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

Arsenic is a toxic metalloid that continues to contaminate the water and food sources of millions of people globally. Among the numerous health effects of arsenic exposure are urothelial toxicity and cancer. In recent years, small extracellular vesicles (SEVs) have been shown to be vital in intracellular communication and have been used in clinical studies as biomarkers of disease. The overall goal of this thesis is to understand the mechanisms of cell communication during arsenic exposure and to develop minimally invasive biomarkers for the toxic responses. The specific objectives are to: a) determine if SEVs released from arsenic exposed urothelial cells are responsible for mediating urothelial toxicity; and b) assess the application of urinary SEVs as novel biomarkers of arsenic exposure in an exposed population. The hypothesis leading this research is that the biology and protein packaging profile of urothelial SEVs are altered following arsenic exposure because of the induction of cell stress signaling pathways. I also hypothesize that urinary SEV proteins can be used as biomarkers of arsenic exposure because they are positively correlated with urinary arsenic concentrations in an exposed population. SVHUC1 human urothelial cells were dosed with sodium meta arsenite (1, 2, and 5 uM) for 48 hours. T24 urothelial carcinoma cells were also grown in parallel to compare for carcinogenicity. A label-free quantitative proteomics approach was used to assess the differentially expressed proteins in the cell lysate and the SEVs extracted from the culture media to determine the mechanistic pathways involved and how well the protein profiles in SEVs correlate with those in the cell lysate. SEVs were isolated from the archived urine samples of participants (n=36) enrolled in the Yellow Knife Health Effects Monitoring Program (YKHEMP) and two potential biomarkers, transforming growth factor beta receptor 1 (TGFBR1) and ribonuclease inhibitor 1 (RNH1), were measured by an enzyme linked immunosorbent assay (ELISA). SEVs in all samples were successfully characterized based on their size (50-200 nm) and positive antibody array for eight protein markers indicating their endosomal biogenesis. The total number of SEVs was not shown to increase following arsenic exposure in the in vitro study. However, the cancerous T24 cells had nearly four times higher numbers of SEVS compared to the non-cancerous SVHUC1 cells. The changes in the protein profiles in SEVs released following arsenic dosage indicated activation of pathways important for cell survival, viability, and migration and inactivation of pathways related to cell death and necrosis which were also observed in the paired cell lysate samples. Comparison between paired SEV and cell lysate samples, however, indicated selective SEV packaging of proteins which may be for the purpose of intracellular communication. Comparative assessment of SEVs from T24 and arsenic exposed SVHUC1 cells showed similar activation of cancer related pathways including those responsible for malignant tumors and increased proliferation rates. From the in vitro study results, we identified 8 potential SEV biomarkers. Of which, TGFBR1 showed the most promising association, having been positively associated with both inorganic arsenic and cadmium concentrations in urine samples. This thesis showed that SEVs are important mediators of arsenic exposure in urothelial cells and highlighted the comparability of SEV and cell lysate analysis. Furthermore, TGFBR1 was identified as a promising biomarker of arsenic exposure for its positive association with increased arsenic both in vitro and in human biomonitoring analysis.

Mesenchymal stem/stromal cells (MSCs) release Extracellular vesicles (EVs) that are believed to play a major role in nerve regeneration after stroke. However, a major complication when trying to transition MSC-EVs from a pre-clinical to clinical setting is the convenient long-term storage of MSC-EVs. Therefore, we developed a strategy to freeze dry MSC-EVs to store them for more practical clinical applications. We first determined the optimal trehalose concentration for freeze drying the MSC-EVs, and we subsequently investigated the optimal freezing conditions. It was determined that 100 mM of trehalose and freezing temperature at -20°C were the optimal conditions to freeze dry the EVs. The therapeutic capabilities of the freeze-dried MSC-EVs was tested via tube formation assay and co-culturing them with neural stem/progenitor cells (NSPCs). It was found that human vein umbilical endothelial cells (HUVECs) treated with rehydrated MSCEVs promoted tube formation suggesting the trophic factors in the MSC-EVs survived the freeze-drying process. As for the NSPC co-culture, all treatments involving rehydrated MSC-EVs protected by trehalose during the freeze-drying process promoted proliferation and did not affect their ability to differentiate into oligodendrocytes, astrocytes, or neurons. Determining the optimum freezing-drying conditions allows us to stockpile a large amount of MSC-EVs at room temperature for on-demand applications.

Microbiota-released extracellular vesicles (MEVs) have emerged as key players in intercellular signaling in host-microbiome communications. However, their role in gut-brain axis signaling has been poorly investigated. Here, we performed deep multi-omics profiling of MEVs generated ex-vivo and from stool samples to gain insight into their role in gut-brain-axis signaling. Metabolomics unveiled a wide array of metabolites embedded in MEVs, including many neurotransmitter-related compounds such as arachidonyl-dopamine (NADA), gabapentin, glutamate, and N-acylethanolamines. To test the biodistribution of MEVs from the gut to other parts of the body, Caco-2, RIN-14B, and hCMEC/D3 cells showed the capacity to internalize labeled MEVs through an endocytic mechanism. Additionally, MEVs exhibited dose-dependent paracellular transport through Caco-2 intestinal cells and hCMEC/D3 brain endothelial cells. Overall, our results revealed the capabilities of MEVs to cross the intestinal and blood-brain barriers to delivering their cargo to distant parts of the body.

