Background: Affecting one or both eyes, retinoblastoma is the most common intraocular neoplasm occurring in children under the age of 5 years. The most frequent presenting signs of retinoblastoma include leukocoria and strabismus, whilst symptoms of pain or discomfort generally only manifest with advanced intraocular or extraocular disease and systemic spread. Although the management of retinoblastoma is largely determined by the stage of disease at presentation, fewer treatment options are available with late diagnosis. Moreover, diagnostic delay is associated with negative outcomes, including severe vision impairment, enucleation or death. As the keenest observers of their children, parents or primary caregivers are the most likely to notice the earliest signs of retinoblastoma. However, in Australia, parents are not currently provided with any information, nor are there any specific strategies in place, to alert them to the early signs of not just retinoblastoma but any paediatric eye disease. Purpose: The overall aim of this doctoral research project is to develop and evaluate the effectiveness of an evidence-based, theory-informed pamphlet to improve parents’ awareness of the early signs of significant paediatric eye disease. A mixed methods approach, including both qualitative and quantitative methods, was utilised to achieve this aim. It is hypothesised that providing parents with paediatric eye health information will improve knowledge and change help-seeking intention. Research Design and Methods: Aim 1: Develop a brief information pamphlet educating parents to recognise and respond to notable signs of significant paediatric eye disease An exploratory qualitative study investigated and identified existing knowledge gaps and determined factors that influence parents’ help-seeking behaviour for ocular signs or symptoms that they may observe in their child. Using an inductive thematic approach, qualitative data were analysed to ascertain overarching themes and concepts. The qualitative data supported the development of a paediatric eye health awareness information pamphlet for parents, which was guided by established guidelines for writing health information and a theoretical framework for behaviour change. Aim 2: Evaluate the information pamphlet A single-centre, double-blind randomised controlled trial (RCT) was conducted to evaluate the effectiveness of the pamphlet developed in Aim 1. Pregnant women’s awareness and understanding of early signs of paediatric eye disease and their hypothetical help-seeking intentions were measured using a pre-/post-test design. Results: Aim 1: Development of the information pamphlet Analysis of evidence gathered from 18 parents during five focus groups informed the development of a theory-driven information pamphlet specifically addressing knowledge gaps that were identified, as well as barriers and enablers for parents to seek advice for paediatric eye disease. Using a theoretical framework for behaviour change, persuasive messages were designed by linking information obtained from the exploratory qualitative research study specifically aimed at improving knowledge, boosting motivation and prompting parental monitoring of ocular health and action if they observed eye signs of concern. Aim 2: Evaluation of the information pamphlet A total of 518 women consented to participate in the clinical trial and were randomised following completion of the baseline survey. Of these, 395 women completed the follow-up survey, which was sent two weeks after baseline. Using multiple logistic regression analysis, compared to the control group, participants in the intervention group were more likely to be concerned if leukocoria was observed, (OR 1.711, [CI 1.176-2.497] p=0.005) and less likely to delay seeking help (OR 0.560, [0.382-0.817] p=0.003). Whilst participants in the intervention group were no more concerned if they observed strabismus than the control group, they were less likely to delay help-seeking (OR 0.318, [0.125-0.806] p=0.016) at follow-up. Conclusion: Motivated by delayed diagnosis of retinoblastoma reported in the literature and my own clinical experience in Victoria, this doctoral study explored a potential strategy to minimise such delays in the broader context of paediatric eye disease. If parents are provided with relevant information, messages to motivate and guidance on behaviour skills, their knowledge and help-seeking intention if they observe leukocoria or strabismus can improve. Systematic dissemination of useful health information to parents may reduce diagnostic delay for retinoblastoma, or indeed other, more common paediatric eye diseases.

Glaucoma is one of the leading causes of irreversible blindness and early detection and treatment are key to preventing or slowing the progression of vision loss. An objective, non-invasive method to detect early functional changes in glaucoma would be invaluable, and a potential surrogate marker of retinal ganglion cell health may lie in the photopic negative response (PhNR) of the electroretinogram (ERG). Purpose: To develop and validate a reliable testing protocol for the PhNR recorded using a portable ERG device (RETeval®) that can be easily implemented in clinical practice. Methods: Normal controls and glaucoma participants were recruited for the study. Responses were elicited using a red stimulus on a steady blue background with the RETeval®. A comparison of baseline detrending methods as well as electrode performance was evaluated between the groups. The PhNR stimulus-response function was characterised, and the correlation between PhNR parameters and current measures of retinal structure and function were assessed. The sensitivity of the PhNR to detect glaucomatous changes was evaluated in glaucoma participants receiving treatment for intraocular pressure lowering. The PhNR was also used to follow glaucoma participants over 12-months to assess its relative ability to detect disease progression. Results: A method of baseline detrending was developed which improved the test-retest repeatability of the PhNR without compromising its clinical utility in glaucoma. Sensor strip electrodes provided a viable alternative to conventional Dawson, Trick and Litzkow (DTL) electrodes, although had inferior signal-to-noise performance that may reduce the sensitivity to detect subtle changes in disease. Measuring the PhNR across a range of stimuli to determine the maximum response provided reliable data and may be more appropriate than a single stimulus strength. However, the maximum response showed only weak correlation with standard automated perimetry and retinal nerve fibre layer thickness in glaucoma. With the optimised protocol, short-term changes in the PhNR were detected after intraocular pressure lowering in a subset of participants. Disease progression was also suggested by monitoring the PhNR over 12-months in a pilot study. Conclusion: This body of work developed a standardised, reliable method for PhNR recording with a portable ERG device which can complement current tests in monitoring glaucoma progression and evaluating treatment response in the clinic.

Background Age-related macular degeneration (AMD) is a complex multifactorial disease that affects the elderly. In the early stages of disease, dark adaptation problems are more significant for patients than visual acuity impairment. For decades, studies of rod function related to dark adaptation issues suggested that rod function could be useful as a functional marker for differentiating AMD severity and monitoring disease progression. However, there remains many unanswered questions about how best to investigate rod function in the early stages of AMD. Earlier studies were not able to phenotype AMD cases to the extent we can today as they relied only on colour fundus photographs to determine AMD subgroups. We now have the opportunity to look at different AMD phenotypes using multimodal imaging techniques. These advances have added clarity to the phenotyping of AMD, as high-risk features such as reticular pseudodrusen (RPD), hyperreflective foci (HRF), and nascent geographic atrophy (nGA) can now be identified. At the same time as the advances in detecting anatomical changes were made, instruments that could measure rod function in a more thorough way were improving. Previously, instruments measured rod function at only a single retinal location, or could only detect large losses in sensitivity due to a lack of dynamic range. Recent advances have seen perimeters that are able to measure rod function at multiple locations in the one setting and also have a larger dynamic range to detect subtle rod dysfunction. I have used one of these new tools in my studies. Aims Two-color dark-adapted chromatic perimeter (DACP) is a novel device, designed and manufactured in Melbourne, Australia. It was first used in 2015 when I commenced my PhD. This perimeter measures rod function at multiple locations with sufficiently large range of stimulus intensities to detect subtle changes seen in early AMD. My thesis aimed to investigate the ability of the DACP to reliably detect and record rod sensitivities. I then utilized this new perimeter to investigate the ability of rod function to act as a robust functional biomarker that could be used to determine AMD disease severity and to monitor disease progression, and to compare rod function in AMD cases to normal control participants. I was also able to separate my AMD cohort into those with or without RPD, a high-risk AMD phenotype, to investigate the impact of this phenotype on rod function. Both static and dynamic rod functional changes were recorded at baseline visits and then again at 6- and 12-months. The results of this study will contribute to our understanding of functional loss in AMD and help clinicians and researchers when designing research protocols aiming to evaluate rod functional impairment before vision loss.   Methods This study was conducted between 2015 and 2018 at the Macular Research Unit, Centre for Eye Research Australia. During that time, three main studies were completed. An initial assessment of test-retest reliability of the DACP was followed by a cross-sectional study comparing the severity of rod dysfunction based on point wise sensitivity (PWS) and point wise sensitivity difference (PWSD) of two color sensitivities. AMD cases were divided into an AMD group with traditional drusen only and an RPD group, and rod function was evaluated at multiple ring eccentricities within 24º of the central macula. A further 12-months evaluation of static (PWSD) and dynamic rod functional changes (rod intercept time (RIT) and rod recovery rate (RRR)) within the central 12º was conducted in participants with intermediate AMD with and without RPD as well as normal control eyes. Results The DACP did not have ceiling or floor issues during the intrasession and intersession testing, with a coefficient of repeatability of ± 5 dB. Intermediate AMD (iAMD) with the presence of RPD was found to be associated with poorer rod function compared with iAMD without RPD and normal control eyes, but only within the central macular 8º. Rod dysfunction was more significant if the eyes were prebleached before undertaking the sensitivity measurements. In a longitudinal study, dynamic rod functional changes, ie. RRR, was the only parameter that changed over 12 months, and only in the iAMD without RPD group. Change was found only at the peripheral 12º ring eccentricities. Conclusion The DACP offers a great opportunity to evaluate topographic rod function when eyesight is still normal. The findings from this study support that concept of rod function being impaired in the early stages of AMD. The presence of RPD in cases of iAMD were associated with further reductions in rod function. Measuring dynamic rod function offers a more sensitive functional marker in determining severity of AMD within the central 8 degrees of the macula, but changes over time were only detected at more peripheral locations. Longer follow-up will be beneficial to determine how rod function deteriorates over time and correlates with progression to vision loss.  

The overwhelming majority of the world’s vision impaired reside in low-income settings. Cataract is the leading cause of blindness and the second-leading cause of moderate to severe vision impairment (MSVI). Proxy measures of cataract disease burden and access to cataract surgery services include cataract surgery rate (CSR) and cataract surgery coverage (CSC) rates. The present research aims to evaluate and model associations between the scale of vision loss and burden of cataract disease with socioeconomic indices, drawing comparisons with other countries globally to identify key country policies, practices and initiatives that have resulted in significant improvements to service delivery. A meta-analysis was conducted using CSR data between 2005 and 2014 from published literature and unpublished literature. A systematic review of Rapid Assessment of Avoidable Blindness (RAAB) studies from 1995 to 2015 extracted information on the prevalence and causes of blindness, rate of cataract blindness, cataract surgery coverage (CSC), and visual acuity outcomes of cataract surgery (CSO). HDI and GDP data were collected from the United Nations Development Program (UNDP) and World Bank repositories respectively. Estimates of age-standardised prevalence data of vision impairment and blindness were collected from the 2010 Global Burden of Disease Study (GBD). CSR was found to be closely associated with GDP and GNI using data throughout the study period. HDI levels accounted for the majority of global variance in the prevalence of moderate-severe VI and blindness. Of the three HDI components, education was identified as the most influential factor, accounting for two-thirds of the global variance in MSVI and blindness prevalence. Lower total health expenditure per capita, and as a proportion of total GDP, were associated with a higher prevalence of moderate-severe VI and blindness. A strong association exists between socioeconomic factors as indicated by GDP, GNI and HDI and the quality and quantity of cataract surgery. This suggests there are strong relationships between resource availability and healthcare delivery. Low-cost and innovative services as well as strategic investment in capacity development is important to address cataract surgery needs in low-resource settings.

