Retinal hypoxia and inadequate oxygen delivery have been implicated as causal for the development of several eye diseases, including diabetic retinopathy, glaucoma, and retinopathy of prematurity. The imaging of oxygen tension in the retina, generated from a measure of the phosphorescence lifetimes of bolus-injected palladium-porphyrin probes, has been used successfully to study retinal oxygen dynamics in numerous animal models. However, the specific parameters for applying this technique in the mouse have not been thoroughly investigated. The goals of this project were to calibrate a newly-constructed phosphorescence lifetime imaging instrument and data analysis software against known oxygen concentrations, to determine specific parameters for probe excitation and image collection and analysis in the mouse eye, and to assess any damage caused to the eye by the technique using histological analysis. An in vitro system was developed for calibration of the probe and for estimation of power of excitation light and camera settings necessary to produce acceptable oxygen maps. In vivo experiments were then performed, and plots indicating camera settings necessary for producing varying qualities of oxygen maps were constructed. Trypsin digestion of retinal tissue was used in an attempt to assess any damage to experimental subjects, but this histological technique was deemed inadequate for analyzing the capillary structures of the mouse eye. Alternatively, damage was assessed using the instrument itself to calculate changes in oxygen tension during the experimental process. The results of this work will allow the phosphorescence lifetime imaging system to be used in the mouse to study how changes in retinal oxygen tension correlate with the progression of eye diseases where oxygen is implicated, including diabetic retinopathy.

The electrocardiogram (ECG) is a graphical representation of the electrical activity of the heart. The 12-lead ECG shows this activity in 12 "views" called "leads", relative to the location of sensors attached to the body surface. The ECG is a routinely applied cost effective diagnostic medical test, utilised in healthcare settings around the world. Although more than three hundred million ECGs are recorded each year, correctly interpreting them is considered a complex task. Failure to make correct interpretations can lead to injury or death and costs vast sums in litigation payments. Many automated attempts at interpreting ECGs have been implemented and continue to be developed and improved. Despite this, automated methods are still considered to be less reliable than expert human interpretation. As ECG interpretation is both a cognitive and visual task, eye-tracking holds great potential as an investigative methodology. This thesis aims to identify any cues in visual behaviour that may indicate differences in subsequent ECG interpretation accuracy. This is the first work that uses eye-tracking to analyse how practitioners interpret ECGs as a function of accuracy. In order to investigate these phenomenon, several experiments were carried out using eye-movements captured from clinical practitioners that interpret ECGs as part of their usual clinical role. The findings presented in this thesis have advanced research in the understanding of ECG interpretation. Specifically: Clinical history makes a difference to how people look at ECGs; different gaze patterns are often found in accurate and inaccurate interpretation groups. Grouping data to account for within ECG lead behaviour (eye-movement patterns within a lead) is more revealing than analysis at the level of the lead (eye-movements between leads). Findings suggest analysing visual behaviour at this level is crucial in order to detect behaviour in ECG interpretation. Further to this the thesis presents eye-tracking techniques that can be applied to wider areas of task performance. These methods work over complex stimuli, are able to deal post hoc with differently sized groups and generate appropriate areas of interest on a stimulus. These methods detect important differences in eye-movement behaviour between groups that are missed when applying standard inferential statistical techniques.

