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.

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.