator to elicit ERGs from non-dark adapted participants with normal and compromised rod or cone function. Results: The results from the rod experiments showed that ERGs elicited by rod isolating silent substitution stimuli exhibit low-pass temporal frequency response characteristics with an upper response limit of 30Hz and saturate beyond 1000ph Td. Responses are optimal between 5 - 8 Hz and between 10-100 photopic Td. There is a significant correlation between the response amplitudes obtained with the silent substitution method and current standard clinical protocols. The results from the cone experiments showed that the L-, M- and S-cone stimulation produced ERGs with very different morphologies. L- and M-cone stimulation is of limited use as an objective measure of colour vision deficiency. Conclusion: Silent substitution provides an effective method for the isolation of human rod and cone photoreceptor function in subjects when stimuli are used within appropriate parameter ranges.

The bestrophinopathies are a set of inherited retinal degenerations caused by mutations in BEST1, and include Best vitelliform macular dystrophy (BVMD), autosomal dominant vitreoretinochoroidopathy (ADVIRC) and autosomal recessive bestrophinopathy (ARB). The corresponding protein, bestrophin-1, is localised to the basolateral membrane of the retinal pigment epithelium (RPE), where it is thought to function as a Ca2+-activated Cl- channel. Currently, there are no treatments for these conditions. In recent years, gene therapy has emerged as an exciting treatment option for inherited retinal disorders (IRDs). Gene delivery to retinal cells using a recombinant adeno-associated virus (rAAV) has produced positive results in several IRDs. Given the recessive nature of ARB, this thesis proposes that the rAAV-mediated delivery of bestrophin-1 to the RPE could represent a potential therapy. The aims of this thesis were to produce and compare rAAV vectors in vitro and in vivo for protein expression, localisation following transduction, restoration of chloride conductance in vitro and safety following sub-retinal injection in vivo. Following the production of two rAAV vectors expressing bestrophin-1, western blots confirmed bestrophin-1 protein expression following transduction of HEK293 cells in vitro. Immunocytochemistry (ICC) revealed bestrophin-1 expression that was localised to the cytosol. Whole-cell patch-clamping revealed a significant increase in chloride conductance in HEK293 cells transduced with AAV-BEST1 vectors which was then ablated upon the removal of chloride from the buffers. Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) indicated that the bestrophin-1 protein was successfully transcribed and translated from the BEST1 coding sequence (CDS). Sub-retinal injections of AAV-BEST1 produced bestrophin-1 expression in the RPE of wild-type C57BL/6 mice however significant retinal thinning was seen at higher doses of vector. In conclusion, rAAV-mediated transfer of bestrophin-1 to the RPE has potential to be a future therapy for ARB, however safety issues need to be addressed and an RPE-specific promoter could be more suitable.

Late-onset retinal macular degeneration (L-ORMD) is a fully penetrant autosomal dominant macular degeneration resulting from a Ser163Arg substitution in the gene encoding the protein C1QTNF5. Clinically L-ORMD results in dark adaptation delay in the fifth decade, central visual loss in the sixth decade and further progressive visual field loss in successive decades of life. Pathologically the disease results in thick sub-retinal deposits, which have a similar composition to drusen seen in AMD, retinal pigment epithelial (RPE) loss, and neuro-retinal atrophy. The function of C1QTNF5 is incompletely understood however within the eye it is expressed most strongly by the RPE cells. An in vitro model for L-ORMD was developed using human induced pluripotent stem cells (hiPSCs) derived from patients and with stem cells from patient’s unaffected siblings used as controls. The hiPSCs were differentiated to RPE (hiPSC-RPE). L-ORMD hiPSC-RPE shared baseline characteristics with sibling control hiPSC-RPE. In order to model in vivo conditions hiPSC-RPE were grown on permeable supports in human serum enriched media. Case hiPSC-RPE cell lines were found to activate the complement pathway resulting in increased deposition of the terminal complement complex (TCC) C5b-9 when compared to control hiPSC-RPE. Using depleted serum, deposition was not affected by depletion of classical and lectin pathway components but was reduced by depletion of alternative complement pathway components. Depletion of complement components C3 and C5 abolished TCC deposition. The addition of a monoclonal antibody against C5 also reduced TCC deposition. The role of complement dysregulation in L-ORMD pathogenesis was confirmed by immunostaining of L-ORMD and age-matched control human donor retinal sections. L-ORMD retinal sections displayed increased C3d and C5b-9 deposition. Using mutant and wild type-protein generated from a bacterial expression system it was found that the mutant protein was less stable than the wild-type. In addition the wild type protein formed multimers whilst the mutant was mainly monomeric. A surface plasmon resonance (SPR) study showed an increased affinity of wild-type C1QTNF5, especially in multimeric form for complement factor H (CFH), a key regulator of the alternative complement pathway when compared to mutant protein. Taken together these studies implicate dysfunction of the alternative complement pathway in L-ORMD disease mechanism and have suggested a role for C1Q TNF5 in the extracellular matrix. The studies also show that L-ORMD and AMD share a pathogenic and clinical similarities.