In the study of alphaA-crystallin (CRYAA), G98R CRYAA was cloned into a mammalian expression vector pcDNA6-His/myc version B and the sequence was confirmed by direct sequencing. Following lipophilic transfection to lens epithelial B3 cells, the recombinant mutated CRYAA protein was highly insoluble upon 0.5% Triton X-100 (Tx) extraction. It was retained and formed aggregation, and distributed in the endoplasmic reticulum (ER) along with the ER resident protein (protein disulfide isomerise). The wild-type (WT) CRYAA was found to be soluble and diffusely distributed in the cytoplasm. The accumulation of G98R mutant induced ER stress, and the affected cells were prone to apoptosis. After treatment with a small chemical molecule, the natural osmolyte trimethylamine N oxide (TMAO), the Tx insolubility of mutant protein was reduced in dose- and time-dependent manners. It was also prone to be degraded via ubiquitin proteasome pathway (UPP). In mutant-expressing cells, the mutant protein aggregation was decreased after treatment. The ER stress and the rate of apoptosis were also alleviated, probably mediated by heat shock response, as demonstrated by the effect of TMAO on heat shock protein 70 expression. / The third eye gene model was myocilin (MYOC ), the first identified gene responsible for primary open angle glaucoma. The aim of this study was to investigate if glaucoma-causing MYOC variants, including D384N MYOC, could be correctable. D384N MYOC was identified in a Chinese family diagnosed with high tension juvenile-onset primary open-angle glaucoma. Disease causing mutations in MYOC (R82C, C245Y, Q368X P370L, T377M, D380A, D384N, R422C, R422H, C433R, Y437H, I477N, I477S and N480K) were cloned into mammalian expression vector p3XFLAG-myc-CMV"-25 and the sequences confirmed by direct sequencing. Following lipophilic transfection to human trabecular meshwork (HTM) cells, the Tx solubility and secretion of MYOC and cell apoptosis were examined in the presence or not with small chemical treatments. 4-PBA, TMAO and deuterium oxide (D2O), reduced the portion of insoluble fractions to various extents in the mutant proteins. The osmolytes TMAO and D2O were more effective than 4-PBA in improving MYOC solubility. TMAO was further shown to improve the secretion and ER-Golgi trafficking of D384N MYOC, thereby reducing the ER stress and rescuing cells from apoptosis. (Abstract shortened by UMI.) / The truncated G165fsX8 gammaD-crystallin ( CRYGD) variant was studied to further examine the effects of small chemical-assisted protein rescue of a CRYGD mutant that causes congenital cataract. G165fsX8 CRYGD was identified in a Chinese family with nuclear type of congenital cataract. The mutation was cloned into a mammalian expression vector p3XFLAG-myc-CMV"-25 and sequence was confirmed by direct sequencing. Following lipophilic transfection to COS-7 cells, the G165fsX8 CRYGD mutant protein was significantly insoluble upon 0.5% Tx extraction and was mistrafficked to the nuclear envelope with co-localization with nuclear lamins, whereas WT protein was Tx soluble and nuclear located. Treatment with small chemical sodium 4-phenylbutyrate (4-PBA) substantially reduced the Tx insolubility and reversed the mutant protein to nuclear localization. This correction has resulted in better cell survival, probably via a heat-shock response, as demonstrated by heat-shock protein 70 up-regulation. / To date, many genes and mutations are identified to cause various ocular diseases. Some of them result in a disruption of protein folding, an important cause of disease pathogenesis and progression. In my laboratory, novel mutations of crystallins and myocilin have been identified to segregate with congenital cataract and primary open-angle glaucoma, respectively. In this thesis, I reported molecular investigations of the resultant protein variants and their altered cellular functions in relation to the clinical phenotypes that contributed to new understanding of the roles of these genes in ocular tissues. / Gong, Bo. / Adviser: Chi-Pui Pang. / Source: Dissertation Abstracts International, Volume: 73-02, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 163-188). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