Glaucoma is a group of optic neuropathies that may be characterized by gradual degeneration of retinal ganglion cells (RGCs) and their axons leading to irreversible vision loss [1, 2]. Glaucoma is the second leading cause of blindness worldwide [3-5] and it is estimated that the number of people affected by the disease will reach 80 million by 2020, while more than 11 millions will be bilaterally blind [6]. In this project I focus on primary open-angle glaucoma (POAG), as it accounts for the majority of glaucoma cases worldwide. So far, multiple risk factors for glaucoma have been identified; however, the exact mechanism causing RGC loss in patients remains elusive. Furthermore, examination of RGCs affected in POAG is difficult pre-mortem due to their anatomical location. To overcome this problem, somatic cells can be reprogrammed into patient-specific induced pluripotent stem cells (iPSCs), which can be then differentiated into cell type of interest, i.e. RGCs. This PhD project consisted of several steps. First, I assessed the feasibility of transferring the iPSC culture into the automated platform. Using automation was essential to generate large number of samples required for analysis. The transition to automation was successful, as evidenced by maintenance of iPSC morphology, expression of pluripotency markers and ability to differentiate into derivatives of three germ layers. I also demonstrated that incorporating automation into human (h) iPSC culture allows standardization of maintenance and passaging procedures reducing inter-sample variability and human error. I subsequently used the platform to generate over 300 hiPSC lines for POAG modelling. In parallel, I optimized RGC differentiation protocol to obtain sufficient number of cells for their examination with single cell RNA sequencing (scRNA-seq). Next, iPSC-derived RGCs were subjected to scRNA-seq to gain in-depth information about transcriptomic differences between healthy controls and POAG patients. Understanding mechanisms underlying RGC function, maintenance of homeostasis and those conferring susceptibility to POAG is crucial to discover new therapeutic targets and commence the process of drug discovery.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss and blindness in elderly people in the developed world. The two main vision threating subtypes are geographic atrophy (GA) and neovascular AMD (nAMD). The latter is responsible for 90% of vision loss attributed to the disease. Currently, the treatment for nAMD is through the use of intravitreal injections of anti-Vascular endothelial growth factor (anti-VEGF) agents. Randomized controlled clinical trials and retrospective studies conducted for anti-VEGF treatment in nAMD patients have shown good improvements in visual outcomes in the majority of patients but due to unknown reasons, a broad range of outcome responses have also been reported. Approximately 5-10% of patients typically show no improvement in the readout of visual acuity (VA) (loss of > 15 Early Treatment Diabetic Retinopathy Study (ETDRS) letters) and exhibit a continuous decline in VA over the course of long term anti-VEGF therapy. A number of studies have indicated factors that might influence the response to treatment, which includes demographic, clinical and genetics factors. Among these, genetic factors are considered as one important determinant which likely result in a pharmacogenetic anti-VEGF treatment response. Genetic factors have been investigated for their association with change in VA following anti-VEGF treatment but these have been based on VEGF pathway genes as well as known AMD risk genes including CFH, ARMS2/HTRA1, VEGFR2, EPAS1 and APOE. Currently, findings from these genetic studies appear inconsistent with regard to their associations with anti-VEGF treatment outcomes (detailed in Chapter 2). This indicates that it is possible that other genetic factors are involved in determining response to anti-VEGF treatment in nAMD patients. Identifying these potential genetic variants across the human genome requires the use of sophisticated genetic techniques such as genome wide association studies (GWAS) and whole exome sequencing (WES). The objective of my PhD is to undertake pharmacogenetic studies to identify genetic variants which could influence anti-VEGF treatment outcome in nAMD patients. To accomplish this, I have used strategies which include candidate gene analysis and the next generation genetic techniques: GWAS and WES. Firstly, in Chapter 4, I assessed the genetic association of several previously described single nucleotide polymorphisms (SNPs) rs4576072 (VEGFR2), rs6828477 (VEGFR2), rs2070296 (NRP1) and rs9679290 (EPAS1) with regard to treatment outcome in nAMD after anti-VEGF treatment. A total of 207 nAMD patients were genotyped for these 4 SNPs with readout outcome measures of change in visual acuity (VA), retinal fluid clearance and central macular thickness (CMT) as measured by optical coherence tomography (OCT) following up-to 1 year of anti-VEGF treatment. Statistical analysis revealed no significant association between the genotypes of these four variants with change in VA at 3, 6 and 12 months of anti-VEGF treatment (P > 0.05 for each). Furthermore, there was no evidence of an association between each of the four variants with retinal fluid clearance or CMT change at 3 months of treatment (P >0.05 for each). A systematic search of Pubmed, Web of Science and Embase in August 2016 revealed two previous studies that had reported on the association of SNPs rs4576072 and rs682847 in the VEGFR2 gene with treatment outcomes (reported using only change in VA) following anti-VEGF therapy in nAMD. A meta-analysis of my own study with the two previously published studies showed no evidence of association between the genotypes of rs4576072 or rs682847 with mean change in VA at 3 or 12 months of treatment (P >0.05 each). In conclusion, there was insufficient evidence to ascribe a genetic association between the variants rs4576072, rs682847, rs2070296 and rs9679290 and anti-VEGF treatment outcome in nAMD patients. Chapter 5 describes a cost effective pooled DNA based GWAS on 285 anti-VEGF treated nAMD patients. The primary outcome measure was change in VA in ETDRS letters after 6 months of anti-VEGF treatment: patients who lost ≥5 (ETDRS) letters were classified as non-responders while all remaining patients were classified as responders. GWAS analysis identified 44 SNPs associated with change in VA at 6 months of anti-VEGF treatment. In a technical validation phase, individual genotyping of these 44 variants showed three SNPs (rs4910623 p = 5.6 × 10-5, rs323085 p = 6.5 × 10-4 and rs10198937 p = 1.30 × 10-3) that remained suggestively associated with VA response at 6 months of anti-VEGF treatment. Replication of these three SNPs in an independent cohort of 376 anti-VEGF nAMD patients of European origin followed by meta-analysis of both cohorts (673 samples) confirmed association of the SNP rs4910623 in the Olfactory Receptor Family 52 Subfamily B Member 4 (OR52B4) gene with poor VA response after 3 months (p = 1.2 × 10-5) and 6 months (p = 9.3 × 10-6) of anti-VEGF treatment in nAMD patients. In Chapter 6, I have undertaken a multi-centre GWAS on anti-VEGF treated nAMD cohorts identified via the International AMD Gene Consortium (IAMDGC) custom exome chip. In the discovery GWAS phase, a total of 678 anti-VEGF treated nAMD patients from 5 different clinical sites were included and the main outcome measure was change in VA at 3 months of anti-VEGF treatment. Linear regression analysis was undertaken on imputed genetic variants for each cohort with change in VA, followed by meta-analysis. Six SNPs rs241692, rs12138564, rs13002976, rs242939, rs2237435 and rs927203 revealed suggestive association with change in VA (p<1x10-5) at three months of treatment. Replication of these six variants was undertaken in an additional 6 anti-VEGF treated cohorts from outside the IAMDGC and subsequent overall meta-analysis of both the discovery + replication cohorts (n=2058) was undertaken. A variant, rs12138564 in the Chaperonin Containing TCP1 Subunit 3 (CCT3) gene remained associated with change in VA (p = 1.3x10-5) after three months of anti-VEGF treatment. The gene based burden test for non-synonymous rare variants (MAF<0.01) showed significant association of three genes Chromosome 10 Open Reading Frame 88 (C10orf88), Unc-93 homolog B1 (UNC93B1) and Cytoskeleton associated protein 2 like (CKAP2L) with change in VA associated (corrected p = 4.2 x10-7, p=6.0x10-7 and p = 1.3x10-6 respectively) at three months of anti-VEGF treatment. In addition pathway analysis revealed aldosterone signalling and G protein pathway as the top biological processes associated with change in VA (p<1x10-4) following treatment. Chapter 7 presents a Whole Exome Sequencing (WSE) study on 54 anti-VEGF treated AMD patients to identify genetic variants that influence the qualitative measure of retinal fluid presence (non-responders) relative to the retinal fluid dryness (responders) measured on OCT after 3 monthly doses of anti-VEGF injections. After downstream quality control and statistical analysis of the WES data, five SNPs; rs8786, rs36078271, rs2275123, rs1753430 and rs3801790 showed evidence of association with fluid presence (p ≤ 1x10-4) after 3 months of anti-VEGF treatment. Replication of these top variants in an independent anti-VEGF treated cohort (n=161) followed by meta-analysis of both cohorts (n=215) revealed association of rs1753430 and rs3801790 in the olfactory receptor family 6 subfamily J member 1 (OR6J1) and dedicator of cytokinesis 4 (DOCK4) genes with presence of retinal fluid after three months of anti-VEGF treatment (p = 0.002, and p = 0.01, respectively). Furthermore, the gene enrichment pathway analysis revealed the G-protein coupled receptor (GPCR) pathway as the top functional process (corrected p=8x10-15), suggesting involvement of cell signalling molecules in anti-VEGF treatment response in nAMD patients. In conclusion, I was able to undertake candidate gene, GWAS and WES in anti-VEGF treated nAMD patients, identifying variants in four novel genes; OR52B4, CCT3, OR6J1 and DOCK4, associated with varying treatment outcomes in nAMD. Findings from this thesis indicate multiple genetic factors are likely involved in varied response to anti-VEGF therapy and by investigating the biological role of these four novel genes in abnormal angiogenesis. It will aid in the future identification of new drug targets as potential alternatives or adjunctive treatments to existing anti-VEGF therapy. Additionally, it is the first step towards optimizing and personalizing treatment based on a pharmacogenetic approach for patients with nAMD.

