The glycoprotein CD36 is expressed in the plasma membrane (PM) of many cell types that surround or contact arteries, including macrophages, myocytes, and endothelial cells. CD36 binds oxidized low density lipoprotein (oxLDL), which promotes atherosclerosis, and fatty acids (FA), which promotes their cellular uptake. To gain insights into the molecular mechanisms of uptake, HEK293 cells expressing CD36 were studied by cell biological and fluorescence methods. To test our hypothesis that the PM is not an impermeable barrier to FA and that FA move into cells by diffusion via their uncharged form, we first applied biophysical fluorescence spectroscopy to directly measure transmembrane FA movement and membrane fluidity. Expression of CD36 in HEK293 cells did not increase either transport across the PM or the fluidity of the PM compared to HEK293 cells without CD36; however, CD36 enhanced intracellular FA esterification. Furthermore, the widely used “inhibitors” of FA transport did not alter either the rapid FA transmembrane diffusion in HEK293 cells or diffusion in control experiments with protein-free phospholipid bilayers. To gain new insights into the physiological relevance of FA binding to CD36, we applied surface plasmon resonance (SPR) to quantify FA and oxLDL binding to the ectodomain of CD36. Structurally distinct FA [saturated, monounsaturated (cis and trans), polyunsaturated, ω-3, ω-6, and oxidized FA] were pulsed in a solubilized form (bound to methyl-β-cyclodextrin) across SPR channels, generating real-time association and dissociation binding curves. With the exception of the oxidized FA hydroxyoctadecadienoic acid (HODE), all FA tested bound to CD36 with rapid association and dissociation kinetics similar to human serum albumin. In addition, FA increased oxLDL binding to CD36. To investigate whether FA affect CD36-mediated oxLDL uptake in live cells, we monitored fluorescent oxLDL (Dii-oxLDL) uptake using confocal microscopy. Addition of exogenous FA to serum-free media enhanced dose-dependent oxLDL uptake. Exceptions were ω-3 FA, which bound to CD36, and HODE, which did not bind to CD36, demonstrating FA structure-specific effects on a major function of CD36 and a new mechanistic link between atherosclerosis and high levels of FA in obese and Type-II diabetic individuals.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第23751号 / 医博第4797号 / 新制||医||1055(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 寺田 智祐, 教授 江藤 浩之, 教授 髙折 晃史 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

