Micro- and nanofabricated sample environments are useful tools for characterizing diffusion in confined aqueous environments. The steady-state diffusion and adsorption of unilamellar lipid vesicles in arrays of hydrophilic micropillars is investigated. Gradients in the coverage of fluorescently labeled, pillar-supported lipid films, formed from vesicle fusion, are determined from 3D z-stack images using confocal microscopy. The gradients are the result of preferential adsorption of vesicles near the tops of the pillars, which progressively deplete them from solution as they diffuse toward the base of the array. However, the increased propensity for vesicle adsorption near the pillar tops compared to the confined spaces between pillars results in the formation of confluent supported lipid bilayers at the pillar tops that resist the adsorption of additional vesicles while leaving the pillar surfaces below available for binding. This results in a reduction in the numbers of depleted vesicles compared to what one would anticipate based on diffusive fluxes. The resulting inhomogeneous spatial profiles of lipid structures on the pillars are the result of the system being maintained in a dissipative, nonequilibrium steady state during incubation of the pillar arrays in the vesicle solution, which is ultimately quenched by rinsing away the unbound, freely diffusing vesicles.

The thesis introduces two developed methods to quantify extracellular vesicles (EVs) isolated from cancer cell lines and healthy human saliva by using capillary zone electrophoresis. In the first chapter, the importance of EVs, as well as the existing EV isolation, characterization, and quantification methods, are described. The general principle of capillary electrophoresis (CE) is explained for a better understanding of these two methods. Chapter II describes the idea and concepts of Extracellular Vesicles quantitative Capillary Electrophoresis (EVqCE). The method evolved from the previous study carried out in our research group for the quantification of viruses. After the isolation of EVs from different cell lines, the characterization and quantification of EVs were performed using nanoparticle tracking analysis (NTA) and flow cytometry. EVqCE consists of four steps for EV quantification. In this study, EVqCE was employed to know the concentrations of EVs in unknown samples, followed by calculation of the average mass of the RNA present in EVs. In the next chapter, one of the human body fluids, i.e., saliva, was chosen for the quantification of EVs. Salivary EVqCE was developed in a similar way as EVqCE for cell lines since the general theories and procedures are practically the same. However, human saliva contains an abundant amount of viscous proteins and ribonuclease (RNase), that were the major obstacles for salivary EVs detection and quantification. The method for the isolation of EVs from the saliva was optimized, and the quantification was performed successfully. The average mass of RNA in saliva EVs was also calculated and analyzed. The concentration of saliva EVs in unknown samples were compared with the results from NTA and flow cytometry to validate the salivary EVqCE. In the last chapter, I described the application of EVqCE to study the quality control of EVs. The method calculates the degradation level of EVs samples under different conditions, providing a potential way for real-time monitoring of the EVs status in the body fluid sample.

In the presented studies, chitin synthase containing nanoparticles (chitosomes) from the yeast Saccharomyces cerevisiae lacking the chs3 gene were investigated. Two step centrifugations using sucrose gradients led to considerable purity of the chitosomal complexes. Chitin synthase I activity was determined via a previously described ELISA based WGA assay and a novel assay using the Streptomyces chitin binding protein CHB1, which provided good tools to follow the purification procedure. In collaboration, it could be shown that the complexes produce fibers in the presence of the substrates uridine-diphosphate-N-acetylglucosamine (UDP-GlcNAc) and N-acetylglucosamine (GlcNAc) and this reaction was inhibited by addition of chitin synthase inhibitor nikkomycin Z. These results demonstrate for the first time that CSI containing chitosomes can be gained. Investigation of the purified nanocomplexes with CSI activity led to the additional conclusion that proteins of the glycolytic pathway such as glyceraldehyde-3-phosphate (GAPDH isoform Tdh3), enolase (Eno1), pyruvate decarboxylase (Pdc1) and pyruvate kinase (Pyk1) are also concentrated around the peak of CSI activity. The presence of these proteins in the pure chitosomes was further verified via testing for their individual enzymatic activities and by antibody studies. The relative levels of GAPDH, Pdc1 and Pyk1 were found to be higher in comparison to enolase; however GAPDH and Pdc1 proteins had a broad distribution across the purification gradient and were also found in neighboring fractions of peak of CSI activity. In addition to these, two high molecular weight proteins showing similarity to glucan synthase and fatty acid synthase were also found in such fractions as analyzed via MALDI-MS. In future it will be worthwhile to ascertain the active functional relationships among the different proteins found in chitosomal preparations using immuno fluorescence co-localization studies.

abstract: Extracellular Vesicles (EVs), particularly exosomes, are of considerable interest as tumor biomarkers since tumor-derived EVs contain a broad array of information about tumor pathophysiology including its metabolic and metastatic status. However, current EV based assays cannot distinguish between EV biomarker changes by altered secretion of EVs during diseased conditions like cancer, inflammation, etc. that express a constant level of a given biomarker, stable secretion of EVs with altered biomarker expression, or a combination of these two factors. This issue was addressed by developing a nanoparticle and dye-based fluorescent immunoassay that can distinguish among these possibilities by normalizing EV biomarker level(s) to EV abundance, revealing average expression levels of EV biomarker under observation. In this approach, EVs are captured from complex samples (e.g. serum), stained with a lipophilic dye and hybridized with antibody-conjugated quantum dot probes for specific EV surface biomarkers. EV dye signal is used to quantify EV abundance and normalize EV surface biomarker expression levels. EVs from malignant (PANC-1) and nonmalignant pancreatic cell lines (HPNE) exhibited similar staining, and probe-to-dye ratios did not change with EV abundance, allowing direct analysis of normalized EV biomarker expression without a separate EV quantification step. This EV biomarker normalization approach markedly improved the ability of serum levels of two pancreatic cancer biomarkers, EV EpCAM, and EV EphA2, to discriminate pancreatic cancer patients from nonmalignant control subjects. The streamlined workflow and robust results of this assay are suitable for rapid translation to clinical applications and its flexible design permits it to be rapidly adapted to quantitate other EV biomarkers by the simple swapping of the antibody-conjugated quantum dot probes for those that recognize a different disease-specific EV biomarker utilizing a workflow that is suitable for rapid clinical translation. / Dissertation/Thesis / Doctoral Dissertation Biomedical Engineering 2019

