(Abstract shortened by UMI.) / The conclusion of the studies reported herein can be summarized as follows: (1) PI in TCD correlates well with WMC volume and helps to differentiate those with and without WMC in stroke patients. (2) Post-stroke cognitive complaints are not related to severity of WMC among lacunar stroke patients. (3) The ARWMC scale correlates with objective cognitive performances and the operational definitions of ARWMC scale improves inter-rater reliability on CT. (4) Cognitive impairment in patients with confluent WMC is mediated by global and frontal cortical atrophy. Predictors for cognitive progression are cortical atrophy, absence of hyperlipidemia, low BP, and low cognitive scores. / With an aging population, prevalence of dementia is expected to escalate in the coming decades. The burden is especially great in developing countries like China. Similar to Alzheimer's pathology (e.g. amyloid plaque), age-related white matter changes (WMC) are important substrates of dementia. Since WMC are considered to be of ischemic origin, dementia related to WMC is believed to be more preventable than Alzheimer's disease. Yet, studies focusing on WMC have been relatively few. The thesis will cover 4 aspects of WMC and cognitive impairment. / Xiong, Yunyun. / Adviser: Vincent Mok. / Source: Dissertation Abstracts International, Volume: 73-06, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 198-244). / 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; some appendixes in Chinese.

過敏性疾病（如過敏性哮喘和過敏性皮炎）發病率在香港及世界均呈上升趨勢。過敏性哮喘是一種慢性反復發作的炎症疾病，而過敏性皮炎是一種慢性皮膚炎症。呼吸道細菌及金黃色葡萄球菌可分別加重過敏性哮喘病人氣道炎症及過敏性皮炎患者的炎症反應。人體對細菌的先天免疫反應主要通過模式識別受體(PRR)介導。NOD樣受體(NLR)和Toll樣受體(TLR)是兩種重要的PRR。 NLR 家族成員NOD2幾乎識別所有細菌中結構保守的胞壁醯二肽(MDP)。而LTR2識別範圍廣泛的病原相關分子模式，如革蘭氏陽性菌中的肽聚糖(PGN)和脂磷壁酸(LTA)，以及人工合成的脂蛋白Pam3CSK4。 / 本研究包括：NOD2配體MDP，哮喘相關的腫瘤壞死因子家族成員LIGHT對共培養的人嗜鹼性粒細胞和人支氣管上皮細胞的活化作用；熱滅活的金黃色葡萄球菌（HKSA），MDP，TLR2配體PGN，LTA，以及Pam3CSK4對共培養的人嗜鹼性粒細胞和人皮膚成纖維細胞的活化作用；在體內NLR配體對卵清蛋白(OVA)致敏的哮喘小鼠的作用。 / 研究發現，在共培養體系中，MDP能顯著增強嗜鹼性粒細胞與支氣管上皮細胞表面粘附因子（細胞間粘附因子ICAM-1 及血管細胞粘附因子VCAM-1）的表達。同時，MDP能顯著促進共培養體系中炎症相關細胞因子IL-6，趨化因子CXCL8及抗菌肽β-防禦素2的釋放。在MDP刺激下，支氣管上皮細胞是共培養體系中釋放IL-6，CXCL8及β-防禦素2的主要細胞。在MDP刺激下，嗜鹼性粒細胞中包括胞核因子-kappaB（NF-κB）在內的幾個核轉錄因子的表達上升。ICAM-1，VCAM-1，IL-6，CXCL8，及β-防禦素2的表達被信號分子化學抑制劑所抑制，結果表明，嗜鹼性粒細胞與支氣管上皮細胞的相互作用受不同的信號通路（NF-κB, p38 MAPK 及 JNK）調節。OVA致敏小鼠實驗表明，NLR配體能增加分泌粘蛋白的杯狀細胞在肺氣管中的數量，使小鼠支氣管下皮結締組織纖維化並增厚。NLR配體進而提高過敏性哮喘小鼠支氣管肺泡灌洗液中CCL5與IL-13 的表達水平。 / 研究表明，在嗜鹼性粒細胞和皮膚成纖維細胞的共培養體系中，HKSA，MDP，PGN，LTA，或Pam3CSK4顯著誘導ICAM-1, IL-6, CXCL8, CCL2 和 CCL5 的表達。而嗜鹼性粒細胞與皮膚成纖維細胞的直接相互作用是釋放IL-6, CXCL8, CCL2 與 CCL5 所必需的。嗜鹼性粒細胞與皮膚成纖維細胞的相互作用並釋放細胞因子與趨化因子受p38 MAPK 及 NF-κB信號通路調控。 / 在嗜鹼性粒細胞與支氣管上皮細胞共培養體系中，LIGHT 可能通過受體HVEM 與 LTβR顯著增強支氣管上皮細胞表面粘附因子的表達，提高細胞因子IL-6, CXCL8 與 MMP-9的釋放。 / 研究結果表明，在過敏炎症中，通過與組織細胞（如支氣管上皮細胞，人皮膚成纖維細胞）相互作用，嗜鹼性粒細胞有利於組織細胞對病原相關的分子模式作出反應。因此，研究結果對細菌介導的先天性免疫應答與過敏炎症的加重之間的聯繫作出了新的解釋。以上結果也增強了我們對LIGHT在氣道重塑中的免疫病理作用及其作為氣道重塑治療靶標的認識。 / The incidences of allergic diseases such as allergic asthma and atopic dermatitis (AD) are increasing in Hong Kong and worldwide. Allergic asthma is a chronically relapsing inflammatory pulmonary disease, while AD is a chronic inflammatory skin disorder. Respiratory bacterial and Staphylococcus aureus (S. aureus) infection can provoke allergen sensitization and subsequently amplify and sustain inflammation in allergic asthma and AD, respectively. The innate immune system recognizes bacterial infection through pattern recognition receptors (PRRs), two important PRRs involving in inflammatory and immune responses are nucleotide-binding oligomerization domain-like receptors (NLRs) and Toll-like receptors (TLRs). NOD2 is one member of the NLR family, which senses