Wang Chi Chiu. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1997. / Includes bibliographical references (p. 294-336). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

acase@tulane.edu / TAp63, a member of the p53 family, has been shown to regulate energy metabolism. Here, we report coiled coil domain-containing 3 (CCDC3) as a new TAp63 target. TAp63, but not ΔNp63, p53 or p73, induces the expression of CCDC3 mRNA level by directly binding to the p63 consensus DNA binding sequence within the CCDC3 enhancer region. The CCDC3 expression is markedly reduced in TAp63-null mouse embryonic fibroblasts and brown adipose tissues and by tumor necrosis factor alpha that reduces p63 transcriptional activity but induced by metformin, an anti-diabetic drug that activates p63. Also, the expression of CCDC3 is positively correlated with TAp63 levels, but inversely with ΔNp63 levels, during adipocyte differentiation. Interestingly, CCDC3, as a secreted protein, targets liver cancer cells and increases long chain polyunsaturated fatty acids, but decreases ceramide in the cells. CCDC3 alleviates glucose intolerance, insulin resistance, and fatty liver (steatosis) formation in transgenic CCDC3 mice on the high-fat diet by markedly reducing hepatic PPARγ expression and consequently leading to a drastic decrease of the PPARγ target gene, CIDEA, and other genes involved in de novo lipogenesis and of lipid droplets formation in their livers. Similar results are reproduced by hepatic expression of ectopic CCDC3 in mice on high-fat diet. Altogether, these results demonstrate that CCDC3 modulates liver lipid metabolism by inhibiting liver de novo lipogenesis as a downstream player of the p63 network. / 1 / Wenjuan Liao

Glycosylation of recombinant proteins can change during the culture of animal cells. Since lipid-linked oligosaccharides (LLOs) are the carbohydrate donors in N-linked glycosylation, their availability is postulated to influence the extent of glycosylation. To test this hypothesis, relative LLO and glycosylation levels in Chinese hamster ovary (CHO) cells were monitored over the course of batch culture for corresponding changes. Radiolabelling studies reveal that throughout the length of culture, intracellular LLO levels remained within a 2-fold range and overall CHO protein glycosylation varied by less than 30%. The implications of these results and hypotheses to explain the findings are discussed. / Singapore-MIT Alliance (SMA)

Purpose: To examine the variables that influence lipid deposition on conventional and silicone hydrogel contact lens materials and to build a physiologically relevant in vitro model of lipid deposition on contact lenses. Methods: Lipid deposition on contact lens materials can lead to discomfort and vision difficulty for lens wearers. Using a variety of radiochemical experiments and two model lipids (cholesterol and phosphatidylcholine), a number of clinically significant parameters that may influence lipid deposition were examined. • The optimization and characterization of a novel artificial tear solution (ATS) was examined (Chapter 3) • Optimization of an extraction system to remove deposited cholesterol and phosphatidylcholine from various contact lens materials (Chapter 4) • The influence of different tear film components on lipid deposition was researched (Chapter 5) • The efficiency of hydrogen peroxide disinfecting solutions to remove deposited lipid from contact lenses was investigated (Chapter 6) • The effect of intermittent air exposure on lipid deposition was examined through the use of a custom built “model blink cell” (Chapter 7) Results: A novel complex ATS designed for in-vial incubations of contact lens materials was developed. This solution was stable and did not adversely affect the physical parameters of the contact lenses incubated within it. An efficient extraction protocol for deposited cholesterol and phosphatidylcholine was optimized based on chloroform and methanol with the addition of water and acetic acid for phosphatidylcholine extraction. Overall, cholesterol and phosphatidylcholine deposition is cumulative over time and found to deposit in greater masses on silicone-containing hydrogels. Cholesterol and phosphatidylcholine deposition is influenced by the composition of the incubation medium and air exposure which occurs during the inter-blink period. Hydrogen peroxide disinfecting solutions were able to remove only marginal amounts of lipid from the contact lenses, with the surfactant containing solution removing more. Conclusion: This thesis has provided hitherto unavailable information on the way in which lipid interacts with conventional and silicone hydrogel contact lens materials and the in vitro model built here can be utilized in various ways in the future to assess other aspects and variables of lipid and protein deposition on a variety of biomaterials.

