Osteopontin (OPN) plays an important role in left ventricular (LV) remodeling after myocardial infarction (MI) by promoting collagen synthesis and accumulation. This study tested the hypothesis that MMP inhibition modulates post-MI LV remodeling in mice lacking OPN. Wild-type (WT) and OPN knockout (KO) mice were treated daily with MMP inhibitor (PD166793, 30 mg/kg/day) starting 3 days post-MI. LV functional and structural remodeling was measured 14 days post-MI. Infarct size was similar in WT and KO groups with or without MMP inhibition. M-mode echocardiography showed greater increase in LV end-diastolic (LVEDD) and end-systolic diameters (LVESD) and decrease in percent fractional shortening (%FS) and ejection fraction in KO-MI versus WT-MI. MMP inhibition decreased LVEDD and LVESD, and increased %FS in both groups. Interestingly, the effect was more pronounced in KO-MI group versus WT-MI (P < 0.01). MMP inhibition significantly decreased post-MI LV dilation in KO-MI group as measured by Langendorff-perfusion analysis. MMP inhibition improved LV developed pressures in both MI groups. However, the improvement was significantly higher in KO-MI group versus WT-MI (P < 0.05). MMP inhibition increased heart weight-to-body weight ratio, myocyte cross-sectional area, fibrosis and septal wall thickness only in KO-MI. Percent apoptotic myocytes in the non-infarct area was not different between the treatment groups. Expression and activity of MMP-2 and MMP-9 in the non-infarct area was higher in KO-MI group 3 days post-MI. MMP inhibition reduced MMP-2 activity in KO-MI with no effect on the expression of TIMP-2 and TIMP-4 14 days post-MI. Thus, activation of MMPs contributes to reduced fibrosis and LV dysfunction in mice lacking OPN.

Macrophages are central players during atherosclerosis. Especially, macrophage cholesterol efflux, which promote the removal of free cholesterol from foam cells, are crucial to prevent lipid accumulation and reverse atherogenesis. microRNAs (miRNAs) are important regulators of various pathways involved in atherosclerosis. During inflammation, macrophages secrete extracellular vesicles (EVs) carrying miRNAs to communicate signals to nearby cells. However, the role of macrophage-derived EVs in atherogenesis is not known. In the first study, we find that EVs derived from cholesterol-loaded macrophages can inhibit macrophage migration in vitro and in vivo. This effect appears to be mediated by the transfer of several miRNAs, including miR-146a, to recipient macrophages where they repress the expression of specific pro-migratory target genes Igf2bp1 and HuR. Our studies suggest that EV-derived miRNAs secreted from atherogenic macrophages may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall. Understanding macrophage communication via EVs provided the rationale for the design of nanoparticles (NPs) that mimic macrophage EVs to deliver beneficial miRNAs to the atherosclerotic plaque. While cationic lipid/polymer-based NPs have been employed as systemic delivery vehicles of siRNA, none of these have been used to deliver miRNAs to macrophages in vivo. In the second study, we developed a chitosan NP platform for effective delivery of miRNAs to alter macrophage function in vivo. We showed that our NPs made using a cross-linked chitosan polymer can protect as well as transfer miR-33 to naïve macrophages and regulate the expression of its target gene (Abca1) as well as cholesterol efflux in vitro and in vivo. Finally, almost all miRNAs that have been characterized are efflux-repressing miRNA, thereby accelerating atherosclerosis. miR-223 is one of a few miRNAs whose overexpression can promote cholesterol efflux, modulate the inflammatory response, and thus, be antiatherogenic. However, its contribution to the pathogenesis of atherosclerosis in vivo and the mechanism underlying its effects has not been thoroughly characterized. We herein find that miR-223 is capable of suppressing plaque development via modulating cholesterol efflux and inflammatory responses, thus may serve as a potential therapeutic to reduce atherosclerosis. These effects of miR-223 appear to be dependent on the inhibition of its target gene, the transcription factor Sp3. Overall, this thesis highlights the importance of both endogenous and extracellular miRNAs in controlling different aspects of atherogenic response.