the conserved structural component muramyl dipeptide (MDP) in almost all bacteria. TLR2 recognizes a wide range of pathogen-associated molecular patterns (PAMPs) including peptidoglycan (PGN) and lipoteichoic acid (LTA) from Gram-positive bacteria and synthetic triacylated lipoprotein N-palmitoyl-S-[2,3-bis (palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S] -lysine (Pam3CSK4). / In the present study, we investigated the effect of NOD2 ligand MDP, asthma-related tumor necrosis factor (TNF) family member LIGHT on human basophils co-cultured with human bronchial epithelial cells and the effect of heat-killed S. aureus, MDP, TLR2 ligands PGN, LTA and Pam3CSK4 on basophils co-cultured with human dermal fibroblasts, and the underlying intracellular mechanisms. The in vivo effect of NOD ligands on ovalbumin (OVA)-sensitized allergic asthmatic mice was also studied. / It was found that MDP could significantly enhance the cell surface expression of adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on basophils and primary human bronchial epithelial cells (HBE) in the co-culture system (all p < 0.05). MDP could further enhance the release of inflammatory cytokine interleukin (IL)-6, chemokine CXCL8, and epithelium derived anti-microbial peptide β-defensin 2 in the co-culture. HBE cells were the major source while basophils were the minor source to release IL-6, CXCL8 and β-defensin 2 in the co-culture upon MDP stimulation. The activities of several nuclear transcription factors, including NF-κB, were up-regulated in human basophils upon MDP stimulation. The cell surface expression of ICAM-1 and VCAM-1 and the release of IL-6, CXCL8 and β-defensin 2 were suppressed by the signaling molecule inhibitors, implying that the interaction between basophils and primary human bronchial epithelial cells could be differentially regulated by the NF-κB, p38 MAPK and JNK pathways. The animal study showed that iE-DAP and MDP could increase the number of mucin-secreting goblet cells, the thickness and fibrosis of the bronchial subepithelial tissue of airways from the OVA-sensitized mice. The iE-DAP and MDP could further promote the levels of CCL5 and IL-13 (all p < 0.05) in bronchoalveolar lavage fluid (BALF) of allergic asthmatic mice. / It was found that the induction of ICAM-1, IL-6, CXCL8, CCL2 and CCL5 was significantly promoted upon the interaction between human basophils and dermal fibroblasts activated by heat-killed S. aureus, MDP, PGN, LTA or Pam3CSK4. The release of IL-6, CXCL8, CCL2 and CCL5 might depend on the direct interaction of basophils and dermal fibroblasts. The p38 MAPK and NF-κB pathways should be involved in the release of the cytokines and chemokines upon the interaction of basophils and human dermal fibroblasts. / LIGHT could significantly promote the cell surface expression of adhesion molecule, the release of IL-6, CXCL8 and MMP-9 from human bronchial epithelial cells upon the interaction with basophils, probably through the receptors HVEM and LTβR. / The results suggest that, through the interaction with tissue-resident cells such as bronchial epithelial cells and dermal fibroblasts, basophils may facilitate the activation of tissue-resident cells in response to the PAMPs in allergic inflammation. The results therefore provide a new insight of the crucial link between the bacterial-mediated innate immune response and the exacerbation of allergic inflammation. The above results also enhance our understanding on the immunopathological roles of LIGHT in airway remodeling, and the potential therapeutic target for airway remodeling. / 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. / Qiu, Huaina. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 165-196). / Abstract also in Chinese. / Acknowledgements --- p.i / Abbreviations --- p.iii / Abstract --- p.vi / 摘要 --- p.ix / Publications --- p.xi / Table of Contents --- p.xiii / Chapter Chapter 1: --- General Introduction --- p.1 / Chapter 1.1 --- Asthma and atopic dermatitis (AD) --- p.1 / Chapter 1.2 --- Human basophils in allergic inflammation --- p.3 / Chapter 1.2.1 --- Development and morphology of basophils --- p.3 / Chapter 1.2.2 --- Receptors and products of basophils --- p.4 / Chapter 1.2.3 --- Cell surface markers on basophils --- p.7 / Chapter 1.2.4 --- Basophils in allergic inflammation --- p.7 / Chapter 1.3 --- Human bronchial epithelial cells in airway inflammation --- p.10 / Chapter 1.4 --- Human fibroblasts in AD --- p.11 / Chapter 1.5 --- Staphylococcus aureus (S. aureus) in AD --- p.12 / Chapter 1.6 --- NOD2 and TLR2 in allergic inflammation --- p.14 / Chapter 1.7 --- IL-33 in allergic inflammation --- p.18 / Chapter 1.8 --- IL-6 in allergic inflammation --- p.18 / Chapter 1.9 --- CXCL8 in allergic inflammation --- p.20 / Chapter 1.10 --- CCL2 in allergic inflammation --- p.21 / Chapter 1.11 --- CCL5 in allergic inflammation --- p.22 / Chapter 1.12 --- β-defensin 2 (HBD-2) in allergic inflammation --- p.23 / Chapter 1.13 --- ICAM-1 and VCAM-1 in allergic inflammation --- p.25 / Chapter 1.14 --- LIGHT and airway remodeling in allergic asthma --- p.25 / Chapter 1.15 --- Signal transduction pathways in allergic inflammation and pharmacological inhibitors --- p.26 / Chapter 1.15.1 --- Signal transduction pathways in allergic inflammation --- p.26 / Chapter 1.15.2 --- Signaling molecule inhibitors as new drugs for inflammatory diseases --- p.31 / Chapter 1.16 --- Aims and scope of the study --- p.32 / Chapter Chapter 2: --- Materials and Methods --- p.35 / Chapter 2.1 --- Materials --- p.35 / Chapter 2.1.1 --- Reagents and buffers for the purification of human basophils --- p.35 / Chapter 2.1.2 --- Primary cells and cell lines --- p.36 / Chapter 2.1.3 --- Heat-killed Staphyloccocus aureus (HKSA) --- p.38 / Chapter 2.1.4 --- Ligands for NLR and TLR2 --- p.39 / Chapter 2.1.5 --- Recombinant human cytokines --- p.39 / Chapter 2.1.6 --- Reagents and buffer solutions for flow cytometry --- p.40 / Chapter 2.1.7 --- RNA extraction, reverse transcription-polymerase chain reaction (RT-PCR), and real-time quantitative PCR (qPCR) --- p.45 / Chapter 2.1.8 --- Cytometric Bead Array (CBA) Kits --- p.48 / Chapter 2.1.9 --- MILLIPLEX® MAP Human Cytokine/Chemokine Magnetic Bead Panel Kit --- p.49 / Chapter 2.1.10 --- Enzyme-linked immunosorbent assay (ELISA) kits --- p.49 / Chapter 2.1.11 --- Procarta Transcription Factor Assay kit --- p.50 / Chapter 2.1.12 --- Signal transduction inhibitors --- p.50 / Chapter 2.2 --- Methods --- p.50 / Chapter 2.2.1 --- Purification of primary human basophils and basophil culture --- p.50 / Chapter 2.2.2 --- Culture of KU812 cells --- p.51 / Chapter 2.2.3 --- Culture of primary human bronchial epithelial cells --- p.51 / Chapter 2.2.4 --- Culture of BEAS-2B cells --- p.52 / Chapter 2.2.5 --- Culture of human dermal fibroblasts --- p.52 / Chapter 2.2.6 --- Co-culture of primary human bronchial epithelial cells/human bronchial epithelial cell line (BEAS-2B) cells and basophils/KU812 cells --- p.52 / Chapter 2.2.7 --- Co-culture of human dermal fibroblasts and basophils/KU812 cells --- p.52 / Chapter 2.2.8 --- Co-culture of fixed primary human bronchial epithelial cells and basophils --- p.53 / Chapter 2.2.9 --- Co-culture of human dermal fibroblasts and basophils in the