The accumulation of pro-inflammatory macrophages in the inflamed vascular wall is a critical step in atherogenesis. The mechanism of macrophage retention within the site of inflammation is not understood yet. High adhesion that prevents macrophage migration is one of the potential mechanisms. Previous research in our laboratory showed that integrin αDβ2 is upregulated on pro-inflammatory macrophages, promotes macrophage retention, and contributes to atherogenesis. However, a key ligand for αDβ2 within the tissue is yet to be identified, since αDβ2 does not interact with major ECM proteins, collagens, and laminins. We recently found that during acute inflammation, the oxidation of docosahexaenoic acid (DHA) leads to the generation of end product carboxyethylpyrrole (CEP), which forms an adduct with fibrinogen and albumin via ε-amino group of lysines. There is evidence that macrophages adhere to CEP-modified albumin in αDβ2-dependent manner. We continued the advancement of the proposed hypothesis that non-conserved, basic amino acids of integrin αDβ2 located near the MIDAS site of the I-domain are responsible for binding to CEP. αD I-domain and generated I-domain mutants: H272(D), K297(Q) and K309(N) were used to map the ligand binding site between integrin and CEP. Using site-directed mutagenesis, mutant αD I-domains were generated with minimal amino acid substitutions. Protein-protein binding reveals that the generated mutation of K297(Q) on the I-domain demonstrates the strong reduction of binding, while H272(D) and K309(N) did not have a significant effect on integrin binding properties. Therefore, lysine 297 located in I-domain of integrin αD, is a critical amino acid for αDβ2 binding to CEP-modified proteins. The identification of a binding site for CEP-modified proteins within αDβ2 will help to develop a blocking reagent for the treatment of the inflammatory component of atherosclerosis.

Obesity is a significant health care problem, affecting more than one third of the United States population and is an important risk factor for Type 2 Diabetes Mellitus (T2D). Adipose tissue expansion results in the recruitment and accumulation of macrophages, which secrete proinflammatory cytokines that impair insulin signaling. Adenosine regulates inflammation by signaling through G-protein coupled receptors (GPCRs), such as the A2b adenosine receptor (A2bAR). Recently a role for adenosine receptors has been described in the differentiation of osteoblasts and adipocytes. This thesis tests the hypothesis that the A2bAR regulates adipose tissue dynamics at the level of preadipocyte differentiation and macrophage inflammation. This thesis showed that activation of the A2bAR inhibited preadipocyte differentiation. A2bAR-induced adipocyte inhibition was dependent on the expression of Krüppel-like factor 4 (KLF4), which is important for stem cell maintenance and renewal. A2bAR knockdown enhanced adipogenesis in vitro and A2bAR knockout (KO) mice had more adipocytes as compared to wild type (WT) mice, suggesting enhanced adipogenesis in the absence of the A2bAR. The translational potential of this work is strengthened by the previous finding of elevated A2bAR expression in adipose tissue of obese individuals as well as our new finding of a close correlation between the expression of A2bAR and KLF4 in adipose tissue of obese individuals. A2bAR KO mice have impaired insulin resistance, in part due to reduced levels of insulin receptor substrate-2 (IRS-2). Proinflammatory cytokines have been shown to reduce IRS-2 levels. Given the role of the A2bAR in regulating inflammation, the contribution of A2bAR signaling in macrophages to insulin resistance was elucidated. Transgenic mice that express A2bAR only in macrophages were generated. Intriguingly, restoration of A2bAR signaling in macrophages ameliorated insulin resistance, glucose tolerance, and fat and liver tissue insulin signaling. As expected, tissue and plasma proinflammatory cytokine levels were reduced to that of WT mice. This suggested that the protective effect of A2bAR signaling on insulin resistance was due in large part to A2bAR control of macrophage cytokine expression. This thesis highlights the importance of A2bAR signaling in adipogenesis and in regulating inflammation in the setting of obesity and T2D.

Obesity has reached epidemic proportions in recent years. The World Health Organization estimated in 2008 that 1.4 billion people were overweight of whom 500 million were obese. Obesity associates with a wide range of conditions, such as cardiovascular diseases, cancer, diabetes, and neurological disorders, and causes approximately 2.8 million deaths each year. Many studies have established that obesity strongly impacts the normal function of the immune system: it dysregulates production of inflammatory and anti–inflammatory cytokines, alters numbers of immune cells, and causes an overall weaker immune response. Developing therapies that aim to improve the immune response is crucial in order to increase the quality of life of obese subjects and to reduce their ever–increasing healthcare-related costs. The long-term objective of this work is to contribute to the development of therapies that can increase the immune response in obese macrophages. In particular, gene modifications adjusting the response to infection in obese macrophages closer to that of lean macrophages are desired. To this end, the present work focused on the Toll-like Receptors (TLRs), which play an essential role in the detection of pathogens and the initiation of both innate and acquired immune responses. Genes essential to the transmission of the infection signal were first identified using a model of the TLR signaling pathways. These genes provided the basis for reconstructing a gene regulatory network that not only accounts for information coming from the TLRs, but also regulates key reactions within the pathways. The topology and regulatory functions of this network were identified by applying novel computational techniques to time-series gene-expression datasets. The TLR signaling and gene-regulatory networks were then integrated to develop a modeling framework for macrophage that predicts the time behavior of several markers for infection. Finally, formal verification techniques were used to demonstrate that the model satisfies several properties characteristic of the response to infection in macrophage. The work detailed in this dissertation offers a suitable platform for developing and testing biological hypotheses that aim to improve responses to infection.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第19774号 / 農博第2170号 / 新制||農||1040(附属図書館) / 学位論文||H28||N4990(農学部図書室) / 32810 / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 植田 充美, 教授 栗原 達夫, 教授 矢﨑 一史 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21622号 / 医博第4428号 / 新制||医||1033(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 妻木 範行, 教授 松田 秀一, 教授 渡邊 直樹 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Thesis advisor: Tim van Opijnen / Streptococcus pneumoniae is a prominent gram-positive commensal and opportunistic pathogen which possesses a large pan-genome. Significant strain-to-strain variability in genomic content drives the use of varied pathways to perform similar processes between strains. Considering this variation, we employ a set of 36 strains, representative of 78% of total pan-genome diversity, with which to perform functional studies. We previously determined the set of genes required by 22 of the 36 strains to maintain successful infection in a host, or the virulome. In this work, we sought to parse from the virulome the genes required specifically for nasopharyngeal adhesion, a crucial step in S. pneumoniae colonization and transmission, and often a precursor to invasive disease, as well as gene requirements for subversion of the macrophage. We performed in vitro attachment Tn-seq in the 22 strains to D562 human nasopharyngeal epithelial cells, identifying thirteen factors that exhibit requirements for adhesion, and preliminarily validated a proposed universal requirement for survival of the macrophage by a killing assay using J774A.1 murine migratory macrophages. / Thesis (BS) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: A&S Honors. / Discipline: Biology.

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京都大学 / 新制・課程博士 / 博士(医学) / 甲第24137号 / 医博第4877号 / 新制||医||1060(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 上野 英樹, 教授 森本 尚樹, 教授 寺田 智祐 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第24140号 / 医科博第141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 金子 新, 教授 椛島 健治, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM