A growing interest in macular pigment has highlighted the need for further investigation into environmental and genetic factors which may influence individual density and whether these levels can be increased. This thesis has aimed to address these fundamental questions through a systematic review of the current literature, a cross-sectional analysis of environmental and genetic factors associated with macular pigment and a randomised, placebo-controlled trial using a lutein and zeaxanthin supplement. Environmental and genetic factors affecting individual ability to respond were also investigated. Integral to macular pigment research is the ability to accurately measure macular pigment in vivo. This thesis also aimed to assess repeatability of the widely used HFP method, and to compare this technique to fundus reflectometry using the Viscucam 200. A statistically significant increase in serum lutein and macular pigment in response to supplementation was demonstrated by both the systematic review and randomised, placebo-controlled trial within this thesis. Both studies also show great variability in the response of macular pigment. When associations with macular pigment and serum carotenoid concentrations were examined cross-sectionally, established relationships were replicated with regards to baseline and environmental determinants of serum lutein and macular pigment. PTe taster status was not significantly associated with dietary intake of lutein and zeaxanthin. With regards to measurement of macular pigment, measures taken by the. Visucam 200 were found to be repeatable but not comparable to HFP using the MPS 1000. This thesis demonstrated good repeatability of the MPS 1000 over both short and longer-term periods making it useful for cross-sectional and longitudinal studies . To conclude, environmental and genetic factors found to be significantly associated with baseline macular pigment in previous studies have been replicated in the current study and new association identified, including environmental and genetic factors associated with macular response. Newly identified associations warrant further investigation.

Ageing is the main risk factor for the sight threatening disease, age-related macular degeneration (AMD) which causes central vision loss. Although the exact pathogenic mechanism underlying this disease is not well known, recent advances in understanding of how gene expression is regulated suggest a role for small nOI1- coding regulatory RNAs called microRNAs. The main aims of this PhD project were to characterise the endogenous microRNAs in retinal tissues and to investigate how they change with age and in a model of AMD. In addition, how do specific microRNAs regulate retinal pigment epithelium (RPE) function. Firstly, I demonstrated that the small RNA population in three month-old C57BLl6J wild type mice retina and RPE/choroid is extremely complex, including novel orthologs and microRNAs, isomiRs, microRNAs synthesized via non-canonical via Drosha-independent pathways and other small RNAs. Secondly, expression of these microRNAs was changed with age. Half of the microRNAs expressed in polycistronic clusters change with age in the same direction. MicroRNAs altered with age were predicted to be targeting age-related pathways, retina specific functions and inflammation-related pathways, especially TGF -β signalling. Thirdly, miR-26a was highly expressed in RPE/choroid, down-regulated with age and was involved in the regulation of TGF-β signalling. Inhibition of miR-26a caused a change in YEGF expression, cell proliferation and migration . Finally, microRNAs altered in the CCLT/-/ CX3CR 1 GrP/GFP animal model of geographic atrophy were predicted to be targeting genes involved in pathways related to AMD pathology. MicroRNAs altered at before an overt phenotype in this model animal also targeted some of these pathways and may be a prelude to this disease pathology. Some of the microRNA changed with age overlap with this animal model suggesting that microRNA changes in retina contribute to .disease progression. Alhough miR-26a expression changes in this model are not significant. Functional changes with miR- 26a inhibition suggest its potential as a future biomarker for ageing and a therapeutic approach for age related macular degeneration. Thus, microRNAs playa major role in ageing and age-related retinal degeneration.