The emergence and rapid spread of antimicrobial resistance (AMR) is a phenomenon that extends beyond clinical settings. AMR has been detected in multiple environmental compartments, including agricultural soils and water bodies impacted by wastewater discharges. The purpose of this research project was to evaluate what factors could influence the environmental persistence of antibiotic resistance genes (ARGs), as well as to identify potential strategies employed by human pathogens to survive in secondary environment outside the host. The first part of this dissertation describes the incidence of the New Delhi metallobeta lactamase gene (blaNDM-1) – an ARG conferring resistance to last resort antibiotics – in the influent of a wastewater treatment facility processing municipal wastewater from Jeddah, Saudi Arabia. Detection of blaNDM-1 was followed by the isolation of a multi-drug resistant strain of E. coli (denoted as strain PI7) at a frequency of ca. 3 x 104 CFU/m3 in the untreated municipal wastewater. Subsequently, we described the decay kinetics of E. coli PI7 in microcosm experiments simulating biological treatment units of wastewater treatment plants. We identified that transition to dormancy is the main strategy prolonging the persistence of E. coli PI7 in the microcosm experiments. Additionally, we observed slower decay of E. coli PI7 and prolonged stability of extracellular DNA in anoxic/anaerobic conditions. In the last chapter of this thesis, the fate of extracellular DNA is further explored. Using as a model Acinetobacter baylyi ADP1, we describe the stimulation of natural transformation frequencies in the presence of chlorination disinfection byproducts (DBPs). Moreover, we demonstrate the ability of BAA to stimulate transformation is associated with its capacity to cause DNA damage via oxidative stress. Overall, this dissertation addresses important knowledge gaps in our current understanding of ARB and extracellular ARG persistence in the environment. The results from this project highlight the importance of retrofitting the existing water treatment process with advance membrane filtration units, and the need to relook into the current disinfection strategies. Wastewater treatment technologies should be assessed for their efficacies in not only inactivating ARB and ARGs, but also whether unintended consequences such as stimulated horizontal gene transfer would occur.

While enteroviruses, including poliovirus, are conventionally released upon cell lysis, recent studies show that phosphatidylserine-enriched infectious extracellular vesicles (IEVs) shed by infected cells can transport clusters of enteroviruses from cell to cell, resulting in increased infectivity. Combining structural and biochemical analyses, we focused on IEVs shed from poliovirus-infected cells, a classical prototype for studying enteroviruses. Transmission cryo-electron microscopy, cryo-electron tomography and computational reconstruction, present the first three-dimensional structures of well-preserved IEVs and purified exosomes. We observed that single-membraned IEVs present a wide size range in diameter. Clusters of virions can be either densely packed within a protein-coated irregularly shaped IEV, or concentrated at one or both ends of an IEV, forming a polar structure. In addition to virions, IEVs often contain internal vesicles, “ramen-noodle”-like structures with strong density, and partially assembled virion-like structures. Viral replication complex components, including viral proteins polymerase 3D, 3CD, 3A, 3AB, 2BC, 2C and (+) and (-) stranded RNAs were detected in IEVs. Furthermore, (-) stranded RNA templates are protected by the IEVs, not packed in viral capsids. The transported viral replication components (viral proteins and RNAs) and virions within IEVs initiate a stronger and faster viral replication in recipient cells than free virions. Both cryo-electron tomographic and mass spectrometry data also showed that virions and “ramen-noodle”-like structures were also observed in purified CD9 positive exosomes from poliovirus-infected cells. Viral protein 3AB, detected on the membrane of IEVs, can invaginate membranous structures to engulf large proteins into a closed lumen. Our study demonstrates that IEVs can transport viral replication complex components to initiate a rapid onset of viral replication, as part of a novel viral transmission mechanism. Viral protein 3AB may contribute to forming IEVs throughout the infection. / 2019-06-12T00:00:00Z

There is a strong correlation between aging and risk of fractures that is independent of changes in bone quantity. Although the underlying processes that dysregulate the structural health and integrity of bone tissue without affecting bone-quantity with aging are unknown, one hypothesis is that changes affecting extra-cellular components of bone, particularly components of the mineralized matrix, may contribute to the decline in bone quality with aging. Citrullination is a post-translational modification where a peptidyl arginine is enzymatically modified to a peptidyl Citrulline by enzymes knowns as peptidyl arginine deiminases (PADs). Increased citrullination of proteins is associated with altered protein function and a loss of structural stability. We propose that citrullination could have negative effects on the structural integrity of bone matrix proteins by inducing a loss of proteostasis, a conditional loss of protein homeostasis. To address this, we examined whether the levels of citrullinated bone matrix proteins differs between young and old mice. Our results show that there is a profound increase in citrullination of extractable bone matrix with ageing. To assess if changes in citrullination contribute to bone mass and/or mechanical strength, we utilized an existing murine model in which citrullination is increased. Mice deficient in Protein Tyrosine Phosphatase Nonreceptor 22 (PTPN22), an inhibitor of PAD4, and thus of citrullination, have been shown to have increased histone H3 citrullination in macrophages derived from Ptpn22-/- mice compared to WT controls. We assessed several properties of bone in both young and aged Ptpn22-/- animals. In young mice, deficiency in PTPN22 leads to an increase in bone matrix citrullination. However, we did not find a link between increased citrullination and bone loss, as measured by formation of bone resorbing osteoclasts. Further, no difference in bone mass or structural parameters was seen in either young or old Ptpn22-/- mice compared to wild-type littermates. Interestingly, there was also no clear relationship between degree of citrullinated bone matrix and genotype in older mice, possibly due to overall increases in bone matrix citrullination with aging. However, as it is clear that bone matrix citrullination increases with age and that this citrullination in younger mice can be modulated by PTPN22 deficiency, we plan to use Ptpn22-/- mice as a model to learn more about the effects of increased citrullination on mechanical strength and bone matrix properties, therefore unearthing new insights into the role of post-translational modification in the function of bone matrix proteins.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17784号 / 医博第3810号 / 新制||医||999(附属図書館) / 30591 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮地 良樹, 教授 松田 秀一, 教授 戸口田 淳也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(人間健康科学) / 甲第19371号 / 人健博第31号 / 新制||人健||3(附属図書館) / 32385 / 新制||人健||3 / 京都大学大学院医学研究科人間健康科学系専攻 / (主査)教授 坪山 直生, 教授 市橋 則明, 教授 松田 秀一 / 学位規則第4条第1項該当 / Doctor of Human Health Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21661号 / 医博第4467号 / 新制||医||1035(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 篠原 隆司, 教授 小川 修, 教授 近藤 玄 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Physical activity (PA) reduces the risk for deleterious outcomes in both mother and fetus during pregnancy and improves health across the lifespan. How these benefits are bestowed remains poorly understood but may involve the placenta, the critical interface responsible for fetal growth and survival during pregnancy. This thesis first aims to determine whether small extracellular vesicles (sEVs), potential biological mediators of cell-to-cell communication, are released into circulation after acute exercise during pregnancy and how this compares in the non-pregnant state. Pregnant women were found to have greater circulating sEVs levels compared to non-pregnant controls after a moderate-intensity treadmill walk. Since exercise-associated sEVs are proposed to mediate tissue cross talk in response to exercise, exercise-associated sEVs were examined for their ability to influence trophoblasts (specialized placental cells) in vitro using the BeWo choriocarcinoma cell line. Exercise-associated sEVs from pregnant and non-pregnant women interacted with trophoblast-like cells but did not alter their proliferation, gene expression of angiogenic growth factors, or production of the pregnancy hormone, human chorionic gonadotropin. Finally, the relationship between differing intensities of maternal PA and fetoplacental vascular density in a cohort of healthy pregnant women followed prospectively from 24 weeks of gestation until term delivery. Using traditional histopathological point-counting techniques, there was no difference in the fetoplacental vascular density of individuals meeting or exceeding recommended 150 min of moderate-to-vigorous intensity PA. However, the analysis revealed unexpected associations between fetoplacental vascular density and lower intensities of PA, and sedentary time. Together, the work presented in this thesis highlight the potential for exercise-associated sEVs to communicate the benefits of PA to mother and fetus and the need to investigate the effects of varying PA intensities on placental vascular development.

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