There are many reports indicating the presence of various emerging contaminants (ECs) in treated wastewater and other water sources. The detection of such contaminants in the environment and the ability of these contaminants to pose potential threats to the environment at very low concentrations have led to a need for more efficient treatment technologies. Cyclodextrins (CDs) have gained significant interest as an alternative adsorbent for water and wastewater treatment because of their unique physico-chemical characteristics and excellent selectivity towards organic compounds. The property of CD to form inclusion complexes with various molecules through host-guest interactions has made it a useful compound for the removal of a number of contaminants from water and wastewater. The overall goal of the study is to identify and develop a novel adsorbent for the removal of ECs of interest. The specific objectives are a) to synthesize various beta-cyclodextrin (BCD) based adsorbents by coating BCD onto different supports such as silica, filter paper, iron oxide, and zeolite, b) to perform batch and column experiments using the developed adsorbents, c) to evaluate the performance of the adsorbents in different water matrices such as MQ water, simulated wastewater in presence of humic acid, and real municipal wastewater, d) to study the regeneration potential of the adsorbents. In this research, various (BCD) based hybrid adsorbents were synthesized and their performances were evaluated based on the removal of selected ECs. At first, chemically bonded BCD onto silica particles as hybrid adsorbents were synthesized by using crosslinking agents and copolymers. Three different methods were used to synthesize 14 different BCD coated silica adsorbents. The adsorbent prepared by reacting BCD with hexamethylene diisocyanate (HMDI) as crosslinking agent and dimethyl sulfoxide (DMSO) as solvent showed best results in removing the ECs studied. The adsorbent showed more than 95% removal of 17β-estradiol (in single component) and more than 90% of most of the estrogens (mixture of 12), more than 99% of perfluorooctanoic acid (PFOA) (in single component) and more than 90% of most of the PFCs (mixture of 10), and a maximum of 90% removal in case of BPA. The adsorption capacity of the developed adsorbent was observed to be higher for the removal of 17β-estradiol and PFOA than that of commercially available activated carbon (F400) in MQ water. In order to represent the real environmental scenario, further batch experiments were conducted for the removal of two PFCs (PFOA and PFOS (perfluorooctane sulfonic acid)), 17β-estradiol, and BPA at environmentally relevant concentrations from wastewater. The adsorbent was effective in removing the ECs that were spiked in the secondary effluent of a municipal wastewater treatment plant. Furthermore, the adsorbent was successfully regenerated with methanol over four cycles without significant loss in its adsorption capacity for the removal of PFOA and estrogens. Ozonation as an alternative method of regeneration was also used and the process was also very effective in regenerating the adsorbent over seven successive cycles for the removal of BPA and 17β-estradiol. The characterization of the adsorbents using FTIR, TGA, and TEM confirmed the coating of BCD onto silica particles. The TGA results showed high thermal stability of the adsorbent (upto 300oC). As an alternative to chemical impregnation, another method of synthesis was developed where various BCD based hybrid adsorbents were synthesized by physically impregnating hydroxypropyl BCD (hpBCD) polymer onto three different supports: iron oxides, zeolite, and filter paper. The hpBCD impregnated filter papers were synthesized by solvent evaporation method and different adsorbents were synthesized by varying the polymer loadings. The polymer loading was optimized based on the performance of the modified (polymer impregnated) filter paper in terms of its filtration capacity as well as adsorption capacity to remove PFOA and BPA. The magnetic adsorbent was prepared by mixing hpBCD polymer with iron oxide (Fe3O4) particles. It was observed that by increasing the mixing time of the support (Fe3O4) with the polymer from 48 to 96 hr, the adsorption capacity of the adsorbent (hpBCD polymer coated Fe3O4) could be significantly enhanced. The same approach was also used to synthesize hpBCD polymer coated zeolite adsorbent and both adsorbents (hpBCD/zeolite and hpBCD/Fe3O4) were effective in removing the selected ECs from MQ water, simulated water, and wastewater. / Civil Engineering

As a response of the need for a natural antimicrobial to replace sodium benzoate's use as a preservative in beverages, twenty eight compounds known to have antimicrobial activity were evaluated to quantify their solubility. Twenty three of the compounds evaluated are components of plant essential oils and the remaining five compounds are alkyl esters of para-hydroxybenzoic acid. The test compounds were evaluated for aqueous solubility as well as their solubility in an acid-based beverage mixture. The compounds were found to be practically insoluble (< 100 mg/L), very slightly soluble (100 mg/L – 1,000 mg/L) or slightly soluble (1,000 mg/L to 10,000 mg/L). o-Methoxycinnamaldehyde, trans,trans-2,4-decadienal, cinnamic acid, and citronellol were complexed with α- and β-cyclodextrin and evaluated through phase solubility analyses. The complexes formed showed improved aqueous solubility for all compounds. Complexation with α-CD resulted in an increase of aqueous solubility of o-methoxycinnamaldehyde by 10-fold, trans,trans-2,4-decadienal by 3.2-fold, cinnamic acid by 6.3-fold, and citronellol by 8-fold. In addition, complexation with β-CD resulted in an increase of aqueous solubility of o-methoxycinnamaldehyde by 1.6-fold, trans,trans-2,4-decadienal by 3.1-fold, cinnamic acid by 1.7-fold, and citronellol by 1.6- fold. The storage stability of the α-CD complexes of o-methoxycinnamaldehyde trans,trans-2,4-decadienal and citronellol were evaluated for 7 days in an acid-based beverage solution by SPME GC-MS. The complexes exhibited varying levels of degradation throughout the duration of the study all. The concentration of o-methoxycinnamaldehyde detected by SPME GC-MS decreased by 61.7%. Similarly, the concentration of trans,trans-2,4-decadienal and that of citronellol decreased by 62.7% and 43% respectively. Additionally, a comparison by UV/Vis of the storage stability of the complexes stored in glass and PET containers was performed. The storage stability comparison proved that absorption into the PET polymer membrane did not occur. / Master of Science