年齡相關性黃斑變性（AMD）是發展國家高齡人群中不可逆盲的首要原因。在AMD患者中，即使在改變生活模式或進行治療后，其滲出性亞型仍導致超過80% 的病例出現嚴重視力喪失及法定盲。息肉狀脈絡膜血管病變 （PCV）是一種與滲出性AMD在臨床表型上存在相同之處的黃斑病變，它的典型病變被定性為眼底血管螢光造影時出現息肉狀的病灶。近年PCV被認為是滲出性AMD亞型中的一種，因為兩者共享相同的基因成份及環境因素。然而，PCV曾經被認為是與滲出性AMD截然不同的一種疾病，由於兩者的臨床表現並不一致。另外，PCV病人相對年輕，多為亞洲人，以及對光動力治療和抗血管內皮生長因子治療存在不同的反應。一個明確的鑒別診斷可以更好的輔助臨床醫生對患有這些疾病的老年病人進行管理，然而兩者是相同還是不同的疾病種類仍是一個具爭議性的議題。 / CFH 基因和ARMS2/HTRA1位點已被全基因組相關性研究及相關的分子學研究定位為AMD候選基因。鑒於FPR1基因的協調吞噬性白細胞激活及遷移的功能，它可能是一個新的AMD候選基因。本論文評估在滲出性AMD和PCV中FPR1作為一個新的疾病基因基因的可能，獲取滲出性AMD和PCV病人中的CFH，ARMS2，HTRA1和FPR1基因檔案，同時研究在ARMS2/HTRA1位點中基因型和疾病表型的關聯性，以此從基因學方面鑑別滲出性AMD與PCV。 / 本研究在滲出性AMD，PCV病例和對照人群中使用聚合酶鏈反應和直接測序法進行ARMS2， HTRA1， CFH 和FPR1基因篩查。本研究發現滲出性AMD和PCV之間存在不同的基因型分佈，關聯模式以及基因效應值。 / 在HTRA1的多態性中，rs11200638，rs2672598, rs1049331 和 rs2293870 在滲出性AMD和PCV之間表現出鑒別性關聯 （p < 0.001）。其中rs11200638 (p = 1.48×10⁻⁴) and rs2672598 (p = 2.27×10⁻³) 在滲出性AMD病人中相互校正后仍保持各自的顯著性，但rs2672598 未能在PCV病人中保持顯著性(p = 0.20)。並且本研究發現攜帶rs11200638和 rs2672598聯合基因型AA-CC 的病人更傾向是滲出性AMD病人，與PCV相比幾率高11.7倍。 / 在ARMS2中，有11個基因多態性與滲出性AMD和PCV存在顯著性的相關。在與rs11200638校正后，rs10490924保持和滲出性AMD的顯著相關性(p = 0.011)，但PCV中未能保持(p = 0.077)。同時，元分析結果顯示ARMS2 rs10490924和HTRA1 rs11200638不同人群的PCV中的等位基因相關性是一致的。 / 在FPR1中，rs78488639與滲出性AMD (p = 0.049, 比值比 (OR) = 2.05, 95% 信賴區間（CI）: 1.014.14)和PCV (p = 0.016, OR = 2.27, 95%CI: 1.154.47)的疾病風險存在顯著的相關性。多態性rs104229的G等位基因純合子和滲出性AMD存在顯著相關(p = 0.039, OR = 2.27, 95%CI: 1.084.74)，但在PCV中未發現相關性(p = 0.24)。多態性rs2070746 AMD (p = 0.021, OR = 0.57, 95%CI: 0.35 0.91)和rs867229 (p = 0.0091, OR = 0.54, 95%CI: 0.340.86) 的雜合子基因型與滲出性AMD相關，但在PCV中未發現相關性。與此同時，本研究在上述多態性中發現滲出性AMD和PCV之間不同的基因型分佈。 / 本研究發現在滲出性AMD和PCV病人中FPR1 rs78488639和CFH rs800292存在顯著的相互作用（ORs > 4）。兩個多態性之間的相互作用提高滲出性AMD和PCV的疾病風險，而不是僅對其中之一起作用。 / ARMS2 多態性 rs10490924 (A69S, 205G>T, pAMD = 1.01×10⁻²⁹ OR = 7.91, 95% CI: 4.93 - 12.67; pPCV = 8.25×10⁻⁷, OR = 3.51, 95% CI: 1.98 - 5.03), HTRA1 多態性rs11200638 (-625G>A, pAMD = 9.88×10⁻²⁸, OR = 6.95, 95% CI: 4.37 - 11.06; pPCV = 8.02×10⁻⁶, OR = 2.82, 95%CI: 1.77 - 4.47), CFH 多態性rs800292 (V62I, 184G>A, pAMD = 9.00×10⁻⁴ , OR = 0.58, 95% CI: 0.42 0.79; pPCV = 0.011, OR = 0.66, 95% CI: 0.49 0.90) and FPR1 多態性rs78488639 (L97M, 289C>A, pAMD = 0.049, OR = 2.05, 95% CI: 1.01 - 4.14; pPCV = 0.016, OR = 2.27, 95% CI: 1.15 - 4.47)代表各自基因的最強相關性。此外，元分析揭示了在不同種族人群PCV中的等位基因相關性顯著並且一致(ORtotal = 2.14, 95% CI: 1.97 2.33, ORtotal = 2.34, 95% CI: 1.98 2.76 and ORtotal = 0.49, 95% CI: 0.44 0.56)。表型-基因型分析發現ARMS2/HTRA1 的風險基因型和較差的治療反應呈正相關性(p = 0.04)。另外，本研究在滲出性AMD中發現HTRA1 rs11200638和吸煙的聯合作用。然而，在PCV中未觀察到次聯合作用，這可能提示兩者間存在不同的疾病機制。 / 本論文提出FPR1基因是一個新的滲出性AMD和PCV候選基因，揭示了ARMS2，HTRA1，CFH和FPR1在滲出性AMD和PCV間顯著並且一致的相關性， 提供鑒別兩者的基因學證據，闡明了ARMS2/HTRA1 的風險基因型和較差的治療反應之間的相關性以及顯示了吸煙在滲出性AMD和PCV之間的不同影響。然而，由於兩者間基因關聯的趨勢一致，目前尚未能清晰界定兩者的不同。因此，要進一步明確鑒別滲出性AMD和PCV，還需要進行不同種族的複製研究，以及更重要的是，尋找特定的PCV基因以鑒別兩個不同疾病。 / Age-related macular degeneration (AMD) is the leading cause of irreversible blindness for the elderly in developed countries. Its exudative subtype accounts for more than 80% of severe visual loss or legal blindness in AMD patients regardless of modified lifestyle and therapeutic treatments. Polypoidal choroidal vasculopathy (PCV) is a macular disorder characterized by typical polypoidal lesions on fundus angiograhpy and sharing similar phenotype with exudative AMD. PCV was suggested as a distinct disease from exudative AMD based on different clinical features in ophthalmic imaging. Furthermore, PCV patients tend to be younger and more prevalent in Asian, and have different responses to photo-dynamic therapy and anti-vascular endothelial growth factor treatments, compared to exudative AMD patients. Howerver, it has also been suggested that PCV could be a subtype of exudative AMD mainly because of their common genetic and environmental factors. Therefore, genetic differentiation between exudtive AMD and PCV might assist clinicans to determine the condition. / The complement factor H (CFH) gene, and age-related maculopathy susceptibility 2 (ARMS2)/high temperature requirement factor A1 (HTRA1) locus have been mapped for AMD by genome-wide association studies (GWAS) and subsequent molecular investigations. The formyl peptide receptor 1 (FPR1) gene, which mediates trafficking and activation of phagocytic leukocytes, is related to the AMD-associated inflammatory condition. This thesis aims to evaluate FPR1 as a novel disease gene for exudative AMD and PCV, to compare the genetic profiles of ARMS2, HTRA1, CFH, and FPR1 in exudative AMD and PCV, to investigate the correlation of ARMS2/HTRA1 genotypes with disease phenotypes, and to differentiate these two disorders throught the genomic compositions. / Case-control association studies were conducted on ARMS2, HTRA1, CFH and FPR1 in exudative AMD and PCV patients of our Hong Kong Chinese cohort using polymerase chain reaction and direct sequencing. We observed different genotypic distributions (p < 0.05), association patterns and effect sizes between these two diseases. / In HTRA1 polymorphisms, rs11200638, rs2672598, rs1049331 and rs2293870 showed differential associations between exudative AMD and PCV (p < 0.001). Both rs11200638 (p = 1.48×10⁻⁴) and rs2672598 (p = 2.27×10⁻³) remained significant after adjusting for each other in exudative AMD, whereas rs2672598 was not significantly associated with PCV (p = 0.20). The joint genotype AA-CC constructed by the risk alleles of these rs11200638 and rs2672598 were prone to exudative AMD, conferring an 11.7-fold higher risk (p = 4.00×10⁻³) when compared to PCV. / In ARMS2, 11 single nucleotide polymorphisms (SNPs) showed significant associations with both exudative AMD and PCV. After adjusting for rs11200638, ARMS2 rs10490924 remained significantly associated with exudative AMD (p = 0.011), but not with PCV (p = 0.077). / In FPR1, SNP rs78488639 significantly increased the risk to exudative AMD (p = 0.049, odds ratio (OR) = 2.05, 95% confidence interval (CI): 1.014.14) and PCV (p = 0.016, OR = 2.27, 95%CI: 1.154.47). The homozygous G allele of rs1042229 was associated with exudative AMD (p = 0.039, OR = 2.27, 95%CI: 1.084.74), but not with PCV (p = 0.24). The heterozygous genotypes of rs2070746 and rs867229 were associated with exudative AMD (p = 0.021, OR = 0.57, 95%CI: 0.35 0.91; p = 0.0091, OR = 0.54, 95%CI: 0.340.86, respectively), but not with PCV. / Significant interaction was identified between FPR1 rs78488639 and CFH rs800292, with joint ORs > 4 folds for both exudative AMD and PCV. Interactions between FPR1 rs78488639 with CFH rs800292 enhance risks to both AMD and PCV, not just one of them. / Overall, the ARMS2 rs10490924 (A69S, 205G>T, pAMD = 1.01×10⁻²⁹, OR = 7.91, 95% CI: 4.93 - 12.67; pPCV = 8.25×10⁻⁷, OR = 3.51, 95% CI: 1.98 - 5.03), HTRA1 rs11200638 (-625G>A, pAMD = 9.88×10⁻²⁸, OR = 6.95, 95% CI: 4.37 - 11.06; pPCV = 8.02×10⁻⁶, OR = 2.82, 95%CI: 1.77 - 4.47), CFH rs800292 (V62I, 184G>A, pAMD = 9.00×10⁻⁴ , OR = 0.58, 95% CI: 0.42 0.79; pPCV = 0.011, OR = 0.66, 95% CI: 0.49 0.90) and FPR1 rs78488639 (L97M, 289C>A, pAMD = 0.0487, OR = 2.05, 95% CI: 1.01 - 4.14; pPCV = 0.0161, OR = 2.27, 95% CI: 1.15 - 4.47) were responsible for the strongest association in each gene. Moreover, meta-analysis revealed a consistent and significant association of the ARMS2/HTRA1 locus with PCV in different ethnic cohorts (OR{U+209C}{U+2092}{U+209C}{U+2090}{U+2097} = 2.14, 95% CI: 1.97 2.33, OR{U+209C}{U+2092}{U+209C}{U+2090}{U+2097} = 2.34, 95% CI: 1.98 2.76 and {U+209C}{U+2092}{U+209C}{U+2090}{U+2097} = 0.49, 95% CI: 0.44 0.56, respectively). The phenotype-genotype analysis implicated a positive correlation between ARMS2/HTRA1 risk genotype and a worse response to treatment (p = 0.04) in our exudative AMD patients. In addition, joint effects between cigarette smoking and HTRA1 rs11200638 was found in exudative AMD group. However, this effect was not significant in PCV group, which might implicate a different disease mechanism. / This thesis attempts to dissect the genetic profiles of exudative AMD and PCV. Results in this thesis suggest FPR1 as a novel candidate gene for exudative AMD and PCV, reveal a significant and consistent association of ARMS2, HTRA1, CFH and FPR1 with both exudative AMD and PCV, provide evidences for genetic differentiation of these two disorders, demonstrate a significant correlation between ARMS2/HTRA1 genotypes and response to treatment, and indicate different influence of smoking in exudative AMD and PCV. However, definite differentiation between exudative AMD and PCV was limited because of the same trend of associations between these two disorders. Therefore, replication studies in other enthic populations are necessary, and identification of PCV-specific genes/polymorphisms could further differentiate PCV from exudative AMD. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Liang, Xiaoying. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-143). / Abstract also in Chinese. / Title page --- p.i / Abstract --- p.iii / 摘要 --- p.vii / Acknowledgements --- p.xii / Table of Contents --- p.xiii / List of Figures --- p.xix / List of Tables --- p.xxi / Abbreviations --- p.xxiv / Publications --- p.xxvii / Conference Presentations --- p.xxviii / Chapter Chapter 1: --- Introduction / Chapter 1.1. --- Normal retinal architecture --- p.1 / Chapter 1.2. --- Age-related retinal changes --- p.3 / Chapter 1.3. --- Age-related macular degeneration (AMD) --- p.7 / Chapter 1.3.1. --- Classification, clinical manifestation and disease course --- p.7 / Chapter 1.3.2. --- Exudative AMD and therapeutic strategies --- p.9 / Chapter 1.3.3. --- Pathology of AMD --- p.10 / Chapter 1.3.4. --- Risk factors and associated pathogenesis --- p.12 / Chapter 1.3.4.1. --- Age --- p.12 / Chapter 1.3.4.2. --- Ethnicity --- p.13 / Chapter 1.3.4.3. --- Oxidative stress --- p.13 / Chapter 1.3.4.3.1. --- Reactive oxygen species and AMD --- p.14 / Chapter 1.3.4.3.2. --- Antioxidants --- p.15 / Chapter 1.3.4.3.3. --- Association of oxidation genes with AMD --- p.16 / Chapter 1.3.4.4. --- Inflammation --- p.16 / Chapter 1.3.4.4.1. --- Complement in AMD --- p.17 / Chapter 1.3.4.4.2. --- The potential role of formyl peptide receptor 1 (FPR1) in AMD --- p.19 / Chapter 1.3.4.5. --- Genetic predisposition --- p.19 / Chapter 1.3.4.5.1. --- Complement factor H --- p.21 / Chapter 1.3.4.5.2. --- The 10q26 locus --- p.22 / Chapter 1.3.4.5.3. --- Phenotype-genotype correlation --- p.23 / Chapter 1.4. --- Comparisons between exudative AMD and Polypoidal choroidal vasculopathy --- p.24 / Chapter 1.4.1. --- History --- p.25 / Chapter 1.4.2. --- Natural course --- p.26 / Chapter 1.4.3. --- Epidemiological factors --- p.27 / Chapter 1.4.3.1. --- Ethnicity --- p.27 / Chapter 1.4.3.2. --- Gender --- p.27 / Chapter 1.4.3.3. --- Age --- p.28 / Chapter 1.4.3.4. --- Risk factors --- p.28 / Chapter 1.4.4. --- Clinical manifestation and histopathological features --- p.29 / Chapter 1.4.5. --- Genetic determinants --- p.29 / Chapter 1.4.5.1. --- Genes with common associations --- p.30 / Chapter 1.4.5.2. --- Genes not have common association --- p.32 / Chapter 1.4.6. --- Response to treatments --- p.32 / Chapter 1.5. --- Objectives and research prospects --- p.33 / Chapter Chapter 2: --- Materials and Methods / Chapter 2.1. --- Polymorphism identification in ARMS2, HTRA1, FPR1 and CFH --- p.39 / Chapter 2.1.1. --- Study subjects --- p.39 / Chapter 2.1.1.1. --- Diagnostic features of AMD and PCV --- p.39 / Chapter 2.1.1.2. --- Control subjects --- p.40 / Chapter 2.1.2. --- Laboratory methods --- p.40 / Chapter 2.1.2.1. --- DNA extraction and quantification --- p.40 / Chapter 2.1.2.2. --- Genotyping --- p.41 / Chapter 2.1.2.2.1. --- Polymerase chain reaction (PCR) and agrose gel electrophoresis --- p.41 / Chapter 2.1.2.2.2. --- DNA sequencing --- p.42 / Chapter 2.1.3. --- Statistical analysis --- p.43 / Chapter 2.1.3.1. --- Genotypic association analysis --- p.43 / Chapter 2.1.3.2. --- Haplotype association analysis --- p.43 / Chapter 2.1.3.3. --- Logistic regression analysis --- p.44 / Chapter 2.1.3.4. --- Joint effect analysis --- p.44 / Chapter 2.1.3.5. --- Meta-analysis --- p.45 / Chapter 2.1.3.6. --- Statistical power calculation and sample size --- p.45 / Chapter 2.2. --- Phenotype-genotype correlation in ARMS2/HTRA1 locus --- p.46 / Chapter 2.2.1. --- Patient recruitment --- p.46 / Chapter 2.2.2. --- Genotyping --- p.46 / Chapter 2.2.3. --- Outcome measurement --- p.46 / Chapter 2.2.4. --- Statistical analysis --- p.47 / Chapter Chapter 3: --- Results / Chapter 3.1. --- The age and gender distribution in study subjects --- p.57 / Chapter 3.2. --- The HTRA1 sequencing in exudative AMD and PCV --- p.57 / Chapter 3.2.1. --- Polymorphism identification and genotypic association --- p.57 / Chapter 3.2.2. --- Haplotype structure and Haplotype-based association analysis --- p.59 / Chapter 3.2.3. --- Joint genotype analysis --- p.59 / Chapter 3.3. --- Differential association of exudative AMD and PCV with the ARMS2/HTRA1 locus --- p.60 / Chapter 3.3.1. --- Genotypic association --- p.60 / Chapter 3.3.2. --- Haplotype analysis --- p.62 / Chapter 3.3.3. --- Logistic regression --- p.63 / Chapter 3.3.4. --- Meta-analysis of ARMS2/HTRA1 association with PCV --- p.64 / Chapter 3.3.5. --- In-position OR plot --- p.64 / Chapter 3.4. --- FPR1 and CFH in exudative AMD and PCV --- p.65 / Chapter 3.4.1. --- Polymorphism identification and genotypic association --- p.65 / Chapter 3.4.2. --- Haplotype analysis of FPR1 --- p.66 / Chapter 3.4.3. --- The association of CFH rs800292 --- p.67 / Chapter 3.4.4. --- Joint effect analysis of the CFH and FPR1 genes --- p.67 / Chapter 3.4. --- Phenotype-genotype correlation in ARMS2/HTRA1 locus --- p.68 / Chapter 3.4.1. --- Distribution of age and bilaterality --- p.69 / Chapter 3.4.2. --- Greatest linear dimension of CNV lesion in exudative AMD --- p.69 / Chapter 3.4.3. --- Response to treatment in exudative AMD --- p.69 / Chapter 3.4.4. --- Recurrence in PCV --- p.70 / Chapter 3.4.5. --- Smoking status --- p.70 / Chapter Chapter 4: --- Discussion / Chapter 4.1. --- Age and gender distribution --- p.104 / Chapter 