Introduction Malaria retinopathy is a set of visible changes in the retina which are specific to falciparum malaria. Studies to date have been mostly limited to comatose African children. Retinal changes in adults with severe malaria and severely unwell patients without malaria have been less well studied and the specificity, pathogenesis, diagnostic and prognostic value of malarial retinopathy in adults are not known. Methods A series of observational studies of retinopathy in Bangladesh, India and Malaysia were done from 2008-2012. The aims were to describe the spectrum of retinal changes in falciparum and knowlesi malaria in adults, determine their specificity for severe falciparum malaria, quantify the impact of malaria retinopathy on visual function, understand its pathogenesis and assess the potential contribution of retinopathy to confirming diagnosis of malarial coma, predicting prognosis and understanding pathogenesis of cerebral malaria. Results 495 patients were enrolled and underwent retinal photography (305 with P. falciparum malaria (112 cerebral, 68 noncerebral severe, 125 uncomplicated), 44 P. knowlesi, 43 sepsis, 41 encephalopathy and 62 healthy). Retinal whitening and white-centred haemorrhages were common and specific to severe falciparum malaria. Retinopathy was most common and severe in cerebral (88%) and fatal (91%) falciparum malaria. Moderate-severe retinopathy was 95% specific for cerebral malaria in comatose patients, and its severity correlated with depth of coma. Vessel whitening was not seen and papilloedema was rare. In noncerebral severe falciparum malaria, retinopathy predicted increased likelihood of later development of coma and death. Retinal findings in Bangladeshi children were similar to those in adults. Optic nerve sheath diameter was mildly increased and brain swelling minimal on MRI. Severity of retinopathy correlated with plasma lactate, serum bicarbonate, sequestered parasite load and red cell stiffness suggesting a central role for microvascular obstruction in the pathogenesis. Severity of retinal whitening correlated with decreased visual acuity. Conclusions Retinal changes seen in severe P. falciparum malaria in Asian adults is similar, but not identical, to that seen in African children. They have potential to help with diagnosis and prognosis of Asian adults with severe falciparum malaria. Microvascular obstruction is prominent in the pathogenesis of retinopathy and coma in adults whereas raised intracranial pressure is not.

Retinal imaging enables the visualization of a portion of the human microvasculature in-vivo and non-invasively. The scanning laser ophthalmoscope (SLO), provides images characterized by an ultra-wide field of view (UWFoV) covering approximately 180-200º in a single scan, minimizing the discomfort for the subject. The microvasculature visible in retinal images and its changes have been vastly investigated as candidate biomarkers for different types of systemic conditions like cardiovascular disease (CVD), which currently remains the main cause of death in Europe. For the CARMEN study, UWFoV SLO images were acquired from more than 1,000 people who were recruited from two studies, TASCFORCE and SCOT-HEART, focused on CVD. This thesis presents an automated system for SLO image processing and computation of candidate biomarkers to be associated with cardiovascular risk and MRI imaging data. A vessel segmentation technique was developed by making use of a bank of multi-scale matched filters and a neural network classifier. The technique was devised to minimize errors in vessel width estimation, in order to ensure the reliability of width measures obtained from the vessel maps. After a step of refinement of the centrelines, a multi-level classification technique was deployed to label all vessel segments as arterioles or venules. The method exploited a set of pixel-level features for local classification and a novel formulation for a graph cut approach to partition consistently the retinal vasculature that was modelled as an undirected graph. Once all the vessels were labelled, a tree representation was adopted for each vessel and its branches to fully automate the process of biomarker extraction. Finally, a set of 75 retinal parameters, including information provided by the periphery of the retina, was created for each image and used for the biomarker investigation.