In response to a need for a natural antimicrobial to replace sodium benzoate, cinnamic acid was chosen. Due to cinnamic acid's solubility issues, α-cyclodextrin was used as a host molecule to form an inclusion complex with the cinnamic acid molecule. The cinnamic acid: α-cyclodextrin inclusion complex was then characterized using phase solubility analysis, proton nuclear magnetic resonance (H-NMR), and solid inclusion. Phase solubility analysis verified the maximum amount of cinnamic acid that α-cyclodextrin was able to host. H-NMR was used to determine the complex association constant, determine the chemical shifts of available protons, and yield a stoichiometry for the complex. The solid inclusion complex allowed for a physical formation of the complex, yielding further information in support of the complex stoichiometry. Microbiological tests were also performed to quantify the antimicrobial abilities of the complex, the guest, and the host against the yeast Saccharomyces cerevisiae and mold Paecilomyces variotii. Results indicated that approximately 990.29 ppm in aqueous solution was the maximum amount of cinnamic acid in the complex. The 2:1 stoichiometry yields an association constant of 21.7 M-1. Results also indicated that the cinnamic acid readily conformed to fit within the α-cyclodextrin host molecule, which remained a rigid structure. An 8.9% weight to weight of cinnamic acid was calculated for the solid inclusion again reinforcing a 2:1 stoichiometry. Microbiological studies showed little to no inhibition power by the complex at varying concentrations against S. cerevisiae and P. variotii. Free cinnamic acid showed greater antimicrobial activity compared with free α-cyclodextrin and the complex. / Master of Science in Life Sciences

Microcosms were constructed from aquifer sediment samples taken from an actively degrading chlorinated solvent contaminated site located in Virginia Beach, Virginia. The objective of this study was to determine if and how the addition of cyclodextrin (CD) affects reductive dechlorination of chlorinated ethenes. After chlorinated solvent degradation rates were established in anaerobic and aerobic microcosms, 100 mg/L of CD solution was added for a period of 21 days. CD was then removed after 26 days to simulate the degradation response of the aquifer in a post CD injection environment. Degradation rates were determined by analyzing PCE, TCE, and cis-DCE concentration data over the various phases of the experiment. Results from this study indicated that chlorinated solvent degradation could be either impaired or facilitated by the addition of CD. CD appeared to stimulate one anaerobic microcosm (IY-2c) where daughter production had not previously occurred. The activity of this microcosm was greatly enhanced by the addition of CD (0 uM/day to 13.89 uM/day). However, biotransformation of PCE in another anaerobic microcosm in which reductive dechlorination was occurring, ceased after the addition of CD (IY-1a). In a third group of microcosms the rate and extent of reductive dechlorination was greatly enhanced by the addition of CD. The effect of adding CD was also found to be highly dependent on the redox conditions in the microcosm, specifically if the conditions were strongly reducing. The most active microcosms, found in the Aerobic Group, also had the lowest ferrous iron concentrations (3.57 mg/L for BY-1a, 2.25 mg/L for BY-1b, and 0.41 mg/L for BY-1c). The microcosm (IY-2b) that showed no daughter production had the highest level of ferrous iron (44.22 mg/L). This study presents a qualitative approach to the affect of CD on MNA. / Master of Science

Yes / There is a need to understand the nature of aggregation of cyclodextrins (CDs) with guest molecules in increasingly complex formulation systems. To this end an innovative application of Taylor dispersion analysis (TDA) and comparison with dynamic light scattering (DLS) have been carried out to probe the nature of ICT01-2588 (ICT-2588), a novel tumor-targeted vascular disrupting agent, in solvents including a potential buffered formulation containing 10% hydroxypropyl-β-cyclodextrin. The two hydrodynamic sizing techniques give measurement responses are that fundamentally different for aggregated solutions containing the target molecule, and the benefits of using TDA in conjunction with DLS are that systems are characterised through measurement of both mass- and z-average hydrodynamic radii. Whereas DLS measurements primarily resolve the large aggregates of ICT01-2588 in its formulation medium, methodology for TDA is described to determine the size and notably to quantify the proportion of monomers in the presence of large aggregates, and at the same time measure the formulation viscosity. Interestingly TDA and DLS have also distinguished between aggregate profiles formed using HP-β-CD samples from different suppliers. The approach is expected to be widely applicable to this important class of drug formulations where drug solubility is enhanced by cyclodextrin and other excipients.