4.2. --- Genetic differentiation in ARMS2/HTRA1 locus --- p.S104 / Chapter 4.2.1. --- SNPs with common association --- p.106 / Chapter 4.2.2. --- SNPs with different association S --- p.106 / Chapter 4.2.3. --- Comparison with previous studies C --- p.107 / Chapter 4.2.4. --- Sample size S --- p.109 / Chapter 4.3. --- The FPR1 gene in exudative AMD and PCV --- p.110 / Chapter 4.4. --- Interaction between FPR1 and CFH --- p.112 / Chapter 4.5. --- Correlation between phenotypes and genotypes --- p.113 / Chapter 4.6. --- Common and rare variants for complex disease --- p.114 / Chapter 4.6.1. --- The debate of common disease common variant versus common disease rare variant --- p.115 / Chapter 4.6.2. --- Candidate gene screening versus geno-wide association study --- p.117 / Chapter 4.6.3. --- Common variants versus rare variants in 10q26 locus --- p.118 / Chapter 4.6.3.1. --- Common variants --- p.119 / Chapter 4.6.3.2. --- Rare variants --- p.120 / Chapter Chapter 5: --- Conclusions and future prospects --- p.122 / Chapter Chapter 6: --- References --- p.124

Driver distraction contributes to approximately 43% of motor-vehicle crashes and 27% of near-crashes. Rapidly developing in-vehicle technology and electronic devices place additional demands on drivers, which might lead to distraction and diminished capacity to perform driving tasks. This situation threatens safe driving. Technology that can detect and mitigate distraction by alerting drivers could play a central role in maintaining safety. Correctly identifying driver distraction in real time is a critical challenge in developing distraction mitigation systems, and this function has not been well developed. Moreover, the greatest benefit may be from real-time distraction detection in advance of dangerous breakdowns in driver performance. Based on driver performance, two types of distraction - visual and cognitive - are identified. These types of distraction have very different effects on visual behavior and driving performance; therefore, they require different algorithms for detection. Distraction detection algorithms typically rely on either eye measures or driver performance measures because the effect of distraction on the coordination of measures has not been established. Combining both eye glance and vehicle data could enhance the ability of algorithms to detect and differentiate visual and cognitive distraction. The goal of this research is to examine whether poor coordination between visual behavior and vehicle control can identify diminished attention to driving in advance of breakdowns in lane keeping. The primary hypothesis of this dissertation is that detection of changes in eye-steering relationship caused by distraction could provide a prospective indication of vehicle state changes. Three specific aims are pursued to test this hypothesis. The first aim examines the effect of distracting activity on eye and steering movements to assess the degree to which the correlation parameters are indicative of distraction. The second aim applies a control-theoretic system identification approach to the eye movement and steering data to distinguish between distracted and non-distracted conditions. The third aim examines whether changes of eye-steering coordination associated with distraction provide a prospective indication of breakdowns in driver performance, i.e., lane departures. Together, the three aims show how that a combination of visual and steering behavior, i.e., eye-steering model, can differentiate between non-distracted and distracted state. This model revealed sensitivity to distraction associated with off-road glances. The models derived for different drivers have similar structure and fit to data from other drivers reasonably well. In addition, the differences in model order and model coefficients indicate the variability in driving behavior: some people generate more complex behavior than others. As was expected, eye-steering correlation on straight roads is not as strong as observed on curvy roads. However, eye-steering correlation measured through correlation coefficient and time delay between two movements is sensitive to different types of distraction. Time delay mediates changes in lane position and the eye-steering system predicts breakdowns in lane keeping. This dissertation contributes to developing a distraction detection system that integrates visual and steering behavior. More broadly, these results suggest that integrating eye and steering data can be helpful in detecting and mitigating impairments beyond distraction, such as those associated with alcohol, fatigue, and aging.