presence of transwell inserts --- p.53 / Chapter 2.2.10 --- CBA assay --- p.53 / Chapter 2.2.11 --- ELISA --- p.54 / Chapter 2.2.12 --- Human Transcription Factor Plex Assay --- p.54 / Chapter 2.2.13 --- Milliplex Human Cytokine / Chemokine Magnetic Panel assay --- p.54 / Chapter 2.2.14 --- Bio-Plex mouse cytokine assay --- p.55 / Chapter 2.2.15 --- Flow cytometric analysis of cell surface expression of target molecules --- p.55 / Chapter 2.2.16 --- Flow cytometric analysis of intracellular expression of target molecules --- p.55 / Chapter 2.2.17 --- Allergic asthmatic mice model --- p.57 / Chapter 2.2.18 --- Statistical analysis --- p.57 / Chapter Chapter 3: --- Muramyl Dipeptide Mediated Activation of Human Bronchial Epithelial Cells Interacting with Basophils: A Novel Mechanism of Airway Inflammation --- p.59 / Chapter 3.1 --- Introduction --- p.59 / Chapter 3.2 --- Results --- p.61 / Chapter 3.2.1 --- Cell surface expression of CD203c on basophils --- p.61 / Chapter 3.2.2 --- Intracellular expression of NOD2 protein --- p.63 / Chapter 3.2.3 --- Cell surface expression of adhesion molecules on basophils and primary human bronchial epithelial cells activated by MDP --- p.67 / Chapter 3.2.4 --- Induction of cytokine, chemokine and β-defensin 2 upon the interaction of basophils and bronchial epithelial cells stimulated by MDP --- p.71 / Chapter 3.2.5 --- Bronchial epithelial cells were the main source for the release of IL-6, CXCL8 and β-defensin 2 in co-culture --- p.74 / Chapter 3.2.6 --- Effects of signaling inhibitors on MDP-induced cytokines and adhesion molecules --- p.77 / Chapter 3.2.7 --- Differential activation of intracellular signaling pathways involved in the interaction of KU812 and BEAS-2B upon MDP stimulation --- p.84 / Chapter 3.2.8 --- In vivo effect of NOD1,2 ligands on IgE and chemokine production in serum and BALF in allergic asthmatic mice --- p.89 / Chapter 3.3 --- Discussion --- p.93 / Chapter Chapter 4: --- NOD2 and TLR2 Ligands Mediated Activation of Basophils Interacting with Human Dermal Fibroblasts in Atopic Dermatitis --- p.100 / Chapter 4.1 --- Introduction --- p.100 / Chapter 4.2 --- Results --- p.102 / Chapter 4.2.1 --- Cell surface expression of adhesion molecules ICAM-1 on human dermal fibroblasts activated by heat-killed Staphyloccocus aureus (HKSA) --- p.102 / Chapter 4.2.2 --- Induction of chemokines upon the interaction of basophils and human dermal fibroblasts stimulated by HKSA --- p.104 / Chapter 4.2.3 --- Expression of NOD2 and TLR2 protein --- p.107 / Chapter 4.2.4 --- Cell surface expression of adhesion molecule ICAM-1 on human dermal fibroblasts activated by MDP, PGN, LTA or Pam3CSK4 --- p.110 / Chapter 4.2.5 --- Induction of cytokine and chemokines upon the interaction of basophils (with or without IL-33 priming) and human dermal fibroblasts stimulated by MDP, PGN, LTA or Pam3CSK4 --- p.112 / Chapter 4.2.6 --- Direct interaction between human dermal fibroblasts and basophils was required for the release of IL-6, CXCL8, CCL2 and CCL5 upon the stimulation of MDP, PGN, LTA and Pam3CSK4 --- p.118 / Chapter 4.2.7 --- Effect of signaling molecular inhibitors on the expression of adhesion molecule ICAM-1 --- p.121 / Chapter 4.2.8 --- Effect of signaling molecule inhibitors on the release of cytokine and chemokines upon the stimulation by NOD2 and TLR2 ligands --- p.123 / Chapter 4.2.9 --- Differential activation of intracellular signaling pathways involved in the interaction of human dermal fibroblasts and basophilic KU812 upon stimulation of NOD2 and TLR2 ligands --- p.127 / Chapter 4.3 --- Discussion --- p.131 / Chapter Chapter 5: --- Effect of Tumor Necrosis Factor Family Member LIGHT on the Activation of Basophils Interacting with Bronchial Epithelial Cells: Potential Therapeutic Target for Airway Remodeling --- p.138 / Chapter 5.1 --- Introduction --- p.138 / Chapter 5.2 --- Results --- p.139 / Chapter 5.2.1 --- Cell surface expression of HVEM and LTβR --- p.139 / Chapter 5.2.2 --- Effect of LIGHT on the expression of ICAM-1 on basophil or BEAS-2B alone or co-culture --- p.141 / Chapter 5.2.3 --- Induction of cytokine and chemokine upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.144 / Chapter 5.2.4 --- Induction of MMP-9 upon the interaction of basophils and BEAS-2B cells stimulated by LIGHT --- p.147 / Chapter 5.2.5 --- Effect of LIGHT on the release of TGFβ-1, histamine and periostin upon the interaction of basophils and BEAS-2B cells --- p.149 / Chapter 5.3 --- Discussion --- p.152 / Chapter Chapter 6: --- Conclusion and Future Perspectives --- p.156 / Chapter 6.1 --- General conclusions --- p.156 / Chapter 6.2 --- Future perspectives --- p.160 / Appendix --- p.163 / References --- p.165

Abstract not available

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is a national priority, with nearly six million Americans affected at an annual cost of $200 billion and no available cure. A better understanding of the mechanisms underlying AD is crucial to combat its high and rising incidence and burdens. Most cases of AD are thought to have a complex etiology with numerous genetic and environmental factors influencing susceptibility. Recent genome-wide association studies (GWAS) have confirmed roles for several hypothesized genes and have discovered novel loci associated with disease risk. However, most GWAS-implicated genetic variants have displayed modest individual effects on disease risk and together leave substantial heritability and pathophysiology unexplained. As a result, new paradigms focusing on biological pathways have emerged, drawing on the hypothesis that complex diseases may be influenced by collective effects of multiple variants – of a variety of effect sizes, directions, and frequencies – within key biological pathways. A variety of tools have been developed for pathway-based statistical analysis of GWAS data, but consensus approaches have not been systematically determined. We critically review strategies for genetic pathway analysis, synthesizing extant concepts and methodologies to guide application and future development. We then apply pathway-based approaches to complement GWAS of key AD-related endophenotypes, focusing on two early, hallmark features of disease, episodic memory impairment and brain deposition of amyloid-β. Using GWAS and pathway analysis, we confirmed the association of APOE (apolipoprotein E) and discovered additional genetic modulators of memory functioning and amyloid-β deposition in AD, including pathways related to long-term potentiation, cell adhesion, inflammation, and NOTCH signaling. We also identified genetic associations to amyloid-β deposition that have classically been understood to mediate learning and memory, including the BCHE gene and signaling through the epidermal growth factor receptor. These findings validate the use of pathway analysis in complex diseases and illuminate novel genetic mechanisms of AD, including several pathways at the intersection of disease-related pathology and cognitive decline which represent targets for future studies. The complexity of the AD genetic architecture also suggests that biomarker and treatment strategies may require simultaneous targeting of multiple pathways to effectively combat disease onset and progression.

Indiana University-Purdue University Indianapolis (IUPUI) / Introduction: During glutamate excitotoxicity, overstimulation of glutamate receptors leads to sustained elevation in cytosolic Ca2+ ([Ca2+]c), or delayed Ca2+ dysregulation (DCD), which is causally linked to cell death. There are two major hypothetical mechanisms for DCD: the continuous activation of N-methyl-D-aspartate-subtype of the ionotropic glutamate receptors (NMDAR) and the reversal of the plasmalemmal Na+/Ca2+ exchanger. However, the contribution of each of these mechanisms in DCD is not completely established. Major results: Neurons exposed to excitotoxic glutamate produced DCD, an increase in cytosolic Na+ ([Na+]c), and plasma membrane depolarization. MK801 and memantine, noncompetitive NMDAR inhibitors, added after glutamate, completely prevented DCD; however AP-5, a competitive NMDAR inhibitor, failed to do so. The NMDAR inhibitors had no effect on lowering elevated [Na+]c or on restoring plasma membrane potential, which are conditions suggesting NCXrev could be involved. In experiments inducing NCXrev, MK801 and memantine completely inhibited Ca2+ dysregulation after glutamate while AP-5 did not. Inhibition of NCXrev, either with KB-R7943 or by preventing the increase in [Na+]c, failed to avert DCD. However, NCXrev inhibition combined with NMDAR blocked by AP-5 completely prevented DCD. Overall, these data suggested that both NMDAR and NCXrev are essential for glutamate-induced DCD, and inhibition of only one mechanism is insufficient to prevent collapse of calcium homeostasis. Based on the data above, we investigated a NMDA receptor antagonist currently in clinical trials for reducing the effects of glutamate excitotoxicity, ifenprodil. Ifenprodil is an activity-dependent, NMDAR inhibitor selective for the NR2B subunit. We found that ifenprodil not only inhibited the NR2B-specific NMDAR, but also inhibited NCXrev. If ifenprodil is combined with PEAQX, a NMDAR inhibitor selective for the NR2A subunit, low concentrations of both inhibitors completely prevent DCD. Conclusion: The inhibition of a single Ca2+ influx mechanism is insufficient in preventing DCD, which requires simultaneous inhibition of both the NMDAR and NCXrev. These findings are critical for the correct interpretation of the experimental results obtained with these inhibitors and for better understanding of their neuroprotective actions.

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

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Dilantin (diphenylhydantoin or DPH) has been given to epileptic mothers to control seizures during pregnancy. Previous research has demonstrated that exposure of human embryos to Dilantin in vivo results in an increased probability of abnormal development and early fetal loss. Preliminary results with cultured 1-cell and 2-cell mouse embryos demonstrated that Dilantin causes mouse embryonic cleavage events to slow during preimplantation development (Chatot et al., unpublished). Dilantin may be responsible for this by inhibiting the rate of DNA synthesis during cleavage or by affecting the expression of proteins that control cell cycle progression. The standard expression pattern of these cell cycle regulatory proteins (cyclins) has not previously been determined in the mouse preimplantation embryo model. In this study, immunolabellingtechniques have been used to determine the expression pattern of cyclins D, A, and B 1 in the first three cell cycles of preimplantation mouse embryo development.This study reveals a unique expression pattern of cyclins D, A, and B1 in the first three cell cycles of preimplantation embryo development. Examination of the beginning of the first cell cycle, or G1, indicated a moderate expression of cyclin B1 and A but no cyclin D expression. During DNA synthesis (S-phase) all cyclin expression was virtually nonexistent. Toward the end of the cell cycle at G2/M, cyclin D expression appeared to be at moderate levels while cyclins A and B 1 exhibited minimal degrees of expression.In G 1 of the second cell cycle, cyclins D and A were minimally to moderately expressed and cyclin B 1 expression was minimal. At S-phase, cyclin D expression dropped to minimal levels whereas cyclins A and B 1 were at minimal to moderate levels of expression. At G2/M of the second cell cycle, cyclin B1 was expressed at minimal to moderate levels and cyclins A and D were both expressed at minimal levels.The third cell cycle began at G 1 with cyclin B 1 being expressed at moderate levels followed by minimal to moderate levels of cyclin D expression and minimal expression for cyclin A. Cyclin D expression increased to moderate levels at S-phase and cyclin A exhibited minimal to