Indiana University-Purdue University Indianapolis (IUPUI) / Aniridia, a congenital ocular disorder caused by haploinsufficiency of transcription factor PAX6, is characterized by complete or partial iris hypoplasia with associated foveal hypoplasia. Brain imaging performed in patients heterozygous for PAX6 mutations often reveal absence of the brain anterior or posterior commissure, absence of the pineal gland, and a present but reduced in size corpus callosum. Renal coloboma syndrome, another autosomal dominant inherited disease, is characterized by hypodysplastic kidneys and optic nerve defects, and is caused by haploinsufficiency of transcription factor PAX2. In the first part of this thesis we investigated the role of these Pax genes in neural development, by generating an allelic series of knock-in models at the Pax6 locus. We showed that Pax6(5a) and Pax2 could not replace Pax6 for its auto-regulation in lens induction or for neural differentiation in retina. In brain development, however, we demonstrated that cell proliferation in the cerebral cortex and dorsoventral patterning of the telencephalon and neural tube was partially rescued in either knock-in mutant. We believe our novel findings not only reveal Pax-protein functional specificity during neural development, but may also be utilized to understand the aberrant molecular mechanism that result in aniridia and/or renal coloboma syndrome. Aphakia (lack of lens) is a rare human congenital disorder with its genetic etiology largely unknown. In the second part of this thesis, we show that homozygous deletion of Nf1, the Ras GTPase gene underlying human neurofibromatosis type 1 syndrome, caused lens dysgenesis in mouse. While early lens specification proceeded normally in Nf1 mutants, lens induction was disrupted due to deficient cell proliferation. Further analysis showed that ERK signaling was initially elevated in invaginating lens placode, but by lens vesicle stage, Ras signaling antagonist Sprouty2 was up regulated, followed by rapid decrease in ERK phosphorylation. Only after intraperitoneal treatment of U0126, an inhibitor of ERK phosphorylation, was lens development restored in Nf1 mutants. Hyperactive RAS-MAPK signaling is known to cause neuro-cardiofacial-cutaneous (NCFC) syndromes in human. As a member of NCFC family genes, Nf1 represents the first example that attenuation of Ras-MAPK kinase signaling pathway is essential for normal lens development.

published_or_final_version / abstract / Anatomy / Doctoral / Doctor of Philosophy

葡萄膜炎是一組複雜的眼內炎性疾病，可導致嚴重的視力損害，約占世界範圍內工作年齡人群組致盲眼病的10%。孫然治療上已取得一定的進步，但找尋安全有效的治療方式仍是一個臨床難題。基於解剖學分類，葡萄膜炎分為前葡萄膜炎、中葡萄膜炎、后葡萄膜炎和全葡萄膜炎。其中，前葡萄膜炎 (AU) 為最常見的臨床形式。此外，基於病因學葡萄膜炎也可被歸類為感染性和非感染性兩大亞型。作為一種炎症性疾病，已有研究表明許多內源性的免疫機制及遺傳因素參與葡萄膜炎的形成。 / 基於對葡萄膜炎疾病進程的深入瞭解，兩條主要潛在的致病通路：T細胞反應途徑和補體系統顯示在分子水平與疾病密切相關。本研究涉及參與上述兩通路的諸多因子，旨在調查葡萄膜炎的遺傳易感性，揭示潛在致病機理，以及發現新的臨床診斷標記物。 / 本研究共納入501名參與者，包括98名AU患者，95名非感染性中後葡萄膜炎 (NIPU) 患者，病例收集自香港眼科醫院及威爾斯親王醫院。此外，308名年齡50歲以上，排除了主要眼科疾患及系統性免疫疾病的健康人被招募為對照組。全面詳細記錄病人資料及臨床信息。進一步剖析葡萄膜炎疾病特點及補體通路參與程度，深入研究補體基因的累加效應及相互作用，以期發現臨床標記物。最後，採用基因型表型相關性分析，探索其與疾病嚴重程度和進展的關係。 / 研究1：系統性綜述在葡萄膜炎基因學研究上的最新發現，研究表明多種基因與葡萄膜炎疾病相關，所涉基因包括白介素、趨化因子、腫瘤壞死因子，以及參與補體和氧化途徑的相關基因。我們廣泛調查了葡萄膜炎的遺傳易感性。基因多態性選擇基於疾病的免疫及炎性特徵。（1）Interleukin與CFH基因，分別參與T細胞反應及補體通路。（2）CFB，CFH的拮抗因子，共同參與了補體旁路的調控。（3）調查C1INH(SERPING1) 因子，闡明補體經典通路在葡萄膜炎形成中的作用。（4）C3和C5基因，分別作為補體系統的“中心“因子及下游調控因子，其與葡萄膜炎的相關性被深入調查。 / 研究 2：首先探索性地調查免疫相關基因的單核苷酸多態性，包括CFH，KIAA1109 與 IL27 基因，我們的結果顯示 CFH 基因多態性 (rs800292，rs1065489) 與前葡萄膜炎顯著相關。更重要的是，CFH-rs1065489 TT基因型被確認為臨床標記物，此基因型攜帶者表現更高的葡萄膜炎復發頻率。此外，易感基因與HLA-B27的交互作用以及性別敏感性差異亦被發現。本研究的第二部份，此三個候選基因在另一個葡萄膜炎亞組NIPU中被進一步調查，CFH基因多態性 (rs800292，rs1065489) 與NIPU相關。KIAA1109-rs4505848也被發現與白塞氏病密切相關。此外，該基因多態性亦表現性別差異，較之對照組，顯性基因型頻率在男性NIPU組表現較高(GG/AG vs. AA)。 / 研究 3：CFB，作為CFH的拮抗因子，共同參與補體旁路的調控，其編碼基因被進一步調查。在AU研究中，位於C2/CFB區域的rs1048709被發現與其密切相關，該遺傳敏感性受HLA-B27影響。