Chronic obstructive pulmonary disease (COPD) is a common respiratory disorder and the third leading cause of mortality. In this thesis we performed a clustering analysis of four specific immune cells in the GSE136831 dataset, using the default recommended parameters of the Seurat package in R, and obtained 16 subclasses with various COPD and cell-type proportions. Clusters 3, 7 and 9 had more pronounced independence and were all composed of macrophage-dominated control samples. The results of the pseudo-time analysis based on Monocle 3 package in R showed three different patterns of cell evolution. All started with a high percentage of COPD states, one ended with a high rate of Control states, and the other two still finished with a high percentage of COPD states. The results of differentially expressed gene analysis corroborated the existence of finer clusters and provided support for their rational categorization based on the similar marker genes. The gene ontology (GO) enrichment analysis for cluster 0 and cluster 6 provided feedback on enriched biological process terms with significant and unique characteristics, which could help explore latent novel COPD treatment directions. Finally, some top-ranked potential pharmaceutical molecules were searched via the connectivity map (cMAP) database. / Graduate / 2023-08-12

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Opioid receptors are members of the superfamily of seven transmembrane G protein-coupled receptors (GPCRs) which share several structural and functional characteristics. There are 3 subtypes of opioid receptors, designated μ (MOR), δ (DOR), and κ (KOR) opioid receptors, have been found in the immune, nervous, gastrointestinal and other tissues. We have attempted to clarify the nature of MOR-induced signal transduction pathways in leukocytes. We found that the activation of MOR leads to a significant induction of ERK phosphorylation in peripheral blood mononuclear cells from normal donors using the MOR-selective agonist DAMGO. We are also interested in determining the role of this signaling pathway in the regulation of the immune response. Recent experiments using selective inhibitors suggests that the activation of ERK involves a pathway composed of Raf, Ras, and MEK1/2 kinases, but is independent of PI3 kinase. After treatment of multiple protein kinase inhibitors we found the PKC inhibitor Go-6983 and PLC inhibitor U73122 could also inhibit ERK phosphorylation in MOR stable line (HEK-MOR). According to the results from the Go-6983 and H-89 inhibitor treatment experiments, we found PKCμ/PKD1, a family member of Protein Kinase D, may be involved in MOR-induced ERK phosphorylation. We also found PKCμ/PKD1 S916 phosphorylation after MOR activation and the PKCμ specific inhibitor CID755673 inhibited the MOR-mediated ERK activation. ERK phosphorylation activated several transcription factors in human monocytes, the activation of transcription factors has been proved to induce miRNA expression. We have initiated a series of experiments to study the regulation of miRNA expression by MOR in human monocytes. We found miR-21, miR-155, miR-29a, miR-20b expression were significantly up-regulated following morphine treatment, and morphine-induced miR-21 expression is down-regulated following pretreatment with the ERK inhibitor U0126 and PKD inhibitor CID755673 in human primary monocytes. The results suggest that morphine-induced MOR activation results in up-regulation of miRNA expression human monocytes and this may regulate monocyte and/or macrophage function thought PKCμ/Ras/Raf/ERK signaling pathway. / Molecular Biology and Genetics

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13603号 / 論医博第2313号 / 京都大学大学院医学研究科医学専攻 / (主査)教授 森田 智視, 教授 佐藤 俊哉, 教授 永井 純正 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

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