This collaborative research project between India and Queens University Belfast funded by the British Council for the Prevention of Blindness was divided into three parts: 1. To determine mutalional load of myoGilin (MYOC) in Indian patients with juvenile open angle glaucoma (JOAG). MYOC was sequenced in 22 JOAG patients from Pune, India and 89 controls. A 32 year old female patient with severe JOAG had one previously reported pathogenic (c.1279G>A; p.Ala427Thr) and one novel MYOC mutation (c.1129A>G; p.Thr377 Ala). Cascade screening has identified at risk family members who are under close clinical surveillance. Myocilin genetic testing should form part of the routine clinical care of JOAG patients and their families. 2. Mutational screening of ANGPTL7 a glaucoma candidate gene in primary open angle glaucoma (POAG) and JOAG. ANGPTL7 was sequenced in 166 patients with POAG from Pune and Dublin, 22 patients with JOAG from Pune and 153 age-matched control subjects. A pathogenic mutation c.299G>T (p. Ser100lle) was detected in one Irish patient with POAG. Molecular modeling predicted a deleterious effect, however further work is required to understand the effect of mutant ANGPTL7 on POAG pathogenesis. 3. Massively parallel sequencing of complete mitochondrial genome to determine whether mutations in mitochondrial DNA (mtDNA) play a role in POAG. The complete mitochondrial genome from 32 POAG patients from India and Ireland and controls was amplified by LR-PCR in two fragments and massively parallel sequencing was performed on the Ion PGM™ Sequencer. All variants were confirmed by conventional Sanger sequencing. 50% of POAG cases had a pathogenic mtDNA mutation. 22 mtDNA mutations consisting of T novel mutations and 8 previously reported mutations were identified. 8/22 (36.4%) of these, were In Complex I mitochondrial genes, supporting the concept that mitochondrial dysfundion , specifically Complex I defects, 'play a significant role in POAG .

Intraocular inflammation encompasses a diverse group of diseases which account for up to 20% of severe vision loss, particularly following pathological neovascularisation in the retina. Nowadays, the use of corticosteroids is still the main armamentarium to manage these devastating conditions. Clearly safer, more target-specific and effective drugs are needed. Experimental autoimmune uveoretinitis (EAU) is an animal model for human intraocular inflammation. It is well known that myeloid derived cells substantially contribute to the pathological activities in this model. The aims of my PhD project were: to understand how myeloid-derived cells contribute to retinal damage in the EAU and to investigate whether modulating these cells can control retinal inflammation and associated retinal angiogenesis. Firstly, we showed that the deletions of both CCL2 and CX3CR1 resulted in reduced retinal inflammation in EAU. In the CCL2/CX3CR1 double knockout EAU mice, the macrophage infiltration was reduced and the inflammation was dominated by neutrophils at the acute stage. A reduction in macrophage infiltration was associated with reduced retinal angiogenesis at the chronic stage of EAU. Secondly, we found that SOCS3 in myeloid cells played an important role in EAU. The LysM-Cre-SOCS3f11f1 mice had an earlier onset and more severe retinal inflammation after immunisation. LysM-Cre-SOCS3f11f1 EAU mice also developed more severe retinal angiogenesis. Inflammation in the LysM-Cre-SOCS3f11f1 EAU mice was characterised by enhanced neutrophil infiltration and greatly increased cytokine expressions. In addition, the bone marrow-derived macrophages from LysM-Cre-SOCS3f11fl mice expressed M2 makers and produced more IL-10 and VEGF-A compared to the cells from wr mice. Finally, we found that blocking CCL2 or VEGF-A alone was not sufficient to suppress chronic inflammation-mediated retinal angiogenesis. However, systemic inhibition of arginase-1 activity was effective in reducing chronic EAU induced retinal angiogenesis. Thus, arginase inhibition could be a novel therapeutic strategy to control retinal neovascularisation related to long-standing uveoretinitis.

Experimental Autoimmune Uveitis (EAU) serves as a model for human non-infectious intraocular inflammation, This CD4+ T cell mediated disease can be induced with peptides from the retinal autoantigen retinol binding protein 3 (RBP-3), Antigen specific CD4+ T cells orchestrate disease leading to a variation in the population of cells infiltrating the retina at different stages of inflammation. Infiltrating activated macrophages cause tissue destruction by release of reactive oxygen species, leading to retinal damage. T cell epitopes can influence effector cells and inflammation, though only a few uveitogenic epitopes have been described in the C57BLl6 mouse. The study of population dynamics of EAU induced with recombinant protein subunits of RBP-3 allowed the identification of a novel uveitogenic peptide in subunit 3 of RBP-3 protein. RBP-3 peptide 629-641 induced EAU in this model, as demonstrated by fundal imaging and retinal cellular analysis by flow cytometry. The C57BU6 model is a mild chronic and persistent inflammatory disease; extensive characterization of infiltrate within the retina in this model is described in this thesis. Post primary peak the infiltrate in the inflamed eye has a significant increase of CD4+ I L-17 produCing cells compared to the spleen. Also demonstrated was a late increase in numbers of CD8+ T cells which expressed low levels of IFNy and IL-17 and lacked the ability to degranulate as determined by CD107a ·expression. Coinciding with the increase in CD8+ T cells was an increase in cytotoxic CD1 07a+ NK cells. This analysis revealed distinct changes in the leukocyte cell populations and microenvironment at the late persistent phase of disease that may regulate or perpetuate chronic EAU.