Giant cell arteritis (GCA) is the most common form of vasculitis in people over 50 years of age. It is characterized by inflammation of the medium and large arteries of the head and neck. When the ophthalmic artery is involved, it can lead to sudden irreversible visual loss. GCA is one of the few true ophthalmic emergencies and has potential life threatening complications such as stroke, aortic dissection and rupture. Diagnosing GCA can be challenging in the acute setting as symptoms and signs are variable often leading to a delay in diagnosis. The gold standard to diagnose GCA is a temporal artery biopsy (TAB), an invasive surgical procedure which confirms the diagnosis histologically. In the absence of specific biochemical markers to identify GCA in the acute setting, cases with a high index of suspicion are treated with high dose corticosteroids. The pathogenesis and genetic architecture of GCA remains largely unknown. The overriding hypothesis of this work is that both genetic and environmental factors contribute to the development of GCA. The work described in this thesis uses multiple techniques including population modelling, analysis of patient cohorts, and modern molecular techniques to provide novel contributions that help define the disease burden, environmental and genetic risk factors as well as the immunological processes occurring in active disease. A systematic review performed at the start of this thesis, predicted that the future worldwide burden of GCA will increase in view of ageing population. By 2050 more than 3 million people will have been diagnosed with GCA and about 500,000 will be rendered visually impaired, highlighting the need to improve our understanding of this disease. Research described in this thesis includes the recruitment of a large patient cohort to investigate the genetic architecture of GCA through a genome-wide association study (GWAS). For complex diseases such as GCA, the number of potential candidate genes is overwhelmingly large. In the last twelve years, GWASs have revolutionised the gene-mapping field. Recruitment of the large cohort of patients with GCA required for GWAS is challenging. It is a rare disease, patients are elderly and many live in residential care, are deceased, or have major medical co-morbidities. Such factors make it difficult to enrol patients in studies where a blood sample is required for DNA extraction. To circumvent this issue, an alternative source of DNA from patients with GCA was investigated; DNA extracted from archived formalin fixed paraffin embedded (FFPE) TAB tissue. An international collaborative group, the Global GCA Genomics Consortium was established and 2084 archived FFPE TAB specimens from 30 pathology centres across four countries were recruited. In addition, 394 blood samples were obtained from patients with biopsy proven GCA over a 3-year recruitment phase. Epidemiological data from this cohort was used to investigate whether there was seasonal variation in the incidence of GCA which could indicate an environmental aetiology. No seasonal preponderance was demonstrated. In addition, an observational study based on the administrative burden involved in setting up a large collaborative nationwide non-interventional study highlighted the need to streamline ethics nationally to prevent duplication of work. For the GWAS, DNA samples extracted from FFPE blocks were genotypes using Illumina SNP microarray chips. After stringent assessment of sample suitability (meeting inclusion criteria), DNA and genotyping quality control, a total of 1,212 DNA samples, provided a genotyping success rate >95%. These 1,212 GCA cases were compared to 9,657 historic controls from the UK Household Longitudinal Study for the discovery phase of the analysis. Association analyses for 8,075,195 SNPs, identified two genetic loci at the genome-wide significance threshold of p<5x10-8, including the HLA region, where the top hit was rs9272417 (p-val = 8x10-51) which is associated to the gene expression of HLA DQA1 in the tibia artery and the aorta. The novel locus outside the MHC region was rs2351254 (OR = 0.52; p-val = 1.54x10-8) on chromosome 15. This SNP is associated with the expression of MFGE8 in the tibial artery and aorta. The association of this locus replicated in two separate cohorts, 177 cases from an independent Australian cohort and 352 cases from the United Kingdom (OR = 0.71; p-val = 0.056 and OR = 0.39; p-val = 0.003). This thesis also describes work to investigate the immunopathogensis of GCA. GCA is presumed to be an autoimmune disease in which T lymphocytes play an important role. High resolution RNA-sequencing (RNA-seq) permits the study of gene expression, giving an insight into the cellular processes driving inflammation during the acute phase of this disease. This technique was used to investigate RNA expression in CD4+ and CD8+ T-lymphocytes from patients with active and quiescent GCA over the course of 12 months. Serial blood samples were collected from 16 patients with GCA and 16 age-matched controls. Studying the circulating expression profiles in these T cells of both patients and controls was used to identify molecular associations with GCA disease status. These could potentially identify a new biomarker of active disease, increase the sensitivity and specificity of haematological monitoring of active disease, and forgo the need of an invasive procedure to diagnose this disease. The results of this RNA-sequencing study show cell type-specific transcript expression profiles, novel gene-phenotype associations, and uncover important biological pathways in GCA. Through monitoring expression profiles of patients over a 12-month period, polynomial modelling analyses identified 179 and 4 statistically significant transcripts with altered expression profiles (FDR < 0.05) between cases and controls in CD4+ and CD8+ populations, respectively. Genes included LMBR1L, UAP1L1, KCNMB4, PTCD2 and THRAP3. Gene correlations were found between both symptoms and biochemical markers used in the acute setting for predicting long-term prognosis. 15 genes were shared across 3 phenotypes in CD4 and 16 across CD8 cells. In CD8, IL32 was common to 5 phenotypes: a history of Polymyalgia Rheumatica, visual disturbance and raised neutrophils at time of presentation, bilateral blindness and death within 12 months. In the acute phase, such gene-phenotype relationships identified could act as disease biomarkers, provide insight to potential disease severity and guide in initiating appropriate patient management. In summary, this work has identified novel gene associations with GCA through a GWAS and has led to the discovery of interesting transcripts profiles through an RNA-seq approach. The results derived from this research have provided the basis for future functional work to investigate the role of these genes in GCA pathogenesis. This research could assist future studies to explore avenues for disease screening, prevention, monitoring and treatment.

