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Background: Between 2005 and 2015 the number of new cancer cases per year in Canada rose by 29% and this number is projected to increase to 277,000 cases per year. Ionizing radiation is used as therapy in the majority of cancer cases; however, it can have long-term detrimental effects on the hematopoietic system. Recent work from our lab, in a preclinical model of radiation damage, demonstrated that endurance exercise training can enhance hematopoietic recovery, while obesity can impair it. Extracellular vesicles (EVs) are a mode of cellular communication that has been implicated in regulating hematopoiesis acutely following radiation exposure. However, the long-term, radiation-induced changes to EVs, and the role of exercise and/or obesity at modulating marrow EVs remains unknown. Thus, the purpose of this project was to determine the extent to which obesity and exercise influence the regenerative potential of bone marrow-EVs following radiation. Method: Mice were randomly divided into control (n=20; CON) or high fat diet (n=20; HF) groups, then subdivided into exercise-trained (EX, n=10) or sedentary (SED, n=10). Mice underwent whole-body exposure to a 3 Gy dose of gamma-radiation at age 13 weeks of age followed by bone marrow collection at 20 weeks of age. EVs were then isolated from the bone marrow by ExoQuick and ultracentrifugation. A non-irradiated, sedentary, control diet group (n=10) was used to determine the effects of radiation alone. Data was evaluated using repeated-measures three-factor (diet, exercise, time) and two-factor ANOVA. Results: High fat diet-induced changes in body weight and composition and altered food consumption (p<0.05). Isolated EVs measured between 78 and 195 nm and western blot confirmed the presence of EV protein markers Alix, TSG101, and Flotillin. No size difference was observed between the groups. The concentration of EVs in irradiated mice was significantly lower compared to EVs from control mice (p<0.01). Radiation, obesity, exercise, or their combination had no significant effect on hematopoietic stem progenitor cells (HSPC) content in co-culture assays. Conversely, EVs from irradiated mice significantly increased the number of CFU-GEMM, CFU-G, and the TOTAL number of colonies compared to EVs from non-irradiated mice (p<0.01). However, EVs from the CON+SED, CON+EX, HF+SED, and HF+EX groups did not have a significant effect on colony formation. Conclusion: Our findings demonstrate that ionizing radiation can diminish the concentration of bone marrow-EVs and that irradiated bone marrow-EVs can increase the total number of myeloid colonies formed in vitro. These results suggest that radiation induces myelopoiesis via a mechanism that includes EVs; however, exercise and obesity induce their effects via a different mechanism.

The exocyst, an octameric protein complex conserved among all eukaryotes, mediates tethering of the vesicle prior to its fusion with the target membrane. Apart from the function of exocyst in exocytosis, new studies from both mammalian and plant fields report its involvement in the cellular self-eating process called autophagy. In land plants the number of paralogs of some exocyst subunits is extraordinarily large. There are 23 paralogs of Exo70 subunit in Arabidopsis thaliana. It is supposed that these paralogs have acquired functional specialization during the evolution - including involvement in autophagy. Using yeast two- hybrid assay it is shown here that Exo70B1 and Exo70B2, but not other Arabidopsis Exo70 paralogs interact with Atg8, an autophagosomal marker. The proximity of these two paralogs and Atg8 in vivo was confirmed by independent Förster resonance energy transfer (FRET) method. Interestingly, interaction of Atg8f with Exo70B2 paralog appears to be stronger than with Exo70B1. Exo70B1-mRUBY expressed under the natural promoter shows punctate membrane structures that are mostly static. That changes after the tunicamycin treatment - movement of some of these dots was induced. Homology modeling of Exo70B1 and Exo70B2 proteins tertiary structure in combination with bioinformatic prediction based...

In the recent years, there has been a growing number of publications indicating at the involvement of plant secretory pathway in defense against phytopathogens. Specifically, roles of plant exocyst complex have been explored in deeper detail in current research. Yet, exactly how exocyst- mediated exocytosis contributes to secretion of antimicrobials and cell wall-based defense remains unclear. In the presented Dissertation, I provide both experimental evidence and devise further hypotheses on selected exocyst's subunits in plant immune reactions. Particularly, I show that EXO70B1 exocyst subunit interacts with immunity-related RIN4 protein. Cleavage of RIN4 by AvrRpt2 Pseudomonas syringae effector protease releases both RIN4 fragments and EXO70B1 from the plasma membrane when transiently expressed in Nicotiana benthamiana leaves. I speculate on how this might have an implication in regulation of polarized callose deposition. In a co-authored opinion paper, we also hypothesize that EXO70B1-mediated autophagic degradation of TN2 resistance protein prevents its hyperactivation and lesion mimic phenotype development. In addition, in collaboration with my colleagues, I present data on EXO70H4's engagement in PMR4 callose synthase secretion, required for silica deposition. Representing a possible...

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22396号 / 薬科博第118号 / 新制||薬科||13(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 髙倉 喜信, 教授 山下 富義, 教授 小野 正博 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

