published_or_final_version / Pharmacology and Pharmacy / Doctoral / Doctor of Philosophy

published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

Congenital cataract is a leading cause of visual disability among children worldwide. It has a heterogeneous genetic basis; the cellular and molecular mechanisms for cataractogenesis remain elusive. A spontaneously occurred autosomal dominant mouse mutant named Secc, which displays small eye, cataract and closed eyelid, has been obtained in our laboratory. By gene mapping and DNA sequencing, we identified a single nucleotide deletion at position 273 of the Cryga gene, leading to a frame-shift from the 3rd Greek Key motif of the A-crystallin (Cryga). The aim of this study is to investigate the pathogenic mechanisms underlying the development of cataract in the Secc mutant, as a disease model for understanding human congenital cataract. Initial phenotype analysis showed that cataract was initiated in E14.5 CrygaSecc mutant embryos, the nuclei of the primary lens fibres were scattered and failed to align in the equatorial region. By E16.5, the secondary lens fibre cells were abnormally arranged with poor lens suture formation. Apoptotic cells were found in the centre of the lens as shown by TUNEL assay, cytoskeleton and cell adhesion in the lens centre were disturbed as shown in immunohistochemistry analysis. Previously by western blotting it was found that mutant -crystallins were enriched in the insoluble fraction. I hypothesized that mutant A-crystallins might be misfolded and protein aggregates were then formed. In this study, aggregation was observed in semi-thin sections stained with toluidine blue. By co-staining using custom-made anti-Secc antibody, CrygaSecc protein was found to be ubiquitinated and was wrapped around by vimentin. Clearly, in the Secc mutant lens, aggresomes were formed for the disposal of the misfolded proteins and to maintain cell survival. However, ultimately cell death would occur in the mutant lens and contributed to cataract formation. It is known that misfolded proteins would trigger unfolded protein response (UPR) and heat shock protein (HSP) responses to facilitate folding and to prevent misfolded proteins from intoxicating the cell. In order to determine which stress response pathway was triggered, gene expression analysis by qRT-PCR was performed. The expression of genes involved in the UPR pathways including BiP, CHOP and spliced variant of XBP-1 were all up-regulated significantly in E14.5 and 16.5 mutant lenses. In addition, among different ER stress related genes, cytosolic chaperones and autophagy related genes, Hsp70 and BiP were upregulated, while Hsp40 and Hsp90aa were downregulated in the homozygotes. The results suggested that both UPR and HSP response pathways were triggered during cataractogenesis in the Secc mutant. In conclusion, mutant A-crystallin appeared to trigger UPR, HSPs and cell death in the fibre cells, while autophagy was not triggered. In the lens fibre cells, the ubiquitin-proteasomal pathway was utilized for the removal of misfolded CrygaSecc proteins. However, the stress perpetuated as the lens grew and produced more mutant proteins. The mutant cells lost their normal cell adhesion, failed to maintain the proper lens architecture, leading to cataract formation. Similar cellular mechanisms could be implicated in human congenital cataract or age-related cataract development. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

Background and objectives: Nonalcoholic steatohepatitis (NASH), which is characterized by concurrent existence of hepatic steatosis and predominantly lobular necroinflammation, represents the more advanced stage in the spectrum of nonalcoholic fatty liver disease (NAFLD). NASH exhibits dramatically increased risk of progression to end-stage liver diseases than simple steatosis. Therefore, the progression of hepatic steatosis to steatohepatitis is the crucial step in the development of obesity-related NASH. Toll like receptor 4 (TLR4), a master regulator of innate immunity, is the principal receptor for endotoxin, which is a central mediator of liver inflammation associated with both alcoholic and nonalcoholic liver disease. However, due to a lack of suitable animal models which fully recapitulate the natural history of obesity-induced NASH, the precise pathophysiological function of TLR4 signaling in the development of this disease remains poorly understood. The objective of this study is to investigate the role of TLR4 in mediating inflammatory responses in obesity-induced NASH using both in vivo and ex vivo approaches, and to unveil cellular and molecular mechanisms responsible for TLR4 actions. Key findings: 1. To address the role of TLR4 in the pathogenesis of NASH, we crossed ApoEdeficient mice (ApoE-/-) with TLR4 mutant mice (TLR4-/-) to generate ApoE-/- /TLR4 wild type mice (ApoE-/-/TLR4-WT) and ApoE-/-/TLR4-/- mice. Noticeably, when fed with high fat high cholesterol (HFHC) diet, ApoE-/-/TLR4-WT mice developed the typical pathology of NASH (hepatic steatosis, lobular inflammation, and hepatocyte ballooning) in the context of obesity and metabolic syndrome, suggesting HFHC-fed ApoE-/- mice as a suitable animal model for NASH. 2. TLR4 inactivation protected ApoE-/- mice against HFHC diet-induced liver injury, as indicated by a significant improvement in liver histology, a a marked reduction in serum ALT activity, a dramatic repression of inflammatory infiltrates, as well as an obvious decrease in hepatic production of pro-inflammatory cytokines. 3. In ApoE-/-/TLR4-WT mice, TLR4 expression was selectively elevated in Kupffer cells in response to HFHC diet feeding. 4. The activation of XBP1, a transcription factor involved in endoplasmic reticulum stress, was markedly elevated in liver of ApoE-/-/TLR4-WT mice fed with HFHC diet, whereas this change was abrogated in HFHC diet-fed ApoE-/-/TLR4-/- mice. 5. In rat primary Kupffer cells, treatment with anti-oxidants blocked endotoxininduced activation of XBP1 and NF-κB, leading to decreased cytokine production. In addition, siRNA-mediated knockdown of XBP1 inhibited NF-κB activation and cytokine production resulted from the treatment with the TLR4 agonist LPS. 6. In ApoE-/-/TLR4-WT mice, adenovirus-mediated expression of dominant negative XBP1 had no obvious effect on HFHC diet-induced hepatic steatosis and ROS production, but markedly decreased lobular inflammation, NF-κB activation, cytokine production in the liver and significantly reduced serum levels of ALT. Conclusions: These findings support the role of TLR4 in Kupffer cells as a key player in mediating the progression of simple steatosis to NASH, by inducing ROS-dependent activation of XBP1. In light of the obligatory role of XBP1 in TLR4-induced liver inflammation and injury, therapeutic interventions that inhibit TLR4/XBP1 activation may represent a promising strategy for treatment of NASH. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

Cardiovascular stents are one of the most widely implanted medical devices, with over 1 million implanted each year in the United States alone. While stent failure modes of restenosis and thrombosis have been widely examined, there is an increasing appreciation of the propensity for stents to fracture and break after implantation. It remains unclear however what causes these fractures, which patients and devices are most susceptible, and whether fracture results in failure of device function. / Engineering and Applied Sciences

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