The degradation of eyesight is a frightening experience for individuals and unfortunately gradual loss of vision with old age is commonplace. One of the most common forms of eye disease which effects vision is Age-Related Macular Degeneration (AMD). AMD is the leading cause of blindness in the developed world and there is currently no cure for the disease. One treatment option available for the neovascular form of AMD is the injection of Bevacizumab [an anti-vascular endothelial growth factor (VEGF) drug] into the eye on a monthly basis. Investigation into the biodegradable polymers poly(L-lactic acid) (PLLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) as a possible drug delivery system with highly uniform and reproducible microspheres was developed. Under optimised parameters Bevacizumab-encapsulated PLLA:PLGA microspheres were successfully prepared with a steady release of Bevacizumab being obtained. Additionally, fibrous scaffolds of methyl methacrylate (MMA) and poly(ethylene glycol) methacrylate (PEGM) were prepared by an electrospinning process. These MMA:PEGM co-polymers were investigated as a possible Bruchs membrane replacement and as the support for retinal pigment epithelium (RPE) transplantation. The MMA:PEGM co-polymers were functionalised with N-succinimidyl which resulted in the fibres forming a gel in vitro. Gel formation was examined further and successful RPE cell attachment and growth onto these gels was observed. Further work on surface active agents was undertaken to improve the cell adhesion, proliferation and growth of RPE cells onto these methacrylate based frameworks. An arginine-glycine-aspartic acid (RGD) peptidomimetic was prepared and reacted onto the surface of the MMA:PEGM co-polymers, however, the peptidomimetic-attached MMA:PEGM fibres offered little improvement in cell growth in comparison with N-succinimidyl–activated MMA:PEGM co-polymer fibres. Additional attachment of the natural proteins laminin, collagen and fibronectin onto the microspheres was achieved. Attachment of these proteins prolonged the release of the dye from the microspheres and showed no cytotoxic effects when examined in-vitro.

Refractive laser eye surgery is a specialised field in ophthalmology which aims to correct the refractive disorder of an eye. The most established technique is LASIK, which has shown good results for the treatment of simple myopia. Complex refractive disorders, such as compound myopic astigmatism, have shown less predictable refractive outcomes, and in some cases the severe over- or under-correction can even worsen the preoperative situation and damage the eye. In its first stage, this research aimed to develop a software system able to present and analyse refractive outcomes. Over 2 prototype stages, this research has led to an operational system named IBRA (Internet Based Refractive Analysis), offering web-based data collection and refractive and vector analysis. In a second stage, Nomogram calculation formulas were developed and integrated into IBRA. These formulas were created from linear regression and best-fit analyses of spherical and cylindrical outcome data stored in IBRA. The purpose of the nomogram calculations was to provide surgeons with adjustment factors that could be used to improve the refractive outcome of patients with complex refractive disorders. Two extensive clinical audits and a randomized controlled trial were performed at Moorfields Eye Hospital to evaluate the IBRA nomogram adjustments. This research showed that IBRA was able to achieve a positive health change. In addition, results from the audits and trial contributed to the knowledge of nomogram adjustments and provided a framework in which future investigations on nomogram and treatment modifications could be performed. In addition to the above clinical studies, two evaluations were performed with the use of IBRA and data logging techniques to investigate users‟ behaviour relating to the management of data entry processes and the use of analysis functions. This research revealed the best method for entering refractive data, and was able to identify the most important analysis methods. Finally, the use of IBRA and its user-interface were investigated with a user satisfaction survey. The results from this questionnaire based study showed a high acceptance of the web-based platform of IBRA and indicated points for improvement (Documentation).

Glaucoma is a leading cause of irreversible blindness and visual impairment. In the clinic, glaucomatous damage can be characterized by structural changes in the optic nerve head (ONH) and retinal nerve fibre layer (RNFL) that can be evaluated by various retinal-imaging techniques such as scanning laser polarimetry and optical coherence tomography (OCT). The structural damage can lead to functional damage in the visual field (VF), normally assessed with standard automated perimetry, which assesses the differential light sensitivity in the field of view. The clinical measurements of retinal structure and visual function play an important role in the detection and management of glaucoma, but the data generated is often complex and highly variable, thus making it hard to clinically interpret. The purpose of this thesis was to investigate knowledge mining procedures for extracting clinically useful information from these measurements. Knowledge mining describes iterative divide-and-conquer type analyses of large-scale questions: solutions to individual smaller problems are used to generate better quality knowledge, which in the case of work reported in this thesis can be translated into clinically useful analysis tools. This thesis describes five knowledge mining procedures specifically developed and applied to structural and functional measurements in glaucoma: (1) probabilistic inference to aid image acquisition of OCT images; (2) a robust and efficient segmentation algorithm to extract features of retina tissue layer structures in large-scale 3-dimensional image volumes acquired by OCT; (3) a predictive structure-function relationship model to bridge the retinal structure and visual function measurements in glaucoma; (4) quantification and visualization of structure-function discordance using the model about structure-function relationship; (5) integration of structural and functional measurements to improve the reproducibility of the measurements. In conclusion the knowledge mining approaches improved the acquisition and/or accuracy of the measurements and provide new clinical analysis tools to detect and manage glaucoma.

