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京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23814号 / 医科博第135号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 村川 泰裕, 教授 松村 由美, 教授 森本 尚樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Rheumatoid arthritis (RA) is a chronic disease characterized by cycles of inflammation and resolution. Previously, it was believed that the resolution of inflammation is simply dissipation of pro-inflammatory signals, although current research indicates that resolution is an active process. Acute inflammation follows defined phases of induction, inflammation and resolution, and resolution occurs by an active process that requires COX-2 activity. This study aims to address whether this paradigm extends to a recognized model of chronic inflammation. We demonstrated in murine collageninduced arthritis that chronic inflammation follows the same sequential course. While there is the normal production of pro-inflammatory cytokines during inflammation and anti-inflammatory mediators such as 15-deoxyΔ12,14PGJ2 (15d-PGJ2) during resolution, interestingly there is sustained production of both COX-2 and the presumably proinflammatory PGE2 during both phases. Blocking COX-2 activity and therefore production of PGE2 during the resolution phase perpetuated instead of attenuated inflammation. Repletion with PGE2 analogs restored homeostasis, and this function is mediated by the pro-resolving lipoxygenase metabolite, lipoxin A4 (LXA4), which is a potent stop signal. Thus, the study provided in vivo evidence for a natural, endogenous link between the cyclooxygenase-lipoxygenase pathways and showed that PGE2 serves as a feedback inhibitor essential for limiting chronic inflammation in autoimmune arthritis. These findings may explain the enigma regarding why COX-2 inhibitors are palliative rather than curative in humans because blocking resolution may mitigate the benefit of preventing induction. / Microbiology and Immunology

Adipose tissue is a type of connective tissue whose purpose was once thought to be limited to fat storage but is now understood to be a key factor in the pathogenesis of different metabolic diseases, including obesity and type-II diabetes. Adipose tissue consists largely of adipocytes, cells responsible for fat and releasing energy in form of lipids. Different classes of fatty acids, such as saturated and unsaturated have different biological effects on adipocytes. Lipid droplets are the primary organelles in adipocytes that store these fatty acids in form of lipids, and the development of engineered adipose tissues would benefit from improved techniques for analysis of lipid droplet composition, distributions, and dynamics based as a function of fatty acid saturation. Conventional microscopic techniques, such as fluorescence microscopy, provides excellent selectivity of lipid-based structures inside adipose tissue cellular structures based on staining with compound dyes. However, fluorescence staining limits multiplex imaging, and requires time consuming steps in preparing the samples for imaging. Therefore, developing a label-free, high resolution imaging platform with sensitivity to lipid composition could enable analysis of structural and compositional differentiation of lipid droplets within adipocytes during differentiation could give valuable insights into the importance of lipid droplets role in metabolism. As an important step towards achieving this goal, a femtosecond based CARS microscopy imaging platform has been developed to perform in vitro, label-free, imaging of fatty acid composition within engineered adipose tissues. / Bioengineering

<p> Gene-targeted studies in mice have shown that the murine scavenger receptor class B type I (mSR-BI) is atheroprotective and plays a key role in the clearance of high density lipoprotein (HDL) cholesterol by the liver. We focused on the analysis of human SR-BI (hSR-BI) and the role of its C-terminal cytoplasmic tail on its localization, lipid uptake activity, and regulation in hepatocytes both in vitro and in vivo. Full length hSRBI and hSR-BI lacking its C-terminal cytoplasmic tail (hSR-BI-DM) localized to vesiclelike structures in the cytoplasm, to juxtanuclear regions and to the cell surface in HepG2 cells. Similar cytoplasmic punctate distribution was observed in transfected human and mouse aortic endothelial cells. </p> <p> In HepG2 cells both hSR-BI and hSR-BI-DM mediated HDL-lipid uptake; however, the truncation mutant displayed only half ofthe activity, suggesting that removal ofthe C-terminal cytoplasmic tail reduced but did not eliminate SR-BI's activity. In HepG2 cells treated with the PKC inhibitor, calphostin C, hSR-BI or hSR-BI-DM mediated HDL-lipid uptake was decreased by 40 and 50%, respectively, indicating that this activity is regulated by PKC. </p> <p> In order to determine the effects of hSR-BI and hSR-BI-DM in vivo, we set out to generate transgenic mice with hepatic overexpression ofeach protein using a bipartite expression system requiring driver and responder transgenes. Mice expressing the responder transgenes, PTREhSR-BI and PTREhSR-BI-DM, as well as a reporter transgene (PTRdacZ), driven by the same bi-directional promoter, were generated and mated to mice with a liver-specific driver trans gene, PMuptTA. The mice were analyzed and showed the presence of a reporter protein, ~-galactosidase, in their livers, but not in other tissues tested. Total and HDL cholesterol levels were not altered in PMuPtTA I PrREhSRBI or PMuptTA I PrREhSR-BI-DM transgenic mice. Further characterization ofthe double transgenic mice revealed that hSR-BI m.RNA transcripts were detected in the livers of PMuPtTA I PrREhSR-BI mice, but not in those ofPMuPtTA I PrREhSR-BI-DM mice. However, neither PMuptTA I PrREhSR-BI nor PMuptTA I PrREhSR-BI-DM mice showed increased expression of SR-BI in their livers. </p> / Thesis / Master of Science (MSc)