Melanopsin (Opn4) is the opsin photopigment of intrinsically photosensitive retinal ganglion cells (ipRGCs). It has a conserved opsin structure and activation mechanism, yet demonstrates unusual functional properties that suggest it will possess unique structure-function relationships. The aim of this thesis was to characterise key OPN4 residues by examining the impact of non-synonymous mutations on melanopsin function. A genotype-driven screen of a chemically-mutagenized mouse archive led to the identification of a novel Opn4 mutant, S310A, located at a known opsin spectral tuning site. Action spectra from ipRGC and pupil light responses (PLR) of Opn4S310A mice revealed no change in wavelength of peak sensitivity. However, Opn4S310A PLR was significantly less sensitive at longer wavelengths, consistent with a short-wavelength shift in spectral sensitivity. This suggests S310A acts as a spectral tuning site in melanopsin. Next, the impact of naturally-occurring missense variants in human melanopsin (hOPN4) was examined in vitro. Fluorescent calcium imaging of 16 hOPN4 variants expressed in HEK293 cells revealed four hOPN4 variants abolished or attenuated responses to light (Y146C, R168C, G208S and S308F). These variants were located in conserved opsin motifs for chromophore binding or hydrogen-bond networks, functional roles apparently shared by melanopsin. Finally, two hOPN4 single nucleotide polymorphisms (SNPs) P10L and T394I, associated with abnormal non-image forming behaviour in humans, were explored in vivo. Using targeted viral-delivery of hOPN4 SNPs to mouse ipRGCs, a range of OPN4-driven behaviours, such as circadian photoentrainment and pupil light responses, were found to be comparable with hOPN4 WT control. Multi-electrode array recordings of ipRGCs transduced with hOPN4 T394I virus had significantly attenuated sensitivity and faster response offset, indicating this site may be functionally important for melanopsin activity but compensatory rod and cone input limits changes to non-image forming behaviour.

Recent advances in retinal imaging have made it possible to take measurements of retinal oxygen saturation noninvasively in humans. This allows studying the supply of oxygen in healthy and diseased retinae, thereby advancing our understanding of both the normal functioning of the retina and of retinal pathologies. However, retinal oximetry is still a research tool only and requires further improvement before being used in a clinical setting. Here, a single-wavelength flickering light was used to increase retinal blood flow in healthy subjects. This increase is revealed by both vasodilation and an increase in retinal oxygen saturation. A flickering light stimulus provides the means to assess the sensitivity of any retinal oximetry system, as such systems should be able to pick up this increase in retinal blood flow. In addition, the flickering light allows for com- parison to be made within rather than between subjects and can be used to examine the activation of the eye. This reduces the influence of potential confounding factors between subjects including differences in fundus pigmentation and illumination. The most commonly used method to measure retinal oxygenation is the optical density ra- tio (ODR) approach. The standard approach is to compute the average ODR for each vessel segment by combining the hundreds of individual ODR readings and then to use the mean of these segment averages as a measure of oxygen saturation. Alternatively, it has been suggested that the peak location of Gaussian functions fitted to histograms of individual ODR readings can be used as an measure of retinal oxygenation. In response to a 10Hz flickering light, the venular diameter increased by 3.44% (SEM: ±0.53%) (n=16, p<0.05) and the arteriolar diameter by 1.87% (±0.72 %) (p<0.05). The optical density ratio, measured with the Gaussian fit, decreased in the venules from 0.713 (±0.015) to 0.694 (±0.015) (p<0.05). No changes in arteriolar optical density ratios were measured. The post-flicker measurement was computed as the average of up to four post-flicker datasets obtained at 10s, 20s, 30s and 40s after onset of flickering. These results suggest that the flickering light increased retinal blood flow. The mean absolute percentage error was lower in venules for the Gaussian fit method than for the gold standard method for datasets taken at 30s and 40s after onset of flickering. Thus, the Gaussian fit method was more robust. All measurements were taken with a custom-made retinal oximeter. The pixel intensity of the blood vessel and the intensity on either side of the vessel had to be extracted to compute the individual optical density ratios. This required the automatic extraction of the retinal vasculature. Two such algorithms were developed and applied to two databases of retinal fundus images: the DRIVE and the novel DR HAGIS database. One algorithm was purely based on the pixel intensities, while the other made use of oriented Gabor filters. These two algorithms segmented the images to a similar accuracy (DRIVE: 94.56% and 94.54%, DR HAGIS: 95.83% and 95.71% for the intensity and Gabor filter based algorithm, respectively) and performed as well as a human expert (DRIVE: 94.73%). These algorithms were of sufficient quality to extract individual segments for the oximetry study and to align fundus images.