MicroRNAs (miRNAs) are master gene regulators that have been widely implicated in many biological processes as well as pathogenesis of many diseases in the eye. The identification of miRNAs that are altered in human diseases as well as in animal models has led to new avenues to develop novel therapeutic targets for ocular disorders. The broad focus of studies described in this thesis is the identification of miRNAs that have altered expression during neovascularization of the retina using a rat model of oxygen-induced retinopathy (OIR). miRNA-Seq studies carried out in rats, mice and humans show that retinal miRNAs in rats and mice are highly homologous with humans. In exploring the altered miRNAs in retinal neovascularization, we found four miRNAs of interest (miR-143, miR-126, miR-150 and miR-145) that were down-regulated in the retina preceding neovascularization in OIR rats. Further investigation suggested that transforming growth factor-beta activated kinase 1 (TAK1), a target gene of miRNA-143, was highly up-regulated in the retina of OIR rats. Selectively pharmacological inhibition of TAK1 activity by 5Z-7-Oxozeaenol led to significant reduction of angiogenic capability of human endothelial cells, as measured by cell proliferation, cell migration and tube formation. Moreover, 5Z-7-Oxozeaenol greatly suppressed vascular spouting of mice aortic explants. In vivo intravitreal administration of 5Z-7-Oxozeaenol profoundly attenuated the retinal neovascularization in OIR rats. Previous studies have reported the down-regulation of miR-126 and miR-150 in OIR mice, and the restoration of these altered miRNAs led to significant suppression of retinal neovascularization in vivo. Here we focus on miR-143 rather than miR-126 and miR-150 to explore its therapeutic potential and underlying mechanisms in retinal neovascularization. Intravitreal administration of a synthetic miR-143 mimic into the eyes of OIR rats suppressed retinal neovascularization by ~50%. Further RNA-Seq identified that a gene cluster mediated by Fos, an inflammatory and stress gene, plays a crucial role in the suppression of retinal neovascularization in OIR rats by the restoration of miR-143. Plasmid vectors of reporter genes and corresponding elements of altered miRNAs such as miR-143, miR-126 and miR-150 were devised. Co-transfecting the devised plasmids with corresponding mimic of the altered miRNAs in HEK293A cells inhibited reporter gene expression. In the context of gene therapy for retinal neovascularization, the results indicate that therapeutic gene expression could be regulated in accordance with the disease activity through altered miRNAs.

Vision impairment (VI) and blindness affect 440 million people worldwide. As most cases can be avoided through evidence-based eye health care interventions, the elimination of avoidable VI and blindness is a global health priority. Nationally-representative population surveys on the prevalence and causes of VI and blindness are required to inform targeted eye health care programs. This thesis documents Australia’s first National Eye Health Survey (NEHS) that aimed to determine the prevalence and causes of vision loss and rates of utilisation of eye health care services for non-Indigenous Australians aged 50 years or older and Indigenous Australians aged 40 years or older residing in all levels of geographic remoteness in Australia. Multistage random-cluster sampling was used to select a nationally-representative sample of 3098 non-Indigenous Australians and 1738 Indigenous Australians from 30 population clusters across all five geographic remoteness strata (Major Cities, Inner Regional, Outer Regional, Remote and Very Remote). Participants underwent an interviewer-administered questionnaire and a series of standardised eye examinations, including visual acuity assessment, anterior segment assessment, perimetry, fundus photography and intraocular pressure measurement. The prevalence of bilateral vision loss was 6.5% (95% confidence interval [CI]: 5.4-7.8) in non-Indigenous Australians and 11.2% (95% CI: 9.5-13.1) in Indigenous Australians. The age- and sex-adjusted prevalence of bilateral vision loss was 2.8 times higher in Indigenous than non-Indigenous Australians (P<0.001). The prevalence of unilateral vision loss amongst non-Indigenous and Indigenous Australians was 16.0% (95% CI: 14.4-17.7) and 14.9% (95% CI: 13.1-16.8), respectively, but after adjustment, unilateral vision loss was 1.4 times more prevalent in Indigenous Australians (P=0.003). Geographic remoteness was associated with a higher prevalence of bilateral (Outer Regional odds ratio [OR]: 2.02) and unilateral (Very Remote OR: 1.65) vision loss in Indigenous Australians, but not non-Indigenous Australians. Uncorrected refractive error and cataract were the leading causes of bilateral and unilateral vision loss, accounting for 70.4%-80.9% of all cases. Age-related macular degeneration was the leading cause of bilateral blindness (71.4%) and the third leading cause of bilateral vision loss (10.3%) in non-Indigenous Australians. Cataract was the leading cause of bilateral blindness (40%), and diabetic retinopathy was the second leading cause of bilateral blindness (20%) in Indigenous Australians. Eighty-two percent of non-Indigenous Australians had undergone an eye examination within the past two years. Forty-seven percent of Indigenous Australians had been examined within the past year, as per national recommendations. Fewer Indigenous than non-Indigenous Australians with diabetes adhered to diabetic eye examination guidelines (52.7% [95% CI: 45.9-59.6] v 77.5% [95% CI: 71.8-83.3], OR: 0.37). Cataract surgery coverage (58.5% [95% CI: 49.8-66.8] v 88.0% [95% CI: 84.5-90.6]) and refractive error treatment coverage rates (82.2% [95% CI: 78.6-85.3] v 93.5% [95% CI: 92.0-94.8) were significantly lower in Indigenous Australians compared to non-Indigenous Australians (OR: 32 and 0.51, respectively). The NEHS has provided nationally-representative data on the prevalence and causes of vision loss and the utilisation of eye health care services for non-Indigenous and Indigenous Australians. This study has shown that the non-Indigenous population of Australia has a lower prevalence of vision loss than other high-income countries, and that there is a significant excess burden of vision loss in the Indigenous population of Australia. Most vision loss in Australia is avoidable and can be eliminated by utilising the findings of this survey to optimise treatment rates of avoidable causes of vision loss. Improvements in the availability and uptake of eye health care services in Indigenous communities, particularly in non-metropolitan areas, are required to close the gap in Indigenous eye health. Marginalised and under-served Indigenous populations in other countries may benefit from the execution of similarly stratified nationwide surveys, the results of which may be used to optimise blindness prevention programs for at-risk population groups.