<i>AIMS:</i> The primary aim of this thesis was to determine what effect modular intramedullary stems of differing lengths have on the initial stability experienced by the tidial tray and the strain magnitude experienced within the proximal tibia due to the differing modular stems in a primary and revision TKA. <i>NULL HYPOTHESIS: </i>Increasing the length of the implant stem has no affect on the micromotion of the tibial tray relative to the bone surface. Adding a modular stem does not affect the strain distribution within the proximal tibia. <i>CONCLUSIONS:</i> A 40mm or 80mm press-fit modular stem does not enhance initial fixation with hybrid or cementless implantation in either primary or T2A revision TKA. The addition of a modular stem when implanting an uncemented tibial tray may well increase the instability of the construct. Cemented implants with no modular stem have better initial fixation compared to all uncemented implants tested. In a primary and revision T2A TKA scenario the addition of a press-fit or fully cemented 80mm modular stem offers no added translational or rotational stability. In the bone impaction grafting group a fully cemented tibial tray with an 80mm modular stem significantly increased the migrational and inducible displacement stability. The use of cemented modular stems in primary TKA and simple revision TKA reduces the strains experienced in the proximal tibia and causes excessive strains within the distal cancellous bone at the stem tip. Press-fit stems do not cause significant stress shielding but do cause localised areas of high strain at the stem tip, (which may be linked to patient pain and discomfort). A cemented long modular stem provided the best strain distribution within the proximal graft in the T1 models.

This thesis presents research conducted at the cognitive level, the neurophysiological level and the psycho-physical level, for examining vision in ASD. The psycho-physical findings suggest that atypical visual perception in ASD is unlikely to have a sub-cortical origin as sub-cortical magnocellular and parvocellular pathway functioning, and low/high spatial frequency detection in adolescents with ASD were found to be no different from typically-developing controls. There was, however, evidence indicating local motion direction perception deficits in the same adolescents with ASD suggesting that atypical motion perception in ASD may have a cortical origin. Electrophysiological investigation of low level visual perception in ASD revealed findings concurring with this latter interpretation. More specifically, whereas visual evoked potentials demonstrated visuo-integrative processes associated with perception of second order and hyperbolic gratings were not atypical in children with ASD, there was increased activity of the visual cortical region. A further gamma power analysis then demonstrated that there may be increased neuro-connectivity within primary visual area V1 in the children with ASD. Atypical low level visual cortical processes may result in locally-biased perceptual style previously observed in individuals with ASD. However, a cross-cultural comparison of perceptual style in children with ASD and TD children from Singapore and England, found evidence suggesting that locally-biased perceptual style in ASD may not be culturally universal. In sum, lower level visual cortical processes may be atypical in ASD, and whether these atypicalities manifest at the higher perceptual level can be determined by cultural variability in attention and response processes.

Ageing of Bruch’s membrane is associated with structural and functional deterioration. Accumulation of normal and abnormal collagen in ageing Bruch’s has led to the hypothesis of diminished matrix degradation mediated normally by a family of protease enzymes called the matrix metalloproteinases (MMPs). Underlying mechanisms leading to diminished MMP activity in ageing Bruch’s remain unknown but functional changes of diminished transport are well documented. Ageing remains the biggest risk factor in AMD with nearly 30% of all individuals reaching the age of 85 years showing some loss of central vision. In the present thesis, the gelatinase system (constituting MMPs 2&9) has been examined resulting in the identification and characterisation of three additional high molecular weight species termed HMW 1&2 and a large macromolecular weight MMP complex (LMMC). HMW1&2 were shown to be covalently bonded homo-and/or hetero- polymers of pro-MMPs 2&9. HMW species in effect sequester pro-MMPs 2&9 reducing the pool available for activation and the age-related increase in HMW species is expected to augment this reduction. In Bruch’s membrane from donors with AMD, levels of HMW1&2 were considerably elevated (p<0.05) with a concomitant reduction in the amount of active MMPs 2&9 (p<0.05). The reduction in activated MMP species therefore underlies the reduced degradative capacity of Bruch’s in these patients. Elution studies demonstrated the existence of a free-bound equilibrium for the gelatinases with the bound forms being retained by hydrophobic, ionic or metal mediated interactions. Since divalent metal ions are deposited in Bruch’s of AMD donors, metal chelation was assessed as a possible means of inducing MMP release. Metal chelation with EGTA resulted in the release of active forms of MMP2 and significantly improved the fluid transport properties of the membrane (p<0.005).