The primary aim of this research is to test the therapeutic potential of certain new generation corticosteroid drugs in order to develop safe and effective treatment for eye diseases that result in oedema, or swelling. The rising incidence of diabetes and the ageing population of developed countries mean that the prevalence of uveitis, diabetic retinopathy and age related macular degeneration will rise. Often, oedema is one of the reasons for vision loss. Corticosteroids are often used to reduce inflammation. Inflammation is one of several sources of oedema. Glucocorticoids, a class of corticosteroids that have anti-inflammatory properties, are thus used to treat ocular oedema. There is an unmet need to support clinical experience of the efficacy of steroids for ocular inflammation and oedema with more substantial scientific evidence. None of the drugs under investigation, with the exceptions of dexamethasone and triamcinolone, have been used for any ocular therapeutic purpose before. This thesis investigates “repurposing” fludrocortisone to the ophthalmic area. 11-Desoxycorticosterone (11D) and Deoxycorticosterone (DCS), other potentially valuable mineralocorticoids, remain completely untested. Lastly, Kenacort ®, or triamcinolone acetonide (TCA), is only used off-label by ophthalmologists. Methods: In the first study, corticosteroids, and especially mineralocorticoids, were investigated for their treatment efficacy in experimental uveitis, or intraocular inflammation (using a model known as endotoxin induced uveitis). In the second study, endothelial cells from choroidal blood vessels in the back of the eye were used in vitro to study whether corticosteroids reduce paracellular (between cells) permeability. Lastly, since endophthalmitis due to frequent injections is a side effect of corticosteroid use, the pharmacokinetics of different size formulations of corticosteroids were studied in an effort to find a formula that would have a prolonged dwell time within the eye.

<p>Since 1986 patients with ocular malignant melanoma have been treated with Ru-106 plaques at S:t Erik Eye Hospital. In 1998 I-125 radioactive seed plaques was presented as an alternative to Ru-106 when treating tumors with an apical height greater than 7 mm. Until June 2005 the doseplanning of these plaques was based on a depth-dose curve made in the dose planning system Cadplan supplied by Varian Medical Systems. In the recent years the capabilities of computerized 3D dose planning system has increased greatly. The number of types of seeds on the market has also increased.</p><p>In order to implement the modern 3D dose planning system Brachy Vision 7.3.10 in planning the I-125 plaques, a review of the dose planning process have been done.</p><p>The ultra sound equipment used by the ophthalmologist to determine the apical height of the tumor has been investigated in terms of accuracy. A phantom has been developed for this task.</p><p>As new seeds entered the market a comparision have been made comparing the Amersham 6711 seed with the Bebig I25.S06 seed. A method for measuring the activity of the single seeds has also been developed.</p><p>The dose planning system Brachy Vision 7.3.10 have been compared to the old dose planning method, and an implementation of the plaques into Brachy Vision have been made.</p><p>The ultra sound equipment was accurate in the regions of interest. It was also discovered that the Bebig I25.S06 seed gave slightly higher dose compared to the Amersham 6711 with the same activity. The difference between the seeds is however small. The results indicate that the old dose planning method gave a slight underdosage.</p>

Dichoptic presentation of vernier stimuli, i.e., one segment to each eye, yielded three times higher thresholds than binocular presentation, mainly due to uncorrelated movements of both eyes. Thresholds allow one to calculate an upper estimate for the amplitudes of uncorrelated eye movements during fixation. This estimate matches the best results from direct eye position recording, with the calculated mean amplitude of eye tremor corresponding to roughly one photoreceptor diameter. The combined amplitude of both correlated and uncorrelated eye movements was also measured by delaying one segment of the vernier relative to its partner under monocular or dichoptic conditions.