To study diseases affecting the retina, we rely on appropriate models to investigate causes of degenerative onset and progression. Human pluripotent stem cells (hPSCs), and in particular patient-specific induced pluripotent stem cells (iPSCs), provide an excellent source of material for generating retinal cells that can be used as disease models to explore disease-causing mutations, genotype-phenotype relationships, therapeutic drug screening and potentially cell replacement therapy. I developed a rapid, one-step differentiation protocol to differentiate hPSCs to the retinal pigment epithelium (RPE), the outermost monolayer of cells in the retina that are damaged in age-related macular degeneration (AMD). Using a simple growth factor treatment regime, functional RPE could be differentiated from hPSCs in 60 days for use in disease modelling and basic fundamental research. Given the protocol’s simplicity, it was easily adapted to an automated platform, enabling the generation of hundreds of patient-specific RPE lines simultaneously for large-scale disease modelling purposes. Retinal degeneration may be mediated by the loss off the outer blood retinal barrier (BRB) integrity, caused by injury of the retinal pigment epithelium. Lysophosphatidic acid (LPA) and its producing enzyme autotaxin (ATX) have been implicated in inflammation, angiogenesis and apoptosis. Given that ATX is a signature marker of RPE, I hypothesized that it plays an important role in maintaining the microenvironment of the outer retina, including the BRB. Using the differentiation protocol I developed, hPSCs were differentiated into RPE and were shown to express high levels of ATX and LPA receptors (LPARs) by quantitative real time polymerase chain reaction (qRT-PCR). Measurements of endogenous LPA and ATX were determined using liquid chromatography mass spectrometry (LC-MS) and western blot respectively, and indicate that hPSC-RPE cells secrete low basal amounts of LPA (0.1-0.4 nM), but secrete large amounts of functional ATX at the apical surface, towards the photoreceptor layer. It is possible that the low detectible amounts of LPA were a result of the short half-life of LPA in culture or the potential lack of the lipid substrate for LPA synthesis in RPE monocultures. It is more plausible that in vivo, given the high expression of ATX, the amount of local LPA produced is much higher. Addition of exogenous LPA to hPSC-RPE cultures resulted in increased expression of tight junction proteins ZO-1 and Occludin at the cell junction, which was validated at the functional level by increased transepithelial resistance (TER), indicative of increased barrier function and reduced paracellular permeability. This data suggests that RPE-derived LPA acts in an autocrine manner to maintain the tight junction barrier, one of the key function of RPE in vivo. To assess the potential paracrine actions of RPE-derived ATX and LPA, the mouse 661W photoreceptor line and optic cup-derived CD73 photoreceptors were treated with different doses of LPA. At high doses (>10 µM), cytoskeletal changes consistent with cellular retraction were observed, as indicated by myosin light chain (MLC) and its phosphorylated form (p-MLC). These results indicate the RPE-derived ATX and LPA act in a paracrine manner, and dysregulation of this pathway could play a role in photoreceptor dysfunction. Endothelial cells that line the outside of the BRB may also potentially contribute to its destruction. RPE-ATX was also secreted basally, although at much lower levels, and therefore it’s highly plausible that the amount of local LPA produced in this microenvironment have an effect on BRB maintenance. Treatment of CD31+ iPSC-endothelial cells with LPA indicated that high doses of LPA caused reduced tubule formation. This indicates that in the retina, LPA may function to suppress angiogenesis. Furthermore, I showed that treatment of mouse-derived CD4+ and CD8+ T cell with LPA resulted in reduced activation, again suggesting RPE-derived LPA may protect against loss of BRB by supressing inflammatory mediators. In conclusion, this Thesis has highlighted a potential novel role for RPE-derived ATX and LPA in the maintenance of the BRB, and that possible dysregulation of this signalling axis may be involved in retinal disease onset and progression where the BRB is compromised.