Age-related macular degeneration and diabetic retinopathy, in which the cells at the back of the eye degrade due to age and diabetes respectively, are prevalent causes of vision loss in adults. We formulate mathematical models of retinal metabolic regulation to investigate defects that may be responsible for pathology. Continuum PDE models are developed to test whether rod photoreceptors, light detecting cells in the eye, may regulate their energy demand by adapting their length under light and dark conditions. These models assume photoreceptor length depends on the availability of nutrients, such as oxygen, which diffuse and are consumed within the photoreceptor. Our results suggest that the length is limited by oxygen and phosphocreatine shuttle-derived ATP under dark and light conditions respectively. Parameter sensitivity analysis indicates that lowered mitochondrial efficiency due to ageing may be responsible for the damage to and death of photoreceptors that are characteristic of age-related macular degeneration. In the latter part of this thesis we shift our focus to the inner retina and examine how metabolite levels in the tissue surrounding the neurons (highly sensitive, excitable cells that transmit electrical signals) are regulated by glial cells. For instance, stimulated neurons activate their neighbours via the release of the neurotransmitter glutamate, while glial cells regulate neuronal activity via glutamate uptake. Diabetes produces large fluctuations in blood glucose levels, and eventually results in neuronal cell death, causing vision loss. We generate an ODE model for the exchange of key metabolites between neurons and surrounding cells. Using numerical and analytical techniques, we use the model to show that the fluctuations in blood glucose and metabolic changes associated with diabetes may result in abnormally high glutamate levels in the inner retina, which could lead to neuronal damage via excitotoxicity (unregulated neuronal stimulation).

Uveal melanoma (UM) is the most common intraocular malignancy in adults. Despite successful ocular treatment, about 50% of patients succumb to metastatic dissemination, which occurs haematogenously and mainly affects the liver. On the basis of clinical, histopathological and genetic features of the primary tumour it is possible to predict if the individual patient is at high risk (HR) or low risk (LR) of developing metastases. However, the mechanisms responsible for the development of metastatic disease in UM are still largely unknown; therefore no adjuvant treatment is currently offered to HR patients to prevent development of fatal disease. As the time to discovery of clinically detectable metastases can range from months to decades, a secreted biomarker(s) that could be routinely tested in blood is much needed. The scope of the work presented in this thesis was to use proteomics as a tool to identify potential novel, UM-specific biomarkers. Moreover, the proteomic data acquired would complement genomic and transcriptomic information already generated by the Liverpool Ocular Oncology Research Group, with the ultimate aim of increasing our understanding of UM development and dissemination. The aim of Chapter 2’s project was to compare the proteome of UM tissue samples at HR versus LR of developing metastatic disease using isobaric tags for relative and absolute quantitation (iTRAQ) labelling and mass spectrometry (MS). The quantification of proteins in our samples, proteomic analysis and further validation by immunohistochemistry has led to the identification of two novel prognostic and potentially therapeutic target, S100A6 and the tumour suppressor PDCD4. In Chapter 3 we focused on proteins released in the conditioned medium (secretome) of short-term cultures of HR and LR UM cells, as well as normal melanocytes. Using a label-free quantitative proteomic approach, almost 2000 proteins were identified and quantified, with more than 30% of these identified as secreted and/or previously described in exosomes. Using these data, an 18-protein signature able to discriminate between HR and LR UM was identified. Further validation will be necessary in secretome samples and in the peripheral blood of UM patients, but this has the potential of being translated into a clinically useful assay to detect early development of metastatic disease. As reported in Chapter 4, we also conducted a pilot clinical study on circulating tumour cells (CTC) in UM, using the CellSearch® platform with the novel melanoma kit to enumerate CTC in the peripheral blood of UM patients at LR, HR or with overt metastatic disease. CTC were detected in metastatic and HR tumours and were not present in LR UM, however, the number of CTC detected varied widely, calling into question the clinical value of using this platform in UM patients. The research detailed in Chapter 5 had a direct clinical value, as it addressed the procedures undertaken during the acquisition and processing of prognostic biopsies from UM tumours treated conservatively. The modifications introduced led to a significant improvement of the success rate of such prognostic biopsies for risk stratification, which is essential for clinical management, follow-up and research purposes. In conclusion, the work conducted throughout this PhD has provided further insight into the molecular characteristics that can differentiate between HR and LR UM, identifying novel potential biomarkers that will need validation in the clinical setting.