The retina is the ocular tissue responsible for the detection of light. Its extensive demand for oxygen, coupled with a concomitant elevated supply, renders this tissue prone to both hypoxia and hyperoxia. In this thesis, we construct mathematical models of the retina, formulated as systems of reaction-diffusion equations, investigating its oxygen-related dynamics in healthy and diseased states. In the healthy state, we model the oxygen distribution across the human retina, examining the efficacy of the protein neuroglobin in the prevention of hypoxia. It has been suggested that neuroglobin could prevent hypoxia, either by transporting oxygen from regions where it is rich to those where it is poor, or by storing oxygen during periods of diminished supply or increased uptake. Numerical solutions demonstrate that neuroglobin may be effective in preventing or alleviating hypoxia via oxygen transport, but that its capacity for oxygen storage is essentially negligible, whilst asymptotic analysis reveals that, contrary to the prevailing assumption, neuroglobin's oxygen affinity is near optimal for oxygen transport. A further asymptotic analysis justifies the common approximation of a piecewise constant oxygen uptake across the retina, placing existing models upon a stronger theoretical foundation. In the diseased state, we explore the effect of hyperoxia upon the progression of the inherited retinal diseases, known collectively as retinitis pigmentosa. Both numerical solutions and asymptotic analyses show that this mechanism may replicate many of the patterns of retinal degeneration seen in vivo, but that others are inaccessible to it, demonstrating both the strengths and weaknesses of the oxygen toxicity hypothesis. It is shown that the wave speed of hyperoxic degeneration is negatively correlated with the local photoreceptor density, high density regions acting as a barrier to the spread of photoreceptor loss. The effects of capillary degeneration and treatment with antioxidants or trophic factors are also investigated, demonstrating that each has the potential to delay, halt or partially reverse photoreceptor loss. In addition to answering questions that are not accessible to experimental investigation, these models generate a number of experimentally testable predictions, forming the first loop in what has the potential to be a fruitful experimental/modelling cycle.

Retina is a unique site in the human body where the microcirculation can be imaged directly and non-invasively, allowing us to study in vivo the structure and pathology of the human microcirculation. Retinal images can be quantitatively assessed with computerized imaging techniques, enabling us to measure several different quantitative traits derived from the retinal vasculature. Arterial and venular calibres are the most extensively studied traits of the retinal microvasculature and numerous epidemiological studies demonstrated promising associations with systemic and ocular diseases as well as with disease markers. However, there has been a lack of research into pathophysiological processes leading to retinal vascular signs, and how they link retinal microcirculation with coronary and cerebral microvasculature change. Information about genetic determinants underlying retinal vascular structure is therefore important for understanding the processes leading to microvascular pathophysiology. Two genome wide association studies have been published so far revealing four loci associated with retinal venular calibre and one locus with arteriolar calibre. Here the results from the genome-wide association analysis of 10 different retinal vessel traits in two population based cohorts are presented. Retinal images were measured in non-mydriatic fundus images from 808 subjects in the Orkney Complex Disease Study (ORCADES) and 390 subjects from the Croatian island of Korcula, using the semi-automated retinal vasculature measurement programme SIVA and VAMPIRE. Using pairwise estimates of kinship based on genomic sharing, heritability was calculated for each trait. Estimates of tortuosity measure and fractal dimensions present first published reports of heritability estimates for those traits. In addition correlation analysis with systemic risk factor was also completed, confirming already published results as well as revealing some new associations. A genome wide association analysis of retinal arteriolar width revealed a genome wide significant hit (1.8x10-7) in a region of chromosome 2q32 (within TTN gene). Replication was sought in a further independent Scottish population (LBC) and additional 400 retinal images were graded. The result did not replicate, however the direction of the effect was consistent and a larger sample size is required. Analysis of the remaining traits did not yield genome wide significant result,s and will also require larger sample sizes. Genetic analysis of a binary retinal trait was also explored in a case control study of retinal detachment, which is an important cause of vision loss. A two-stage genetic association discovery phase followed by a replication phase in a combined total of 2,833 RRD cases and 7,871 controls was carried out. None of the SNPs tested in the discovery phase reached the threshold for association. Further testing was carried out in independent case-control series from London (846 cases) and Croatia (120 cases). The combined meta-analysis identified one association reaching genome-wide significance for rs267738 (OR=1.29, p=2.11x10-8), a missense coding SNP and eQTL for CERS2 encoding the protein ceramide synthase 2. Additional genetic risk score, pathway analysis and genetic liability analysis were also carried out.