Bottom rounds were shipped to the Rosenthal Meat Science and Technology Center, ground and enhanced with one of the following predetermined treatments: control; 0.4% sodium phosphates and 0.3% salt; 0.25% sorghum bran; 2.0% sorghum bran; 0.25% sorghum bran, 0.4% sodium phosphates and 0.3% salt; and 2.0% sorghum bran, 0.4% sodium phosphates, and 0.3% salt. The ground beef was formed into 226 g ground beef patties, packaged in an 80% O2 and 20% CO2 gaseous environment, and stored under retail display at 4 degrees for 0, 3, 6, or 9d. Measurements to determine rate and extent of oxidation, rate of discoloration, and sensory characteristics were taken to evaluate the effectiveness of sorghum bran. Patties containing the highest amount of sorghum bran had the lowest TBARS values over 9 days of storage, lower a* values, greater amounts of discoloration, darker lean color, and less cook loss (P<0.05) than control patties. Patties enhanced with the highest level of sorghum bran had lower beefy/brothy and bloody flavor aromatics, higher sorghum flavor, more bitter and burnt aftertaste, and more sandy/gritty textures (P<0.05) when compared to control patties. Patties containing the low amount of sorghum had lower TBARS values (P<0.05), but similar amounts of cook loss as the control patties. Patties containing a low sorghum level, 0.4% sodium phosphates (SP) and 0.3% salt (S) had lower (P<0.05) amounts of cook loss when compared to control patties. Patties containing low amounts of sorghum were similar to control patties in terms of redness while the addition of low sorghum, SP, and S decreased (P<0.05) the degree of redness. Patties containing low amounts of sorghum bran had similar amounts of discoloration compared to control (CONT) patties. Also, these had less bloody flavor aromatics (P<0.05), but were similar in sorghum flavor aromatics and bitter taste when compared to control patties. The addition of sorghum bran at low levels can retard oxidative rancidity in ground beef patties without causing detrimental color changes and negatively affecting sensory attributes, while patties enhanced with 2% sorghum bran have extensive discoloration and undesirable sensory attributes.

The bioorthogonal copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction exhibits complex but well-defined kinetics in aqueous and organic solution for soluble azides, alkynes, and ligand-bound copper(I). The kinetic profile in two dimensions, however, for CuAAC systems within a lipid bilayer membrane, has yet to be defined. The effect of triazole formation with lipid membrane-bound components on membrane properties such as fluidity and permeability is also of interest. Azide- and alkyne-functionalized lysolipids were synthesized and incorporated into non-fluid vesicles, which were then subject to CuAAC. The rate order for membrane-bound lipid substrates in non-fluid vesicles was observed to be comperable to that of the reaction in solution. Reactions between vesicles showed evidence of lipid transfer between non-fluid membranes, which has not been previously reported. For intervesicular and intravesicular reactions in non-fluid membranes, the observed reactivity was found to be opposite that of previously published reactions between nucleophiles and electrophiles in fluid lipid systems. Applications of this work include the potential for novel symmetric membrane leaflet labeling, bioorthogonal manipulation of cell and tissue function, and the creation of membranes with precisely controlled properties that may not be available in naturally-occurring membranes.

Gram-negative bacteria decorate their outermost surface structure, lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from immune surveillance and protects them from the onslaught of host defenses. Many of these changes occur on the lipid A component of lipopolysaccharide, which is crucial for host recognition of Gram-negative infection. Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the TLR4/MD2 receptor of the innate immune system. A less toxic mixture of mono-phosphorylated lipid A species (MPL) recently became the first new FDA-approved adjuvant in over 70 years. Whereas wild-type E. coli LPS provokes strong inflammatory MyD88-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers indicating the efficacy of these molecules as adjuvants. Other types of modification to the lipid A domain, such as altering the length of the fatty acyl chains that anchor LPS to the cell membrane, were found to affect bacterial fitness but not drastically influence detection by the TLR4/MD2 receptor. Overall, this combinatorial approach demonstrates how engineering lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development as well as for a deeper understanding of bacterial membrane biogenesis. / text

Glaucoma is the leading cause of irreversible blindness worldwide with the most prevalent form, primary open-angle glaucoma (POAG), accounting for the vast majority of glaucoma cases. The main risk-factor for POAG is an elevated intraocular pressure (IOP), and is due to an increased resistance to aqueous humor outflow in the conventional outflow pathway at the juxtacanalicular region of the trabecular meshwork (TM) and the inner wall of Schlemm’s canal (SC). Reducing elevated IOP is the most effective method to prevent further loss of vision in glaucoma; therefore, it is important to understand how outflow resistance is regulated in the conventional outflow pathway in order to find effective methods to reduce ocular hypertension. Sphingosine-1-phosphate (S1P) is an endogenous lipid that reduces outflow facility in porcine eyes, thereby increasing resistance. S1P plays a major role in affecting cell migration, endothelial permeability, and junctional formation, processes that are intimately linked and regulated by cytoskeletal dynamics. Due to S1P’s known effect of decreasing endothelial permeability in vascular endothelial cells, the overall hypothesis of this dissertation is that the S1P-induced decrease in outflow facility occurs through a mechanism that involves S1P receptor activation in SC cells. The results from the studies within this dissertation demonstrate the expression of the S1P₁₋₃ receptor subtypes in SC and TM cells and a decrease of outflow facility by S1P in perfused human eyes. Additionally, S1P promotes F-actin formation and myosin light chain (MLC) phosphorylation at the SC cell cortex. The S1P-promoted MLC phosphorylation in both SC and TM cells, in addition to the S1P-induced decrease of outflow facility in porcine and human eyes, were blocked by the S1P₂ antagonist JTE-013. Results from these studies demonstrate S1P to actively regulate actomyosin dynamics in the cells of the outflow pathway through the S1P₂ receptor. S1P₂ also mediates the S1P-induced increase in outflow resistance. Therefore, S1P₂ is a novel pharmacological target in the conventional outflow pathway to reduce elevated IOP in glaucoma patients.