Introduction: A study in 2005 found that Epidermal Growth Factor Kinase Inhibitors (EGFRki) could promote Retinal Ganglion Cell (RGC) axonal regeneration in vivo when delivered to the crushed Optic Nerve (ON). The axon regenerative effects of these EGFRki were attributed to their blockade of the Epidermal Growth Factor Receptor (EGFR), and EGFR activation was hence suggested to lead to growth cone collapse and failed RGC axon regeneration. Aims: To investigate the role of EGFR in RGC axonal regenerative failure, and to elucidate the mechanisms of action by which EGFRki promote RGC axonal regeneration. Methods: Immunohistochemistry and immunocytochemistry to visualize activated EGFR (pEGFR) Primary retinal cultures to examine the actions of EGFRki such as AG1478 on RGC in vitro. ELISA to examine the conditioned media from these cultures for NeuroTrophic Factors (NTF). Intravitreal (ivit) injections of EGFRki (PD168393) into Optic Nerve Crush (ONC) recipient rats to attempt to elicit in vivo regeneration. The implantation of PD168393-impregnated collagen matrices into ONC recipient rats to attempt to elicit in vivo regeneration. PCR on retinal lysates to detect NTF mRNA. Results: No pEGFR was detected on RGC axons, either in the retina or in the ON of any treatment or control group. pEGFR was detected on almost all ON and retinal glial types prior to injury and almost all glial types exhibited increased pEGFR levels post-ONC. A sub population (~30%) of RGC cell bodies were pEGFR+ but this proportion did not change between control and treatment groups. AG1478 was shown to disinhibit RGC in Nogo-P4 inhibited primary retinal cultures but ELISA on conditioned media for various NTF detected none, however PCR detected mRNA for several of these NTF in retinal lysates. Ivit PD168393 failed to elicit RGC survival or axonal regeneration in vivo. Intra ON implantation of PD168393 impregnated collagen matrices appeared to promote significant RGC axonal regeneration post-ONC, but did not affect RGC survival. Discussion: Several models explaining the in vitro regenerative and in vivo neuritogenic actions of EGFRki were developed. These included the abrogation of various harmful glially mediated processes, the stimulation of NTF release by local glia and the stimulation or blockade of several other non-EGFR dependent signalling cascades by EGFRki. Conclusions: The axogenic and neuritogenic actions of EGFRki in vitro and in vivo were confirmed. The identification of numerous other means by which EGFRki could indirectly promote RGC axonal regeneration, including by acting on targets other than EGFR allowed the construction of a combinatorial model of how EGFRki effect axonal regeneration, and the original hypothesis positing EGFR as an intra-axonal component of a growth cone collapsing signalling cascade was disgarded.

This thesis aimed to investigate a novel way to explore changes in gaze behaviour, whilst walking, in frail populations. Initially three studies were conducted to establish how similar gaze behaviour recorded during walking was to that recorded whilst scene viewing. Duration of time and number of times different features were fixated were found to be similar in the three experiments. Older adults were assessed for falling risk and split into higher risk of falling (HROA) and lower risk of falling (LROA) groups. Their gaze behaviour was recorded whilst scene viewing along with a group of young adults. HROA were found to fixate the travel path longer than LROA and younger adults. HROA were slower at completing the incongruent Stroop task, suggesting a relationship between response inhibition and increased falling risk. A group of stroke patients were assessed for falling risk and split according to lesion location (parietal, occipital or frontal-temporal); gaze behaviour was recorded during scene viewing and compared to controls. Observable differences, which related to falling risk and lesion location, were shown in the gaze behaviour of the stroke patients compared to the controls. The findings of this thesis suggest that scene viewing could be used to better inform us about the changes in gaze behaviour which occur in frail populations that led to an increased risk of falling and the cognitive mechanisms which underlie these changes than laboratory studies.