One of the consequences of G-protein-coupled receptor activation is stimulation of phosphoinositol metabolism, leading to the generation of IP 3 and its metabolites 1,3,4,5-tetrakisphosphate (IP4) and inositol 1,2,3,4,5,6-hexakisphosphate (IP6). Previous reports indicate that high inositol polyphosphates (IP4 and IP6) are involved in clathrin-coated vesicular recycling. In this study, we examined the effects of IP4 and IP6 on spontaneous transmitter release in the form of miniature endplate potentials (MEPP) and on enhanced vesicular recycling by high K+ at frog motor nerve endings. In resting conditions, IP4 and IP6 delivered intracellularly via liposomes, caused concentration-dependent increases in MEPP frequency and amplitude. Pretreatment with the protein kinase A (PKA) inhibitor H-89 or KT 5720 reduced the IP4-mediated MEPP frequency increase by 60% and abolished the IP6-mediated MEPP frequency increases as well as the enhancement in MEPP amplitude. Pretreatment with antibodies against phosphatidylinositol 3-kinase (PI 3-K), enzyme also associated with clathrin-coated vesicular recycling, did not alter the IP4 and IP6-mediated MEPP frequency increases, but reduced the MEPP amplitude increase by 50%. In our previous reports, IP3, but not other second messengers releasing Ca2+ from internal Ca2+ stores, is able to enhance the MEPP amplitude. In order to dissociate the effect of Ca2+ release vs. metabolism to IP4 and IP 6, we evaluated the effects of 3-deoxy-3-fluoro-inositol 1,4,5-trisphosphate (3F-IP3), which is not converted to IP 4 or IP6. 3F-IP3 produced an increase then decrease in MEPP frequency and a decrease in MEPP amplitude. In elevated vesicle recycling induced by high K+-Ringer solution, IP4 and IP6 have similar effects, except decreasing MEPP frequency at a higher concentration (10-4 M). We conclude that (1) high inositol polyphosphates may represent a link between IP3 and cAMP pathways; (2) the IP3-induced increase of MEPP amplitude is likely to be due to its high inositol metabolites; (3) PI 3-K is not involved in the IP 4 and IP6-mediated MEPP frequency increases, but may be involved in MEPP size.

Several chlamydial antigens have been detected in the infected epithelial cell cytosol and on the host cell surface prior to their presumed natural release at the end of the 72-96 h developmental cycle. These extra-inclusion antigens are proposed to influence vital host cell functions, antigen trafficking and presentation and, ultimately, contribute to a prolonged inflammatory response. To begin to dissect the mechanisms for escape of these antigens from the chlamydial inclusion, which are enhanced on exposure to antibiotics, polarized endometrial epithelial cells (HEC-1B) were infected with Chlamydia trachomatis serovar E for 36 h or 48 h. Infected cells were then exposed to chemotactic human polymorphonuclear neutrophils not loaded or pre-loaded in vitro with the antibiotic azithromycin. Viewed by electron microscopy, the azithromycin-mediated killing of chlamydiae involved an increase in chlamydial outer membrane blebbing followed by the appearance of the blebs in larger vesicles (i) everting from but still associated with the inclusion as well as (ii) external to the inclusion. Evidence that the vesicles originated from the chlamydial inclusion membrane was shown by immuno-localization of inclusion membrane proteins A, F, and G on the vesicular membranes. Chlamydial heat shock protein 60 (chsp60) copies 2 and 3, but not copy 1, were released from RB and incorporated into the everted inclusion membrane vesicles and delivered to the infected cell surface. These data represent direct evidence for one mechanism of early antigen delivery, albeit membrane-bound, beyond the confines of the chlamydial inclusion.

The ability of cell-penetrating peptides to cross plasma membranes has been explored for various applications, including the delivery of bioactive molecules to inhibit disease-causing cellular processes. The uptake mechanisms by which cell-penetrating peptides enter cells depend on the conditions, such as the cell line the concentration and the temperature. To be used as therapeutics, each novel cell-penetrating peptide needs to be fully characterized, including their physicochemical properties, their biological activity and their uptake mechanism. Our group has developed a series of highly performing, non-toxic cell-penetrating peptides, all derived from the original sequence of transportan 10. These analogs are called PepFects and NickFects and they are now a diverse family of N-terminally stearylated peptides. These peptides are known to form noncovalent, nano-sized complexes with diverse oligonucleotide cargoes. One bottleneck that limits the use of this technology for gene therapy applications is the efficient release of the internalized complexes from endosomal vesicles. The general purpose of this thesis is to reveal the mechanisms by which our in house designed peptides enter cells and allow the successful transport of biofunctional oligonucleotide cargo. To reach this goal, we used both biophysical and cell biology methods. We used spectroscopy methods, including fluorescence, circular dichroism and dynamic light scattering to reveal the physicochemical properties. Using confocal and transmission electron microscopy we observed and tracked the internalization and intracellular trafficking. Additionally we tested the biological activity in vitro and the cellular toxicity of the delivery systems. We conclude that the transport vectors involved in this study are efficient at perturbing lipid membranes, which correlates with their remarkable capacity to transport oligonucleotides into cells. The improved and distinct capacities to escape from endosomal vesicles can be the result of their different structures and hydrophobicity. These findings extend the knowledge of the variables that condition intracellular Cell-penetrating peptide mediated transport of nucleic acids, which ultimately translates into a small step towards successful non-viral gene therapy.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23225号 / 工博第4869号 / 新制||工||1760(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 秋吉 一成, 教授 跡見 晴幸, 教授 大塚 浩二 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

Detection of cancer at the early stages greatly increases the chance for successful treatment and favourable prognosis for patients. However, a liquid-based biopsy has yet to be developed for most cancers. Extracellular vesicles (EVs) are an attractive candidate for early cancer detection since their surface proteome mirrors the cell of origin. Thus, there is a need for the development of reliable probes that can detect cancer derived EVs. In this thesis, the VBS-1 aptamer was developed to selectively bind to triple-negative breast cancer cell line derived EVs. Initially, several EV isolation methods were compared and isolated EVs were validated and characterized. Aptamer clones were developed by Systematic Evolution of Ligands by Exponential Enrichment to EVs isolated by differential ultracentrifugation and their binding was validated by flow cytometry. The binding partner of the selected VBS-1 aptamer was identified by LC-MS/MS to be the transmembrane protein ATP1A1. The presence of an ATP1A1-positive EV population was validated by flow cytometry. The selected aptamer may find further application in biosensors for the detection of EVs as cancer biomarkers in biological fluids.