Physical stress such as the surface roughness of the implants may activate the NFkB signalling pathway in macrophages. This activation is intimately related to the mechanism(s) by which the macrophage interacts with the surface through serum proteins and/or the formation of membrane rafts. This thesis examines the role of surface topography on activation of the NFkB signalling pathway in macrophages. We examined the effect of implant surface topography on activating the NFkB signalling pathway in the RAW 264.7 macrophage cell line. We also examined the effect surface roughness had on the adhesion of the macrophages using the different media. To finish, we observed the effect the different media and the surface roughness had on the morphology of the macrophages by Scanning Electron Microscopy. Activation of the NFkB pathway was surface topography dependent. The Smooth surface showed the highest level of activation followed by the Etched then the SLA. Addition of suboptimal concentrations of LPS mildly enhanced the response by signalling through the Toll receptor. Activation of NFKB occurred in the absence of fetal calf sera, although to a lesser extent. All three surfaces had very few cells with nuclear translocation at the 5 minutes time point with no significant statistical differences between the surfaces. After 30 minutes, translocation reached comparable levels to those surfaces tested with complete medium. Disruption of the lipid rafts affected the triggering and signalling of the NFkB pathway. This inhibitory effect was concentration and time dependent. Smooth surfaces bound more macrophages in the 30 minutes assay. Fetal calf serum appeared to be very critical for adhesion and spreading of the macrophages on the various surfaces examined. Removal of cholesterol did not affect adhesion or spreading on their respective surfaces. We have clearly demonstrated that the lipid rafts along with surface topography play a role in the activation on NFKB. This in-vitro study has demonstrated that surface topography modulated activation of the NFKB signalling pathway in a time-dependent manner. However, at present, it is unclear through which receptor(s) / surface structure the signal pathway is initiated.

The Saccharomyces cerevisiae oxysterol binding protein homologue Kes1/Osh4 is a member of an enigmatic class of proteins found throughout Eukarya. This family of proteins is united by a ?-barrel structure that binds sterols and oxysterols. An N-terminal lid is thought to both sequester sterols inside the core and promote localization of Kes1 to regions of high membrane curvature via a predicted ArfGAP lipid packing sensor motif. Additionally, a phosphoinositide-binding region on a discrete surface of Kes1 has also been identified. In this thesis, structure-function analysis of Kes1 determined that phosphoinositide binding is required for membrane association in vitro, and in vivo phosphoinositide binding is required for localization to the Golgi. Ergosterol, the major sterol in S. cerevisiae, and membrane curvature had minimal effects on membrane association. This study also revealed a role for Kes1 in the regulation of both phosphatidylinositol-4-phosphate and phosphatidylinositol-3-phosphate homeostasis. Phosphoinositide and sterol binding by Kes1 are necessary for it to alter phosphatidylinositol-4-phosphate, but not phosphatidylinositol-3-phosphate homeostasis. Misregulation of phosphatidylinositol-4-phosphate homeostasis by Kes1 manifested itself in an inability of the v-SNARE Snc1 to traffic properly and was consistent with a defect in trans-Golgi/endosome trafficking. I went on to demonstrate a role for Kes1 in regulating the conversion of phosphatidylinositol-4-phosphate to phosphatidylinositol for the synthesis of sphingolipids, and I present a model for the role of Kes1 at the Golgi. Kes1 acts as a sterol sensor that regulates phosphatidylinositol-4-phosphate to sphingolipids metabolism, which ultimately regulates the delivery of proteins that assemble into lipid rafts for their transport from the Golgi to the plasma membrane. I also uncovered a previously unknown role for Kes1 in the regulation of the cytoplasm-to-vacuole and autophagy trafficking pathways, which is mediated by the ability of Kes1 to regulate phosphatidylinositol-3-phosphate homeostasis.