Levels of circulating large extracellular vesicles (L-EVs) are increased in individuals with type 1 diabetes mellitus (T1DM) and associated with increased cardiovascular risk. T1DM in pregnancy induces vascular injury leading to adverse maternal and neonatal outcomes. Conversely, exercise has been shown to improve cardiovascular and metabolic health in pregnancy and may represent a non-pharmacological approach to improving pregnancy outcomes. Assessment of vascular health may aid in the identification of individuals at risk of complications and allow for intervention with strategies to improve the maternal vasculature. Unfortunately, there is a paucity of strategies for assessing vascular health in pregnant women. L-EVs are membrane-encapsulated particles released from stressed/injured cells. They are emerging biomarkers of vascular health. The purpose of this thesis was to assess the impact of T1DM and pregnancy on L-EV levels and protein composition, the relationship between L-EVs and pregnancy outcomes and the effect of exercise on L-EV levels. In aim #1, I observed that high levels of L-EVs are predictive of adverse pregnancy outcomes. In aim # 2, I examined the protein composition of circulating L-EVs in hypertensive, diabetic and healthy mice models. Diabetes-enriched proteins were involved in inflammation, SNARE signaling and NAD+ biogenesis. The changes were found in L-EV protein content were consistent with proteins associated with inflammation, cytoskeletal organization, and angiogenesis. Finally, in aim #3, I examined the changes in plasma L-EVs after an acute bout of moderate-intensity aerobic exercise in healthy pregnant and non-pregnant women. I observed that circulating L-EVs significantly decreased after the acute exercise only in non-pregnant individuals. Taken together, my thesis work advances knowledge on L-EVs in T1DM, pregnancy, and hypertension and sets the stage for future work on L-EVs as predictive biomarkers, for molecular profiling, and for monitoring of vascular health interventions in pregnancy.

京都大学 / 新制・課程博士 / 博士(医学) / 甲第24993号 / 医博第5027号 / 新制||医||1069(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 滝田 順子, 教授 岩田 想, 教授 万代 昌紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

No description available.

High-frequency irreversible electroporation (H-FIRE) is a non-thermal ablation technique that uses intense, short, bipolar electrical pulses to induce cell death in cancerous tissues. It's being studied for treating hepatocellular carcinoma (HCC) in dogs. Previous in vitro research suggests H-FIRE may impact the release of extracellular vesicles (EVs). This study aims to explore how H-FIRE affects peripheral extracellular vesicle (EV) dynamics, potentially providing insights into its broader systemic effects and implications for biomarker development in canine liver cancer treatment. Dogs diagnosed with HCC were enrolled in a clinical trial. H-FIRE was applied to tumors, followed by surgical resection at three different time points. Peripheral blood samples were collected before and immediately after H-FIRE treatment. Plasma was isolated, aliquoted, and stored at -20°C. EVs were enriched from plasma via filtration and ultracentrifugation. Nanoparticle Tracking Analysis (NTA) quantified EV concentration and size distribution. Ten patients provided pre- and post-treatment plasma samples. The median EV concentration in peripheral blood increased from 2.56 x 10^11 particles/ml pre-treatment to 2.68 x 10^11 particles/ml post-treatment (p = 0.0048). The mean EV size decreased from 99.32 nm pre-treatment to 87.82 nm post-treatment (p = 0.007). The mode of EV size decreased from 83 nm pre-treatment to 70.5 nm post-treatment (p = 0.0076). The results of this study raise intriguing questions on the significance of changes in extracellular vesicle size and concentration post-treatment, as well as the potential clinical implications of these changes. / Master of Science / High-frequency irreversible electroporation (H-FIRE) is a new method to destroy cancer cells without using heat. It's being tested for treating liver cancer in dogs. Previous lab studies suggest H-FIRE might affect the release of small structures known as extracellular vesicles (EVs). This study aims to see how H-FIRE affects EVs in the blood of dogs with liver cancer. Understanding these changes could help develop new ways to diagnose and treat the disease in dogs and humans. Dogs with liver cancer were part of a study. They received H-FIRE treatment followed by surgery, and blood samples were taken before and right after treatment. The plasma was separated and stored. EVs were collected from plasma using special methods, including Nanoparticle Tracking Analysis (NTA) to help measure the number and size of EVs.

No description available.

As células-tronco processam e são sensíveis a múltiplos sinais dentro de seu microambiente, os quais podem exercer influências que regulam seu destino e sua função de forma espaço temporal. Neste contexto, células podem exercer seu papel biológico por transferir informação genética e alterar expressão gênica de alvos celulares através de vesículas extracelulares (VEs). MicroRNAs (miRNAs), uma classe de pequenos RNAs não codificantes, podem ser encontrados nestas vesículas e são considerados moléculas efetivas no controle do neurodesenvolvimento por regular genes chaves em tempo controlado. Pouco se sabe sobre como a diferenciação influencia o conteúdo de miRNAs liberados via VEs revelando o papel dos mesmos no microambiente de cada etapa do comprometimento neural. Assim, a proposta deste estudo foi analisar o perfil de miRNAs intracelulares e presentes em VEs envolvidos na diferenciação neural dopaminérgica de células-tronco pluripotentes e identificar os possíveis alvos regulados pelos mesmos como mecanismo de estabelecimento de um destino neural específico. / Stem cells sense and process multiple signals in their microenvironment, which can exert influences that regulate cell fate and function in a time spatial manner. In this context, the stem cells can exert their biological role transferring genetic information and altering the genetic expression of target cells through extracellular vesicles (EVs). MicroRNAs (miRNAs), a class of small non coding RNAs, can be found in those EVs and are considered effective molecules in the control of neurodevelopment and differentiation by regulating key genes in a time specific manner. However, little is known about how the cell differentiation influences the miRNAs content released through EVs, and how these molecules function in the microenvironment of each phase of neural commitment. Thus, the purpose of this study was to analyze the intracellular and EVs miRNAs profiles involved in the dopaminergic differentiation of pluripotent stem cells in attempt to identify possible targets regulated by miRNAs as a mechanism of specific neural fate decision.