moderate levels of expression. Cyclin B 1 was observed at moderate levels of expression at S-phase of the third cell cycle. G2 of the third cell cycle included a drop to minimal levels of expression of cyclin D, while cyclin A expression remained at minimal to moderate levels and cyclin B remained at moderate levels of expression.The cyclin expression pattern for the first three cell cycles in preimplantation mouse embryos is unique compared to known cyclin expression patterns in other species. Cyclin D is expressed in G1 and is known to be necessary for advancement to S-phase in human glioblastoma cell lines (Xiong et al., 1991). Cyclin A is active at S-phase through Win human fibroblasts and xenopus oocytes (Giordino et al., 1991; Minshul et al., 1990). Cyclin B is present at G2 through mitosis in human fibroblasts and xenopus oocytes (Pines and Hunter, 1990; Minshul et al, 1990). / Department of Biology

M.Phil.(Optometry) / The concept of contact lenses was conceived over 500 years ago and has now evolved into a fundamental component of optometric practice. Soft contact lenses have become a convenient, aesthetically pleasing and comfortable alternative to spectacles that are becoming increasingly popular. The use of rigid contact lenses is imperative in the management of conditions such as keratoconus due to spectacles being insufficient in providing adequate vision. Placing a contact lens onto the cornea is an invasive procedure. The contact lens is a foreign body to the eye hence it is expected that the eye would react to that foreign body. Literature has revealed that the general reactions of the eye to contact lens wear are initial tearing, alteration of the tear layer and oedema due to reduced oxygen transmission but these are just a few of the known consequences amongst the multitude of the unknown consequences. What exactly goes on under a contact lens remains an enigma which contact lens researchers have strived to uncover over the past century. The consequence of contact lens wear is a vast area of research and can best be investigated by focusing on one aspect at a time. The aim of this study was to use dioptric power matrices and multivariate statistics to explore the effects of both gas permeable and gas non-permeable rigid contact lenses on corneal curvature. This study involves auto-keratometric measurements of the corneal curvature before and after lens wear to establish if there are any curvature changes induced by the contact lens. Keratometric data was collected with an automated keratometer (Nidek ARK-700) and was analysed correctly and completely using multivariate statistics. This thesis presents the findings of a study done in an effort to establish the short-term effects of rigid contact lens wear on keratometric behaviour by using complete methods of multivariate statistical analysis. Twenty four subjects were equally divided into three groups. One group wore polymethyl methacrylate (PMMA) rigid lenses, another group wore rigid gas permeable (RGP) contact lenses and the third group served as the control. The control group was included in the study to establish a reference for normal diurnal changes in keratometric behaviour. Fifty autoii keratometric measurements were taken before and immediately after three hours of rigid contact lens wear for the experimental groups and 50 auto-keratometric measurements were taken before and immediately after three hours of no lens wear for the control group. Data collected was analysed using multivariate statistical methods that in the past have been used infrequently in this area of research.

p-21-activated kinase 6 (PAK6) is a serine-threonine protein kinase originally identified as an Androgen Receptor (AR) interacting protein. In current study, we determined the subcellular localization of PAK6 through mutational analysis. We have found that the N-terminal CRIB domain is partly responsible for plasma membrane targeting, the region between amino acid residues #292 to #368 is functionally relevant to plasma membrane localization and that amino acid residues #119 through #190 are responsible for nuclear targeting of PAK6, in addition to a stretch of positively charged N-terminal residues (#2-#11) since mutants lacking this sequence mis-localizes to cytoplasm. In junction forming epithelial cells, PAK6 is demonstrated to co-localize with B-catenin at adherens junctions, suggesting that PAK6 is an activation-dependent event and that PAK6 translocates from plasma membrane to the cytoplasm in response activation via the PKA signal pathway. / by Ciny John. / Thesis (M.S.)--Florida Atlantic University, 2012. / Includes bibliography. / Mode of access: World Wide Web. / System requirements: Adobe Reader.