Since 1986 patients with ocular malignant melanoma have been treated with Ru-106 plaques at S:t Erik Eye Hospital. In 1998 I-125 radioactive seed plaques was presented as an alternative to Ru-106 when treating tumors with an apical height greater than 7 mm. Until June 2005 the doseplanning of these plaques was based on a depth-dose curve made in the dose planning system Cadplan supplied by Varian Medical Systems. In the recent years the capabilities of computerized 3D dose planning system has increased greatly. The number of types of seeds on the market has also increased. In order to implement the modern 3D dose planning system Brachy Vision 7.3.10 in planning the I-125 plaques, a review of the dose planning process have been done. The ultra sound equipment used by the ophthalmologist to determine the apical height of the tumor has been investigated in terms of accuracy. A phantom has been developed for this task. As new seeds entered the market a comparision have been made comparing the Amersham 6711 seed with the Bebig I25.S06 seed. A method for measuring the activity of the single seeds has also been developed. The dose planning system Brachy Vision 7.3.10 have been compared to the old dose planning method, and an implementation of the plaques into Brachy Vision have been made. The ultra sound equipment was accurate in the regions of interest. It was also discovered that the Bebig I25.S06 seed gave slightly higher dose compared to the Amersham 6711 with the same activity. The difference between the seeds is however small. The results indicate that the old dose planning method gave a slight underdosage.

Delivering drugs to effectively treat diseases of the back of the eye can be a challenging task. Although pharmacological therapies exist, drug delivery devices and techniques are not very effective at targeting delivery of drugs to the diseased tissues. This work introduces a novel approach to effectively deliver drugs to target tissues such as the choroid and retina. The approach involves a device, a hollow microneedle, to administer the drug formulation into a unique location in the eye, the suprachoroidal space. This new route of administration and a device to accomplish the delivery may provide an effective way to treat diseases of the choroid and retina. The first part of the work determines the ex-vivo feasibility of delivering materials within the suprachoroidal space. The results show that fluids and particles can be delivered into the suprachoroidal space of rabbit, pig and human eyes using a hollow microneedle. It further examines the important parameters for injection of the particles within the suprachoroidal space. The data shows that injection pressure and microneedle length are important parameters for effective delivery of particles. The results lead to a theory on the mechanism by which the particles are delivered into the suprachoroidal space. The second part of the research aims to develop a reliable in vivo delivery device and study the surface area coverage of materials injected into the suprachoroidal space. A hollow glass microneedle device is developed and for the first time shown to be effective in delivering a fluid into the suprachoroidal space in vivo. Up to 100 µL of India ink could be delivered into rabbit eyes in vivo and the spread within the suprachoroidal space is characterized. The results show that a single microneedle injection can cover a significant percentage of the available suprachoroidal space. This is the first study to examine the spread of a material injected into the suprachoroidal space of a live animal. A hollow metal microneedle device is also developed and shown to be effective. The device was able to inject up to 150 µL of latex into suprachoroidal space of fresh human cadaver eyes. The spread of latex is characterized and the results also show that a significant portion of the suprachoroidal space can be covered. The final part of the study examines the clearance of materials injected into the suprachoroidal space of rabbit eyes in vivo. First a comparison of a suprachoroidal injection to a conventional intravitreal injection shows that a suprachoroidal injection is more targeted to the chorioretinal tissues. In addition hollow microneedles are shown to effectively target macromolecules and a therapeutic antibody to the chorioretinal tissues. A study of the clearance kinetics show half lives within the suprachoroidal space on the order of several hours. Nano- and microparticles were also injected into the suprachoroidal space and showed very effective targeting. These non-degradable particles are shown to be present in the suprachoroidal space for months. Basic visual safety assessments identified no adverse effects from the injection of these materials. This represents the first study to compare intraocular clearance kinetics between a suprachoroidal injection and an intravitreal injection. It is also the first study to examine the clearance of a variety of materials from within the suprachoroidal space. Overall this work shows that microneedles have the capability to deliver a variety of materials into the suprachoroidal space of rabbit, pig, and human eyes. The injection can be done in a minimally invasive way with the proper design of an injection device and can target the chorioretinal tissues more effectively than the currently used method. In addition particles have long residence times in the suprachoroidal space, so a particle based drug formulation could provide sustained delivery to the eye. This work represents the first comprehensive study on using the suprachoroidal space as a drug delivery route and also the first study to use hollow microneedles to deliver formulations into the eye in vivo.