此外，我們還發現一個單體型 (AATA) 以及CFH與CFB的疊加效應均可導致AU風險度增高。同時，攜帶rs1048709(AG) 基因型患者傾向于更高程度的前房細胞數及KP比例。在NIPU研究中，類似的與CFB基因上的不同易感位點rs4151657相關性亦被檢測到。 / 研究 4, 5, 6: 在隨後的研究中，參與補體通路的其他三個候選基因 (SERPING1，C3和C5) 被進一步評估，儘管多種深入的分析方法被應用，但均未顯示出與疾病明確的相關性。 / 綜上，我們的結果首次揭示補體系統及其分子因素在葡萄膜炎進程中起著至關重要的作用。參與補體旁路調控的細胞因子 CFH 與 CFB，被確認為疾病風險因素。此外，分別參與補體經典通路或下游調控體系的其他三個候選基因 SERPING1(C1INH)，C3 和 C5， 以及參與T細胞反應通路的IL2_21區域和IL27基因在葡萄膜炎的發生發展中所起作用有限。因此，將來對於葡萄膜炎基因學及免疫學的研究應著眼于補體系統及其旁路途徑。 / Uveitis is a group of heterogeneous ocular inflammatory diseases with complex phenotypes, which causes substantial visual impairment and accounts for about 10% of blindness worldwide among the working age group. Despite considerable progress in treatment, safe and effective management is still a clinical challenge. Uveitis can be anatomically classified as anterior, intermediate, posterior, and panuveitis, anterior uveitis (AU) is the most common form. Based on etiology, uveitis can be also categorized as infectious and noninfectious subtypes. Uveitis is generally accepted as an inflammatory condition and regulated by various endogenous immunological mechanisms. Moreover, uveitis can occur in individuals with genetic predisposition coupled with environmental factors. / Based on our understanding of the critical checkpoints in the uveitis process, two major pathways, T-cell response and complement system, appear to be most related to uveitis in the molecular level. We therefore target on several molecular factors involved in the two major pathways to evaluate the genetic impact on susceptibility to uveitis, reveal potential disease mechanisms, and discover diagnostic markers. / We recruited a total of 501 unrelated Chinese individuals at the Hong Kong Eye Hospital and the Prince of Wales Hospital in Hong Kong, including 98 AU patients, 95 patients with noninfectious intermediate and posterior uveitis (NIPU), and 308 control subjects aged ≥ 50 years without major eye diseases or any systemic immune-related disorders. Clinical information and demographic conditions of the patients were documented. We moved on to depict the disease profile and estimate the contribution of each complement activation pathway in uveitis process. We also conducted interaction analysis among the complement factor genes to reveal the putative clinical markers for uveitis. Genotype-phenotype correlations were performed to explore their relationships with the disease severity and progression. / Study 1: In a systemic review to explore recent genetic findings in uveitis susceptibility, we found several genes persistently associated with uveitis and involved in various pathways. They are genes expressing interleukin, chemokine, tumor necrosis factor, and genes involved in complement and oxidation pathways. Genetic polymorphisms were selected based on the immunological and inflammatory properties of uveitis. (1) Interleukin and CFH genes, involving in the T-cell response and complement system respectively. (2) CFB, as a competitor of CFH, involved in the alternative pathway of complement cascade together. (3) Investigation of C1INH (SERPING1) in uveitis, with a view to elucidating the involvement of the classic pathway of complement cascade in uveitis development. (4) Evaluation of C3 and C5 genes in uveitis, due to their respective role of center component and downstream factor in the complement cascade. / Study 2: Genetic variations in the CFH, KIAA1109 and IL27 genes were examined. Our results showed an association between AU and CFH polymorphisms (rs800292 and rs1065489). The frequency of the CFH-rs800292 184G allele and GG homozygosity was higher in female patients than in controls. CFH-rs1065489 TT genotype was identified as a clinical marker associated with higher uveitis recurrence frequency. Interactions with HLA-B27 status in AU patients and different gender susceptibility were observed. In the second part of this study, these three candidate genes were examined in the other uveitic entity, NIPU, in our study cohort. CFH gene polymorphisms (rs1065489 and rs800292) were associated with NIPU patients. Specific association between KIAA1109-rs4505848 polymorphism and Behçet’s disease was identified. There was also gender specific genetic difference. The dominant genotype of KIAA1109-rs4505848 in male NIPU patients was significantly more frequent than in male controls (GG/AG vs. AA). / Study 3: CFB, a competitor of CFH and participated in the same complement alternative pathway, was investigated. SNP rs1048709 in the C2/CFB region was associated with AU, and this genetic influence was affected by HLA-B27 status. Furthermore, one haplotype block across CFB (AATA) significantly predisposed AU with an increased risk of 1.97 (95% CI: 1.41-2.76; Pcorr=0.0005). Joint effects of CFB-rs1048709 with (CFH-rs800292 and CFH-rs1065489) were identified to be at a risk of 7.48 and 7.0 respectively. In addition, patients carrying rs1048709 (AG) were predicted to develop a higher degree of anterior chamber cells and higher proportion of keratic precipitate (KP) during AU course. For NIPU, association with CFB was detected for a different SNP, rs4151657, in female patients only. / Studies 4, 5, and 6: Three candidate genes (SERPING1, C3 and C5) across the complement cascade were orderly evaluated in the whole study cohort of AU, NIPU and controls. They did not show any significant associations with both two uveitis entities, although multiple in-depth analyses have been performed. / Collectively, our results provide evidence for the involvement of the complement system in the disease pathogenesis of uveitis. CFH and CFB, involved in the complement alternative pathway, are identified as genetic risk factors for uveitis. Other complement pathway genes, SERPING1 (C1INH), C3 and C5, as well as IL2_21 region and IL27 involving in the T-cell response, confer either no or limited risk for the development of uveitis. Future genetic and immunologic investigations in uveitis should therefore be focused on the complement system and the alternative pathway. / 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. / Yang, Mingming. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references (leaves 148-163). / Abstracts also in Chinese. / Title page --- p.i / Abstract --- p.iii / 摘要 --- p.vii / Acknowledgement --- p.x / Table of Contents --- p.xi / List of Tables --- p.xvi / List of Figures --- p.xx / Abbreviations --- p.xxi / Publications and Conference Presentation --- p.xxiv / Awards Received --- p.xxvii / Chapter Chapter 1 --- General Introduction --- p.1 / Chapter 1.1 --- Uveitis-one of the most challenging dilemmas in ophthalmology --- p.1 / Chapter 1.1.1 --- Classification of uveitis --- p.4 / Chapter 1.1.2 --- Clinical characteristics of uveitis --- p.5 / Chapter 1.1.3 --- Epidemiology of uveitis --- p.6 / Chapter 1.1.4 --- Etiology of uveitis --- p.9 / Chapter 1.1.5 --- Current management of uveitis and future perspectives --- p.11 / Chapter 1.2 --- The Descriptive Complexity of Uveitis --- p.13 / Chapter 1.3 --- Uveitis Genetics Research Strategies --- p.14 / Chapter 1.3.1 --- Candidate gene association study --- p.14 / Chapter 1.3.2 --- The identification of new genes --- p.15 / Chapter 1.4 --- Statistical Genetics for Uveitis --- p.16 / Chapter 1.4.1 --- Hardy-Weinberg equilibrium test --- p.16 / Chapter 1.4.2 --- Univariate analysis --- p.16 / Chapter 1.4.3 --- Linkage disequilibrium --- p.17 / Chapter 1.4.4 --- Haplotype analysis --- p.18 / Chapter 1.4.5 --- Multivariable analysis --- p.19 / Chapter Chapter 2 --- Objectives --- p.21 / Chapter Chapter 3 --- General Materials and Methods --- p.23 / Chapter 3.1 --- Overall Study Design --- p.23 / Chapter 3.2 --- Research Ethics --- p.23 / Chapter 3.3 --- Study Subjects Recruitment --- p.23 / Chapter 3.4 --- Demographic and Clinical Characteristics of Patients --- p.24 / Chapter 3.4.1 --- Anterior uveitis (AU) --- p.24 / Chapter 3.4.2 --- Non-infectious intermediate and posterior uveitis (NIPU) --- p.25 / Chapter 3.5 --- General Methods --- p.26 / Chapter 3.5.1 --- Total genomic DNA extraction in study subjects --- p.26 / Chapter 3.5.2 --- Taqman SNP genotyping --- p.27 / Chapter 3.5.3 --- Nested polymerase chain reaction (nPCR) --- p.27 / Chapter 3.6 --- Statistical Analysis --- p.28 / Chapter 3.6.1 --- Hardy-Weinberg equilibrium test --- p.28 / Chapter 3.6.2 --- Individual SNP association analysis --- p.28 / Chapter 3.6.3 --- Pairwise linkage disequilibrium and haplotype analysis --- p.30 / Chapter 3.6.4 --- Genotype-phenotype correlation analysis --- p.31 / Chapter 3.6.5 --- Gene-gene interaction analysis --- p.31 / Chapter Chapter 4 --- Investigation into Genetic Determinants of Uveitis --- p.32 / Chapter 4.1 --- A Critical Review on The Roles of Genetic Factors in Uveitis --- p.32 / Chapter 4.1.1 --- Human leukocyte antigens (HLA) --- p.33 / Chapter 4.1.2 --- Interleukin (IL) genes --- p.36 / Chapter 4.1.3 --- Chemokine and chemokine receptor genes --- p.37 / Chapter 4.1.4 --- Tumor necrosis factor (TNF) genes --- p.39 / Chapter 4.1.5 --- Other genes implicated in susceptibility to uveitis --- p.40 / Chapter 4.1.6 --- Complement system --- p.42 / Chapter 4.1.7 --- Conclusions and directions --- p.43 / Chapter 4.2 --- Interleukin and CFH Polymorphisms in Uveitis --- p.49 / Chapter 4.2.1 --- Introduction --- p.49 / Chapter 4.2.2 --- Study subjects --- p.50 / Chapter 4.2.3 --- SNP selection and genotyping --- p.50 / Chapter 4.2.4 --- Statistical analysis --- p.51 / Chapter 4.2.5 --- Association of interleukin and CFH polymorphisms with AU --- p.51 / Chapter 4.2.5.1 --- Association between SNPs and AU --- p.51 / Chapter 4.2.5.2 --- Association between SNPs and AU stratified by gender --- p.52 / Chapter 4.2.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.52 / Chapter 4.2.5.4 --- Genotype-phenotype correlation analysis --- p.53 / Chapter 4.2.6 --- Association of interleukin and CFH polymorphisms with NIPU --- p.53 / Chapter 4.2.6.1 --- Association between SNPs and NIPU --- p.53 / Chapter 4.2.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.54 / Chapter 4.2.6.3 --- Association between SNPs and NIPU stratified by gender --- p.54 / Chapter 4.2.7 --- Discussion --- p.55 / Chapter 4.2.7.1 --- Association of interleukin and CFH polymorphisms with AU --- p.55 / Chapter 4.2.7.2 --- Association of interleukin and CFH polymorphisms with NIPU --- p.58 / Chapter 4.3 --- C2/CFB Polymorphisms in Uveitis --- p.70 / Chapter 4.3.1 --- Introduction --- p.70 / Chapter 4.3.2 --- Study subjects --- p.70 / Chapter 4.3.3 --- SNP selection and genotyping --- p.70 / Chapter 4.3.4 --- Statistical analysis --- p.71 / Chapter 4.3.5 --- Association of C2/CFB polymorphisms with AU --- p.71 / Chapter 4.3.5.1 --- Association between SNPs and AU --- p.71 / Chapter 4.3.5.2 --- Association between SNPs and AU stratified by HLA-B27 status --- p.72 / Chapter 4.3.5.3 --- Linkage disequilibrium and haplotype association analysis --- p.73 / Chapter 4.3.5.4 --- Genotype-phenotype correlation analysis --- p.73 / Chapter 4.3.5.5 --- Joint-effect analysis between CFH and CFB in AU --- p.73 / Chapter 4.3.6 --- Association of C2/CFB polymorphisms with NIPU --- p.74 / Chapter 4.3.6.1 --- Association between SNPs and NIPU --- p.74 / Chapter 4.3.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.74 / Chapter 4.3.6.3 --- Association between SNPs and NIPU stratified by gender --- p.75 / Chapter 4.3.6.4 --- Linkage disequilibrium and haplotype association analysis --- p.75 / Chapter 4.3.7 --- Discussion --- p.75 / Chapter 4.3.7.1 --- Association of C2/CFB polymorphisms with AU --- p.75 / Chapter 4.3.7.2 --- Association of C2/CFB polymorphisms with NIPU --- p.77 / Chapter 4.4 --- SERPING1 Gene Polymorphisms in Uveitis --- p.94 / Chapter 4.4.1 --- Introduction --- p.94 / Chapter 4.4.2 --- Study subjects --- p.94 / Chapter 4.4.3 --- SNP selection and genotyping --- p.95 / Chapter 4.4.4 --- Statistical analysis --- p.95 / Chapter 4.4.5 --- Association of SERPING1 polymorphisms with AU --- p.95 / Chapter 4.4.5.1 --- Association between SNPs and AU --- p.95 / Chapter 4.4.5.2 --- Association between SNPs and AU stratified by gender --- p.96 / Chapter 4.4.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.96 / Chapter 4.4.5.4 --- Association between SNPs and AU stratified by clinical features --- p.96 / Chapter 4.4.6 --- Association of SERPING1 polymorphisms with NIPU --- p.96 / Chapter 4.4.6.1 --- Association between SNPs and NIPU --- p.97 / Chapter 4.4.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.97 / Chapter 4.4.6.3 --- Association between SNPs and NIPU stratified by gender --- p.97 / Chapter 4.4.7 --- Discussion --- p.98 / Chapter 4.5 --- C3 Gene Polymorphisms in Uveitis --- p.109 / Chapter 4.5.1 --- Introduction --- p.109 / Chapter 4.5.2 --- Study subjects --- p.109 / Chapter 4.5.3 --- SNP selection and genotyping --- p.110 / Chapter 4.5.4 --- Statistical analysis --- p.110 / Chapter 4.5.5 --- Association of C3 polymorphisms with AU --- p.110 / Chapter 4.5.5.1 --- Association between SNPs and AU --- p.110 / Chapter 4.5.5.2 --- Association between SNPs and AU stratified by gender --- p.111 / Chapter 4.5.5.3 --- Association between SNPs and AU stratified by HLA-B27 status --- p.111 / Chapter 4.5.5.4 --- Association between SNPs and AU stratified by clinical features --- p.111 / Chapter 4.5.6 --- Association of C3 polymorphisms with NIPU --- p.111 / Chapter 4.5.6.1 --- Association between SNPs and NIPU --- p.111 / Chapter 4.5.6.2 --- Association between SNPs and NIPU stratified by subtypes and gender --- p.112 / Chapter 4.5.7 --- Discussion --- p.112 / Chapter 4.6 --- C5 Gene Polymorphisms in Uveitis --- p.126 / Chapter 4.6.1 --- Introduction --- p.126 / Chapter 4.6.2 --- Study subjects --- p.126 / Chapter 4.6.3 --- SNP selection and genotyping --- p.127 / Chapter 4.6.4 --- Statistical analysis --- p.127 / Chapter 4.6.5 --- Association of C5 polymorphisms with AU --- p.127 / Chapter 4.6.5.1 --- Association between SNPs and AU --- p.127 / Chapter 4.6.5.2 --- Association between SNPs and AU stratified by gender --- p.128 / Chapter 4.6.5.3 --- Association between SNPs and AU stratified by HLA-B27 status and clinical features --- p.128 / Chapter 4.6.6 --- Association of C5 polymorphisms with NIPU --- p.128 / Chapter 4.6.6.1 --- Association between SNPs and NIPU --- p.128 / Chapter 4.6.6.2 --- Association between SNPs and NIPU stratified by subtypes --- p.129 / Chapter 4.6.6.3 --- Association between SNPs and NIPU stratified by gender --- p.129 / Chapter 4.6.7 --- Discussion --- p.129 / Chapter Chapter 5 --- Conclusions and Future Perspectives --- p.143 / Chapter 5.1 --- General Conclusion --- p.143 / Chapter 5.2 --- Future Research in Uveitis Molecular Genetics --- p.144 / REFERENCES --- p.148

No description available.

Mice bearing the mutant gene Cat:Fr have dominantly inherited congenital cataracts, which are more extensive in the mutant homozygote than in the heterozygote. The biochemical and ultrastructural properties of these lenses were examined and compared with those of lenses in which cataracts were induced with acetaminophen and bleomycin. The Cat mutation induces a dominant, by inheritance, loss of beta-H crystallins, but this change is also seen in the bleomycin cataracts, and in the presence of 1 M sucrose, or at 4(DEGREES)C. There are codominantly inherited alterations in the relative proportions of crystallin and albuminoid components in the inherited cataracts, and in the presence of 1 M sucrose. Changes in amino acid composition of the lens proteins are dominantly inherited in the Cat mutation. There are also abnormalities of amino acid composition in the proteins from the acetaminophen cataracts, but these are different from those caused by the mutation. As to the ultrastructural changes, the inherited cataracts have a relatively normal anterior epithelium, but show marked degeneration of nuclear and deep cortical fibres. The bleomycin cataracts also show extensive nuclear destruction, but in addition, appear to be completely devoid of capsule, and have degenerating anterior epithelium cells, which are not seen in the inherited cataract. The acetaminophen cataracts, by contrast, retain normal overall structural architecture, but the individual fibres become swollen and flaky, and develop an increased number of interdigitating processes. These abnormal biochemical properties not unique to the Cat:Fr mouse are unlikely to be proximal effects of the mutant gene, and may be general consequences of the presence of a cataract, from whatever cause. The differences between ultrastructural abnormalities seen in the drug induced and inherited cataracts suggest that these etiological agents induce cataracts by different mechanisms, a fact that is not always apparent from

As exogenously administered RA suppressed the expression of the RA synthesizing enzymes, further investigation on whether this would lead to deficiency in endogenous RA concentrations was conducted. Results showed that exogenously administered RA significantly reduced the endogenous RA level in the head region with C57 embryos showing a greater reduction than ICR embryos. / In addition, detailed morphological and histological studies were conducted to determine if RA treatment caused early embryonic changes with strain difference. When compared with ICR embryos, C57 embryos exhibited more pronounced responses to RA, including developmental retardation, underdevelopment of the anterior neural plate and absence of or smaller optic pit/optic vesicle formation. However, RA treatment did not cause abnormal apoptosis in the early stages in both strains. / Since the teratogenic effect of RA is highly developmental stage-dependent, it is possible that there is a difference in the developmental stage between these 2 mouse strains at the time of RA injection. Indeed, it was found that the developmental stage of ICR embryos was approximately 6 hours ahead of C57 embryos. However, the role that this factor plays in the differential strain susceptibility to RA can be excluded since C57 fetuses were still 3 times more susceptible to developing anophthalmia/microphthalmia than ICR fetuses that were subject to RA treatment at equivalent developmental stages. Comparison of susceptibility to RA-induced anophthalmia/microphthalmia was also made among heterozygous fetuses obtained from reciprocal matings between C57 and ICR male and female mice, and those in homozygous ICR and C57 fetuses. Results showed that the C57 strain has conferred both genetic predisposition and maternal effects in increasing the embryo's susceptibility to RA-induced ocular defects. / Since the type of RA-induced ocular defects mimic those that developed in Raldh2 null mutant embryos, the effect of RA treatment on the expression of RA synthesizing enzymes, Raldh2 and Raldh3, and the RA-inducible gene Cyp26a1, as well as some early eye development genes were examined. Exogenously administered RA reduced the mRNA expression levels of Raldh2, Raldh3 and Cyp26a1 in the head region, with C57 embryos showing a greater reduction than ICR embryos. / Taken together, results of this thesis suggest that there is a strain difference in susceptibility to RA-induced ocular defects in which exogenously applied RA suppresses the expression of RA synthesizing enzymes and leads to endogenous RA deficiency. This finding may shed light on understanding why both excess and deficiency of RA can lead to similar types of ocular defects. / To determine if there are strain differences in the susceptibility to RA-induced ocular defects, two mouse strains were used. They are C57BL/6J (C57), mice that spontaneously develop ocular defects and ICR mice, which are not prone to developing ocular defects. Detailed time and dose response studies were conducted and eye defects were examined in near-term fetuses. C57 fetuses were found to be significantly more susceptible to RA-induced anophthalmia/microphthalmia than ICR fetuses. / Vitamin A (retinol) and its most active metabolite, all- trans retinoic acid (RA) is essential for vision in the adult and for eye development in the embryo. It is well documented that in humans, excess intake or deficiency of vitamin A or RA is associated with congenital ocular defects such as microphthalmia. However, the underlying mechanism remains unclear. The aim of this study is to examine the pathogenic mechanism of RA-induced developmental ocular defects. / Lau, Wing Sze Josephine. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0240. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 186-211). / 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 Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

The inherited form of primary open angle glaucoma, a disorder characterized by increased intraocular pressure and retina degeneration, is linked to mutations in the olfactomedin (OLF) domain of the myocilin gene. Disease-causing myocilin variants accumulate within trabecular meshwork cells instead of being secreted to the trabecular extracellular matrix thought to regulate aqueous humor flow and control intraocular pressure. Like other diseases of protein misfolding, we hypothesize myocilin toxicity originates from defects in protein biophysical properties. In this thesis, the first preparative recombinant high-yield expression and purification system for the C-terminal OLF domain of myocilin (myoc-OLF) is described. To determine the relative stability of wild-type (WT) and mutant OLF domains, a fluorescence thermal stability assay was adapted to provide the first direct evidence that mutated OLF is folded but less thermally stable than WT. In addition, mutant myocilin can be stabilized by chemical chaperones. Together, this work provides the first quantitative demonstration of compromised stability among identified OLF variants and placing myocilin glaucoma in the context of other complex diseases of protein misfolding. Subsequent investigations into the biophysical properties of WT myoc-OLF provide insight into its structure and function. In particular, myoc-OLF is stable in the presence of glycosaminoglycans (GAGs), as well as over a wide pH range in buffers with functional groups reminiscent of such GAGs. Myoc-OLF contains significant â-sheet and â-turn secondary structure as revealed by circular dichroism analysis. At neutral pH, thermal melts indicate a highly cooperative transition with a melting temperature of ~55°C. A compact core structural domain of OLF was identified by limited proteolysis and consists of approximately residues 238-461, which retains the single disulfide bond and is as stable as the full myoc-OLF construct. This construct also is capable of generating 3D crystals for structure determination. This data, presented in Chapter 3, inform new testable hypotheses for interactions with specific trabecular extracellular matrix components. To gain further insight into the biological function of myoc-OLF, a facile fluorescence chemical stability assay was designed to identify possible ligands and drug candidates. In the assay described in Chapter 4, the target protein is initially destabilized with a chemical denaturant and is tested for re-stabilization upon the addition of small molecules. The assay requires no prior knowledge of the structure and/or function of the target protein, and it is amendable to high-throughput screening. Application of the assay using a library of 1,280 compounds revealed 14 possible ligands and drug candidates for myoc-OLF that may also generate insights into myoc-OLF function. Due to the high â-sheet content of monomeric myoc-OLF and presence of an aggregated species upon myoc-OLF purification, the ability of myoc-OLF to form amyloid fibrils was suspected and verified. The fibril forming region was confirmed to reside in the OLF domain of myocilin. Kinetic analyses of fibril formation reveal a self-propagating process common to amyloid. The presence of an aggregated species was confirmed in cells transfected with WT myocilin, but to a greater extent in cells transfected with P370L mutant myocilin. Both cell lines stained positive for amyloid. Taken together, these results provide further insights into the structure of myocilin and suggest a new hypothesis for glaucoma pathogenesis. Finally, in a related study, small molecule drug candidates were investigated to treat acid â-glucosidase (GCase), the deficient lysosomal enzyme in Gaucher disease, another protein conformational disorder. Three new GCase active-site directed 3,4,5,6-tetrahydroxylazepane inhibitors were synthesized that exhibit half inhibitory concentrations (IC50) in the low millimolar to low micromolar range. Although the compounds thermally stabilize GCase at pH 7.4, only one of the synthesized analogs exhibits chaperoning activity under typical assay conditions. This successful pharmacological chaperone is also one in which GCase is in its proposed active conformation as revealed by X-ray crystallography. Probing the plasticity of the active-site of GCase offers additional insight into possible molecular determinants for an effective small molecule therapy for GD.

Indiana University-Purdue University Indianapolis (IUPUI) / Ciliopathies with multiorgan pathology include renal cysts and eye pathology. Previous studies showed meckelin (MKS3 protein product) are crucial to cilia function and its absence in Wpk rats (with mutated rMks3 gene) causes Leber's congenital amaurosis. Retinal photoreceptors have connecting cilium that joins the inner to the outer segment and plays a role in the transport of molecules necessary for morphological and molecular development and maintenance of the outer segment process. The present study evaluated meckelin expression during normal postnatal retinal development and the consequences of mutant meckelin on photoreceptor development and survival in Wistar-Wpk/Wpk rat. Meckelin was co-expressed in photoreceptors, amacrine, Muller glia and ganglion cells in postnatal day 10 (P10) and P21 wild type retinae. Meckelin was detected in both inner and outer segments of photoreceptors. By P10, both wild type and homozygous Wpk mutant retina had all retinal cell types. In contrast, by P21, cells expressing photoreceptor-specific markers in the Wpk mutant were fewer in number with abnormal expression patterns. Cell death assays confirmed a significant amount of cells undergoing apoptosis in the outer nuclear layer of the mutant rat retina. By electron microscopy, mutant photoreceptors did not develop an outer segment process beyond a connecting cilium and rudimentary outer segment. We conclude that MKS3 is not important for formation of connecting cilium and rudimentary outer segments, but is critical for the elongation and/or maintenance of mature outer segment processes.