Neuro-ophthalmology is a medical discipline focusing on diseases of the central nervous system that may affect the visual sensory or motor systems or pupillary reflexes. Neuro-ophthalmologists are often part of the academic medical team and the eyes may be involved in a variety of medical disciplines. Paediatric ophthalmology adds precision to diagnostic challenges arising from the complexity of foetal and early development of the visual system. The first chapter of this thesis deals with normal neuronal, vascular and immunological development of the retina. The second chapter deals with a selection of visuo-motor disturbances in the neonate and early childhood, including the difficulties faced by the paediatric neuro-ophthalmologist in diagnosis in the context of children, particularly in the early years of life where development adds a profound challenge to the interpretation of conditions and determining whether conditions are progressive or static: • Chapter 2.1 deals with the objective measurement of visual fields by directly recording from the visual (occipital) cortex. • Chapter 2.2 deals with confusion and possible mismanagement of visual path glioma and the importance of objective perimetry in clarifying diagnosis and determining management, particularly in early childhood. • Chapter 2.3 deals with the further problem of regional giantism associated with neurofibromatosis and presenting in early childhood with buphthalmos. • Chapter 2.4 discusses the publication of an early series of cases of optic nerve hypoplasia at a time when there were relatively few reported in the literature, however the condition is now recognised as the commonest cause of blindness at birth. • Chapter 2.5 discusses optic neuropathy and the importance of distinguishing ‘functional’ loss of vision from pathologic conditions such as drug toxicity. • Chapter 2.6 discusses two unusual cases of pathological papilledema in childhood, treated with sheath splitting surgery in its early developmental phase. • Chapter 2.7 deals with a series of cases of Aicardi’s syndrome, the first to appear in the English literature. • Chapter 2.8 deals with strabismus and amblyopia and demonstrates the vulnerability of the developing visual system to abnormal visual environmental influences and motor disturbances. • Chapter 2.9 deals with congenital nystagmus and its differential diagnosis. • Chapter 2.10 discusses the developmental causes of supra-nuclear disturbances of gaze, their differential diagnosis and the importance of excluding tumours of the visual path in early childhood. The final chapter highlights and summarises the major contributions to paediatric neuro-ophthalmology of the topics covered in this thesis and the importance of development adding complexity to the interpretation of diagnosis.

Hypothesis 1: In people who develop a choroidal neovascular membrane (CNVM) secondary to Age-Related Macular Degeneration (AMD), presenting visual acuity varies. Eyes with worse visual acuities at presentation have worse visual acuity outcomes compared to eyes that present with better visual acuities despite treatment. Aim 1: To determine and compare the visual acuity of first and second eyes diagnosed with CNVM secondary to AMD at presentation and through two years of follow-up to determine the long-term visual consequences of poor presenting visual acuity. Methods 1: Retrospective review of clinical notes of patients presenting with newly diagnosed CNVM secondary to AMD in their first eye from the Royal Victorian Eye and Ear Hospital from January 2012 to April 2014. Visual acuity was recorded up to two years. Visual acuity in the second eye developing CNVM was recorded at diagnosis and for a further two years from the time of diagnosis. Results 1: First eyes presented with a visual acuity of 6/52 and second eyes presented with a visual acuity of 6/21. Although this did not reach statistical significance, first eyes tended to have worse visual acuity during the 2 years of follow up. This suggests that there is room to identify and begin treatment earlier once CNVM has developed. Hypothesis 2: Reduced microperimetric macular sensitivity, or a decrease in microperimetric macular sensitivity over time, is associated with an increased risk of developing CNVM in an eye with large drusen whose fellow eye has CNVM secondary to AMD. Aim 2: To determine if reduced macular function, or a decrease in macular function over time, as determined by microperimetry, can predict the development of CNVM in eyes with large drusen whose fellow eye has CNVM secondary to AMD. Methods 2: Participants with unilateral CNVM secondary to AMD in one eye and large drusen in the other eye (study eye, group 1) were recruited prospectively from the Royal Victorian Eye and Ear Hospital, East Melbourne, Victoria Australia between February and May 2014. All participants underwent microperimetry testing at baseline and 12 months study eye developed CNVM or GA. Results 2: Reduced microperimetric sensitivity is associated with the development of CNVM or GA collectively (P=0.045). All eyes that developed CNVM or GA had a mean macular microperimetric sensitivity less than 25dB at baseline. This confirms our hypothesis, and provides further evidence that macular function, as tested by microperimetry, may be a useful biomarker for predicting disease progression. Hypothesis 3: Microstructural changes, such as drusen, EZ disruption, HF, RPD and nGA, are independently associated with decreased retinal function, which partially explain the reduced microperimetric retinal sensitivity seen in eyes that progress to late AMD from Aim 2. Aim 3: To determine if macular structure-function relationships explain the reduced microperimetric macular sensitivity in fellow eyes with large drusen that ultimately go on to develop late AMD of people with unilateral CNVM secondary to AMD in their other eye. Methods 3: Group 1 from Aim 2 was used in Aim 3. Controls with bilateral intermediate AMD in both eyes (group 2) were recruited as a comparison group. All participants from groups 1 and 2 underwent multimodal imaging, including spectral domain optical coherence tomography, fundus autoflourescence, and fundus infrared reflectance. Results 3: Mean microperimetric macular sensitivity is lower in cases compared to controls (P<0.001) at baseline and is independently associated with the development of CNVM or GA collectively (P=0.039), and GA alone (P=0.005) when taking into account microstructural changes. Conclusion: People with CNVM secondary to AMD are presenting with poor vision in an Australian population, which limits their long-term visual outcome despite treatment. Within our study population, all eyes developing late AMD had a baseline mean microperimetric macular sensitivity less than 25dB. Furthermore, microperimetric macular sensitivity is associated with the development of late AMD whose fellow eye already has established CNVM independent of microstructural changes. This provides substantial evidence that microperimetric macular sensitivity, in conjunction with the quantification of structural changes, may prove to be a useful biomarker that could potentially allow for greater risk stratification of those likely to develop late AMD. As such, macular microperimetry has the potential to become a useful clinical tool with practical implications in terms of monitoring.