One of the most significant complications of diabetes is retinopathy. Diabetic retinopathy (DR), a chronic progressive sight-threatening disease of the retinal microvasculature, is the leading cause of treatable blindness in the working age group. Another emerging complication of diabetes is cognitive impairment (Cl). The exact link between diabetes and Cl remains elusive. One theory is that microvascular changes in the brain may be responsible for change in cognition in diabetes. In this study, it was hypothesised that increased severity of DR was associated with impaired cognition (cerebro-microvascular disease) in individuals with Type 2 Diabetes (T2DM). 381 men and women with T2DM recruited to the South East London Diabetic Retinopathy Study were stratified by severity of DR (no/mild retinopathy and proliferative diabetic retinopathy (PDR)) and severity of diabetic maculopathy (non-clinically significant macular oedema (non-CSMO) and CSMO). Each subject underwent tests of cognitive function, psychosocial assessment, ophthalmic and physical examination. Bivariate analysis between categories of DR and maculopathy (ANOVA, Chi square) and ANCOVA for the cognitive scores by DR severity were conducted using SPSS v17. Severity of DR demonstrated an inverse relationship with Cl in patients with T2DM (fully adjusted model). No association was found between severity of maculopathy and Cl. Retinal arteriolar and venular dilation was associated with lower cognition scores in patients with no/mild retinopathy. Decreased levels of serum factor Apo A was associated with decreased cognition. Participants with Cl had consistently elevated risk of stroke compared to participants with no Cl, irrespective of their DR status. Cognition scores also varied by ethnic grouping; participants of ethnic minorities had significantly lower cognition scores than Caucasian participants.

Primary angle-closure glaucoma (PACG) is a common cause of irreversible blindness worldwide but the epidemiology varies significantly between races. This thesis describes the clinical and genetic examination of families with primary angle-closure suspect (PACS) and more advanced disease. Participants were mainly of European origin, from 101 families. Parametric, non-parametric and quantitative trait linkage analyses, a pilot case-control study and targeted gene screening were performed. In the largest PACG family recruited, two promising regions on chromosomes 10 and 13 were identified. Phenotype-genotype correlation highlighted plateau iris to be the most prominent feature in affected individuals, although shorter axial biometry and hyperopic refractive error were also observed. Quantitative trait linkage applied to a number of traits measured by anterior segment optical coherence tomography provided support for the chromosomes 10 and 13 loci. Maximum iris thickness and adjusted axial length at the chromosome 10 region showed LOD score of 1.3 and 1.5 respectively. Adjusted anterior chamber depth and lens vault on chromosome 13 showed LOD scores of 1.7. As the linkage results were suggestive but not conclusive, a pilot case-control study, followed by participation in an international consortium-led genome wide association study (GWAS) was performed. Three single nucleotide polymorphisms, rs1015213, rs3753841 and rs11024102, were found to be associated with cases of acute angle closure (AAC) and PACG. The loci for our family based analyses and the GWAS did not overlap. In 13 families, PAC was found in probands but further clinical and genetic examination of family members identified atypical features. Angle-closure was often observed as part of a more complex ocular phenotype that led to syndromic diagnoses of Noonan, Marfan, Weill-Marchesani, Ehlers-Danlos and Bestrophinopathies. In conclusion, PACG is a multifactorial disease. Phenotypic predisposition such as plateau iris has a small number of genetic contributors, but advanced disease has further contributions from common SNPs.