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23411号 / 医博第4756号 / 新制||医||1052(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 妹尾 浩, 教授 藤田 恭之, 教授 山下 潤 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

Malaria is one of the worlds most deadly infectious diseases and results in almost a million deaths each year, largely in children under the age of five in Sub-Saharan Africa. Outside Africa, malaria is responsible for a large number of cases in the Amazon rainforest of Brazil, Middle East, and in some areas of Asia [37]. According to the World Health Organization, there was an estimated 655, 000 deaths from malaria in 2012. Malaria is caused by a eukaryotic Apicomplexan parasite, Plasmodium, which has three distinct life cycles occurring in the midgut of the female Anopheles mosquito, the liver of the human host, and human erythrocytes. When the parasite infects the erythrocyte, some induced cell host modifications are made in order to accommodate growth. During its intra-erythrocytic life cycle, the malaria parasite traffics numerous proteins to a set of unique destinations within its own plasma membrane including the digestive vacuole, the apicoplast, rhoptries, and micronemes. Vesicular transport is an essential process in eukaryotic cells. This coordinated process is responsible for moving thousands of proteins between compartments within the cell. Essential to the targeting and fusion of protein transport vesicles in eukaryotes are SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors), a family of fusogenic proteins that are localized to distinct intracellular compartments [11]. Studies performed in our laboratory have identified 18 proteins putatively belonging to the PfSNARE family [2]. To date the exact role of PfSNAREs in the unique trafficking pathways of malaria is undetermined. Of particular interest to our study is PfVAMP8. In model eukaryotic organisms, VAMP8 containing vesicles deliver cargo to lysosomes and are involved in endocytosis. The food vacuole of the parasite is very similar to that of lysosomes and is essential to parasite survival. The study aims to identify the organelle(s) to which PfVAMP8 is localized and characterize membrane-association properties of this parasite’s R-SNARE protein. We believe that PfVAMP8 would localize to unique compartments in the parasite protein network flow. An in depth understanding of its mechanisms and localizations could be a key in developing novel anti-malarials. This study aims to identify the organelle(s) to which PfVAMP8 are localized, determine the trafficking determinants of this protein and determine this proteins’ expression and membrane association during the intra-erythrocytic stages of Plasmodium falciparum. Our immunofluorescence studies with known biological markers reveals that, PfVAMP8 passes through the endoplasmic reticulum, Golgi, and localizes to the food vacuole during trophozoite and schizont stage. Further characterization of the membrane association properties of the protein in this study reveals that PfVAMP8 is a soluble integral membrane protein with amphipathic characteristics.

<p>Skeletal muscle is dependent upon its microvasculature to deliver oxygen and substrates to support the metabolic demands of muscle contraction. Skeletal muscle capillary density is determined by a variety of factors including muscle fiber metabolic phenotype and mitochondrial volume as well as prior exercise training status. Additionally, muscle microvascular density and function can diminish with age, contributing to several age-related muscle dysfunctions. Skeletal muscle fibers regulate their surrounding microvasculature through the release of angiogenic and angiostatic signaling factors. A robust increase in angiogenic signaling from skeletal muscle facilitates increases in muscle capillarization following endurance exercise. Extracellular vesicles (EV) are membrane bound signaling factors secreted by every cell type. Skeletal muscle-derived EVs (SkM-EVs) may help facilitate numerous signaling functions of skeletal muscle including between skeletal muscle and its microvasculature.</p> <p>The primary aim of my dissertation research was to determine the signaling roles that SkM-EVs in regulating endothelial cell homeostasis and angiogenesis in states of aging and health. Chapter 1 provides an overview of the relevant literature. Chapter 2 represents an investigation into how age-related cellular senescence impacts the angiogenic potential of skeletal muscle progenitor cells. We found that stress-induced senescence increases release of small EVs and has pro-senescent and angiostatic effects on culture endothelial cells. In Chapter 3 we compared the release, contents, and angiogenic potential of SkM-EVs collected from primarily oxidative or primarily glycolytic skeletal muscle tissue in mice. We found that oxidative muscle tissue secretes more EVs than glycolytic muscle tissue, and the miR contents of EVs differ greatly between the two phenotypes. Additionally, EVs from oxidative tissue enhanced endothelial cell migration and tube formation compared to glycolytic tissue EVs, in a potentially nitric oxide mediated fashion. In Chapter 4, we tested how PGC-1α overexpression effected myotube EV release and angiogenic potential. We found that PGC-1α overexpression did not impact myotube EV release, but increased the angiogenic signaling potential of SkM-EVs. Chapter 5 is a brief summary of the results and limitations of the projects presented in Chapters 2-4, with a short discussion of potential future research directions.</p>

Almost half of patients recovering from open chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that post-operative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent post-operative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis was markedly attenuated by EV pre-treatment; an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of extracellular vesicles at the time of open-chest surgery shows prominent anti-inflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent post-operative AF by reducing atrial inflammation and fibrosis.

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13561号 / 論医博第2290号 / 新制||医||1067(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 武藤 学, 教授 今中 雄一, 教授 村川 泰裕 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM