Imaging cholesterol metabolism and trafficking by stimulated Raman scattering microscopy

Cholesterol is an essential component of mammalian cells which is tightly regulated. However, our understanding of cholesterol transport and metabolism is still incomplete, partly due to lack of suitable tools for studying cholesterol dynamics in living cells and organisms with spatio-temporal information. My dissertation work applied spectroscopic imaging of cholesterol in human tissues, living cells, and model organisms to unravel new insights of cholesterol metabolism and trafficking. Using stimulated Raman spectroscopic analysis of lipid droplets in human prostate cancer patient tissues, we observed an aberrant accumulation of cholesteryl ester in metastatic lesions. Inhibition of cholesterol esterification in prostate cancer cells significantly suppresses the development and growth of metastatic cancer lesions in both orthotopic and intra-cardiac injection mouse models. Gene expression profiling shows that cholesteryl ester depletion suppresses the metastatic potential through upregulation of multiple regulators that negatively impact metastasis. Additionally, Wnt/β-catenin, one of vital pathways for metastasis, is downregulated upon cholesteryl ester depletion. Mechanistically, we found evidence suggesting that inhibition of cholesterol esterification significantly blocks secretion of Wnt3a through reduction of monounsaturated fatty acid levels, which limits Wnt3a acylation. These results collectively validate cholesterol esterification as a novel metabolic target for treating metastatic prostate cancer. My thesis work also developed a new biocompatible cholesterol analog, which enabled real-time imaging of cholesterol metabolism and trafficking in living cells and organisms. Based on quantum chemistry calculations, we designed and synthesized phenyl-diyne cholesterol (PhDY-Chol), which has an extremely large Raman scattering cross section. The phenyl-diyne group is biologically inert and provides a Raman scattering cross section that is 88 times larger than the endogenous C=O stretching mode. Stimulated Raman scattering microscopy offers an imaging speed that is faster than spontaneous Raman microscopy by three orders of magnitude, and a detection sensitivity of 31 μM PhDY-Chol (~1,800 molecules in the excitation volume). Inside living cells, PhDY-Chol mimics the behavior of cholesterol, including membrane incorporation and esterification. In a cellular model of Niemann-Pick type C disease, PhDY-Chol reflects the lysosomal accumulation of cholesterol, and shows relocation to lipid droplets after HPβCD treatment. In living C. elegans, PhDY-Chol mimics cholesterol uptake by intestinal cells and reflects cholesterol storage. Together, this work demonstrates an enabling platform for study of cholesterol trafficking in living cells and organisms

Pulsed Laser Deposition of Rocksalt Magnetic Binary Oxides

Here we systematically explore the use of pulsed laser deposition technique (PLD) to grow three basic oxides that have rocksalt structure but different chemical stability in the ambient atmosphere: NiO (stable), MnO (metastable) and EuO (unstable). By tuning laser fluence, an epitaxial single-phase nickel oxide thin-film growth can be achieved in a wide range of temperatures from 10 to 750 {\deg}C. At the lowest growth temperature, the out-of-plane strain raises to 1.5%, which is five times bigger than that in a NiO film grown at 750 {\deg}C. MnO thin films that had long-range ordered were successfully deposited on the MgO substrates after appropriate tuning of deposition parameters. The growth of MnO phase was strongly influenced by substrate temperature and laser fluence. EuO films with satisfactory quality were deposited by PLD after oxygen availability had been minimized. Synthesis of EuO thin films at rather low growth temperature prevented thermally-driven lattice relaxation and allowed growth of strained films. Overall, PLD was a quick and reliable method to grow binary oxides with rocksalt structure in high quality that can satisfy requirements for applications and for basic research

Applications of pre-open sets

[EN] Using the concept of pre-open set, we introduce and study topological properties of pre-limit points, pre-derived sets, preinterior and pre-closure of a set, pre-interior points, pre-border, prefrontier and pre-exterior. The relations between pre-derived set (resp. pre-limit point, pre-interior (point), pre-border, pre-frontier, and preexterior) and α-derived set (resp. α-limit point, α-interior (point), α-border, α-frontier, and α-exterior) are investigatedJun, YB.; Jeong, SW.; Lee, HJ.; Lee, JW. (2008). Applications of pre-open sets. Applied General Topology. 9(2):213-228. https://doi.org/10.4995/agt.2008.18022132289

Affective Role of the Future Autonomous Vehicle Interior

Recent advancements in autonomous technology allow for new opportunities in vehicle interior design. Such a shift in in-vehicle activity suggests vehicle interior spaces should provide an adequate manner by considering users' affective desires. Therefore, this study aims to investigate the affective role of future vehicle interiors. Thirty one participants in ten focus groups were interviewed about challenges they face regarding their current vehicle interior and expectations they have for future vehicles. Results from content analyses revealed the affective role of future vehicle interiors. Advanced exclusiveness and advanced convenience were two primary aspects identified. The identified affective roles of each aspect are a total of eight visceral levels, four visceral levels each, including focused, stimulating, amused, pleasant, safe, comfortable, accommodated, and organized. We expect the results from this study to lead to the development of affective vehicle interiors by providing the fundamental knowledge for developing conceptual direction and evaluating its impact on user experiences.Comment: 15 pages, 4 figures, 2 table

Link between allosteric signal transduction and functional dynamics in a multi-subunit enzyme: S-adenosylhomocysteine hydrolase

S-adenosylhomocysteine hydrolase (SAHH), a cellular enzyme that plays a key role in methylation reactions including those required for maturation of viral mRNA, is an important drug target in the discovery of antiviral agents. While targeting the active site is a straightforward strategy of enzyme inhibition, evidences of allosteric modulation of active site in many enzymes underscore the molecular origin of signal transduction. Information of co-evolving sequences in SAHH family and the key residues for functional dynamics that can be identified using native topology of the enzyme provide glimpses into how the allosteric signaling network, dispersed over the molecular structure, coordinates intra- and inter-subunit conformational dynamics. To study the link between the allosteric communication and functional dynamics of SAHHs, we performed Brownian dynamics simulations by building a coarse-grained model based on the holo and ligand-bound structures. The simulations of ligand-induced transition revealed that the signal of intra-subunit closure dynamics is transmitted to form inter-subunit contacts, which in turn invoke a precise alignment of active site, followed by the dimer-dimer rotation that compacts the whole tetrameric structure. Further analyses of SAHH dynamics associated with ligand binding provided evidence of both induced fit and population shift mechanisms, and also showed that the transition state ensemble is akin to the ligand-bound state. Besides the formation of enzyme-ligand contacts at the active site, the allosteric couplings from the residues distal to the active site is vital to the enzymatic function.Comment: 35 pages, 14 figures, 3 Table