Since Cohn’s investigation of the cause a of short-sight and the striking evidence which he brought forward as to the part which Education played in connection therewith, we have been accustomed to regard the schools as the "hot bed" of myopia. In view of the apparently conclusive nature of the statistics obtained by Cohn after an examination of over 10,000 scholars in German Schools, supplemented, as these have been, by the work of Snellen, Priestley Smith, Straub (Amsterdam) and many other eminent ophthalmologists, one reads with something of surprise the memoir or Pearson and Barrington “ A First Study of the Inheritance of Vision, and of the relative influence of Heredity and Environment on Sight “. In this memoir, which was issued in 1909 under the auspice of the Eugenic Laboratory of London University, the authors have carefully analysed, by modern statistical methods, much of the evidence adduced in support, of the generally accepted theory that short-sight is largely the result of Educational Environment.

Retinal ganglion cell (RGC) apoptosis and failure of optic nerve (ON) axon regeneration contribute to profound visual loss after traumatic optic neuropathy (TON), for which clinically effective treatments are lacking. Experimental manipulations of cellular signalling have identified phosphoinositide-3-kinase (PI3K) and its downstream mediators mTOR and GSK3\(\beta\) as important regulators of neuronal survival and axon regeneration in an animal model of TON, using targeted siRNA to knock-down key negative regulators of cellular signalling, and to investigate underlying mechanisms using retinal cultures. Intravitreal treatment with siRNA targeting RTP801, a stress-induced inhibitor of mTOR, promoted RGC survival and axon elongation after ON crush (ONC), and potentiated reactive gliosis. In vitro, siRTP801- induced neuroprotection was direct, but required GFAP\(^+\) activated retinal glia to stimulate neurite elongation. siRTP801 also potentiated levels of glial-derived Trk-dependent neurotrophic factors. Knock-down of the axon growth cone/apoptosis regulator GSK3\(\beta\) was also neuroprotective, promoted modest axon elongation after ONC, and increased neurite sprouting in vitro. GSK3\(\beta\) suppression counteracted neurite growth-inhibition induced by CNS myelin-derived Nogo. Combined treatment with siRTP801/siGSK3\(\beta\) augmented axon regeneration after ONC. These findings support a clinically translatable siRNA approach targeting PI3K/mTOR signalling as the basis for development of novel neuroprotective/axogenic therapies.

Diabetes Mellitus (DM) has become one of the most important metabolic diseases that reduces one’s quality of life and doubles the risk of early death. Amongst the major complications linked to DM, diabetic retinopathy (DR) leads to gradual loss of vision and blindness. DR is now the second cause of certifiable blindness among the working age adults in the UK. The lifetime costs to the UK government are calculated to be up to £327,000 per person, with almost 50% of these costs being attributed to loss of productivity caused by visual impairment and blindness. The UK is one of the leading countries in the implementation of DR screening programmes. The latter rely heavily on fundus imaging and grading using trained experts and subsequent referral to hospital for further clinical examination and evaluation depending on the grade of retinopathy. It is now known that subtle, structural changes in the retina that are linked to diabetes can precede detectable vascular changes. The former can affect one’s colour vision and this offers the potential of using changes in chromatic sensitivity as an early biomarker of retinal disease. The first part of this thesis focuses on measuring chromatic sensitivity using the colour assessment and diagnosis (CAD) test in diabetic subjects with varying degrees of retinopathy. The severity of colour vision loss is graded in comparison to other factors that are normally linked to diabetes, such as the type, grade, control methods and duration. The results of this study reveal losses of both red/green and yellow/blue chromatic sensitivity in patients with diabetes, but the correlation with factors, normally associated with high risk of diabetes is low. The results from this study do, however, show that the magnitude of chromatic sensitivity losses correlates with the severity of diabetic retinopathy. The second, related study examines the effectiveness of intravitreal injection of a dexamethasone implant (Ozurdex) in patients with diabetic macular oedema (DMO) in stabilising and reducing loss of visual function and in particular the reduction in chromatic sensitivity up to 24 weeks. This treatment demonstrated efficacy in the treatment of chronic DMO and DMO which is resistant to anti-VEGF treatment. The results show that intravitreal treatment with Ozurdex causes improvement is visual acuity, central retinal thickness and significant improvement in red/green chromatic sensitivity.