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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
11

Mechanistic and Therapeutic Insights of Macrophage MicroRNA in Atherosclerosis

Nguyen, My-Anh 02 October 2019 (has links)
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.
12

Surface Proteome of Extracellular Vesicles and Correlation Analysis for Identification of Breast Cancer Biomarkers

Hüttmann, Nico 25 April 2022 (has links)
Breast cancer (BC) is the second leading cause of death in Canadian women. Detection of the disease at an early stage greatly increases the average 5-year survival rate, however non-invasive early detection methods are not available to-date. Cells release various types of extracellular vesicles (EVs) to mediate intercellular communication by transferring signals in the form of bioactive molecules such as proteins, metabolites, and nucleic acids. Understanding the composition of these biomolecules may shed light on the physiological state of the cell of origin. Therefore, EVs are a promising source of biomarkers for non-invasive detection of BC. However, the surface proteome of EVs is not yet understood well enough to propose BC biomarkers that could be detected directly from biofluids. In this study, small EVs (sEVs) and medium EVs (mEVs) were isolated by differential ultracentrifugation from breast cancer MDA-MB-231 and MCF7, and non-cancerous breast epithelial MCF10A cell lines and analyzed by nano-liquid chromatography coupled to tandem mass spectrometry. EV proteins were analyzed by two approaches: (1) global proteomic analysis and (2) enrichment of EV surface proteins by labelling surface-accessible proteins with a Sulfo-NHS-SS-Biotin reagent. Potential BC biomarkers were obtained from the first approach (1) by identifying the presence of cell line specific sEV proteins, filtering for membrane/surface proteins using UniProt annotations, and predicting the co-localization of proteins on sEVs with known EV marker proteins (CD63, CD9, CD81) by correlation analysis. This resulted in 11 potential BC sEV biomarkers (C8A, AXL, ST14, FAM20B, PROM2, CLDN3, ITGA7, MEGF10, SHISA2, GJC1, IFNGR1); the presence of ST14, CLDN3 and ITGA7 was validated by Western blot analysis. The surface labelling approach (2) enriched proteins previously not identified using the first approach (1). Potential general BC biomarkers were selected from surface proteins commonly identified from MDA-MB-231 and MCF7, but not identified in MCF10A EVs. Annotation with known BC disease associations from DisGeNET yielded 9 and 2 potential surface proteins on sEVs and mEVs, respectively. This study demonstrates the emerging role of EVs as a rich source of known and novel biomarkers which may be used for non-invasive detection of BC.
13

Assessing the Impact of Maternal Physical Activity on Small Extracellular Vesicles and Placental Vascularization During Pregnancy

Mohammad, Shuhiba 11 August 2022 (has links)
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.
14

CIRCULATORY AND SKELETAL MUSCLE EXOSOME RESPONSE IN OLD PARTICIPANTS FOLLOWING A 12-WEEK RESISTANCE TRAINING PROGRAM

Xhuti, Donald January 2021 (has links)
Sarcopenia is the age-related progressive loss of skeletal muscle (SkM) mass, function, and strength. It has been well elucidated that resistance exercise can attenuate the development of sarcopenia. A population of extracellular vesicles, termed ‘exosomes’ (EXO), can contain microRNA and facilitates intercellular communication, including within SkM, though the response to prolonged training is not well understood. Given the potential role of SkM-derived exosomes in the response to exercise, we examined older adults (n = 30, OLD) before (PRE) and after a 12-week (POST), resistance training program. Healthy, young controls (n = 12, YNG) were used for comparison of baseline measures. Exosomes were isolated from platelet-free plasma using size exclusion chromatography in combination with ultracentrifugation (SEC-UC) and characterized via western blotting, nanoparticle tracking analysis and electron microscopy. To assess exosome biogenesis and miRNA synthesis in skeletal muscle, biopsies were taken from the vastus lateralis. Circulating EXO-enclosed and SkM miRNA expression was measured using RT-PCR. In SEC-UC isolates, EXO-markers CD81 and CD9 were significantly lower in PRE compared to YNG (p<0.05) but did not change with training. At baseline, ALIX, TSG101 and CD63 (markers of exosomes) were not altered with aging as compared to YNG; however, their expression significantly increased with training (p<0.05) Circulating EXO-derived mir-1, -133, -23 and -27a were significantly lower in expression of OLD participants as compared to YNG. Following resistance training, their expression significantly increased (p<0.05), returning to a YNG phenotype. Next, we aimed to investigate the contribution of skeletal muscle in the exosome responses. Our data indicate that a small fraction of circulatory exosomes may originate from skeletal muscle. In addition, in biopsy-derived SkM tissue, expression of proteins involved in EXO and miRNA biogenesis (Alix, XPO-5, DICER) were significantly higher in PRE compared to YNG (p<0.05), and further increased with resistance training (POST, p<0.05). Expression of Rab27a, a marker of exosome trafficking, was significantly higher in PRE (p<0.05) but did not respond to training. In conclusion, here we show alterations in circulating EXO content and cargo with age and resistance training partially restores the values to a younger phenotype. / Thesis / Master of Science in Medical Sciences (MSMS) / Aging is the slow and time-dependent process that our organs, down to the cellular level, deteriorate in function reducing the biological fitness of our bodies. Aging specific to skeletal muscle, or sarcopenia, is especially important because skeletal muscle makes up 40% of our weight, is essential for posture, balance, locomotion and breathing. Sarcopenic individuals have low muscle mass, strength, and function and as a result are associated with low independence in activities of daily living and increased risks of falls and fractures. Exercise, and in particular resistance training, has been shown to be beneficial and cost-effective in treating sarcopenia and delaying aging throughout the body. Part of the underlying mechanism regarding how exercise affects us in a multi-systemic manner is not well understood. We know that skeletal muscle releases a multitude of molecular factors during exercise. Amongst them, extracellular vesicles and specifically exosomes are worth investigating because they have been shown to function in intercellular communication by delivering molecular signals, called microRNAs, from origin cells to recipient cells throughout the body. In this thesis project, we investigate exosomes in circulation of older individuals before and after a 12-week resistance training program. We found that aging alters the exosome pool in circulation as well as their miRNA content. After resistance training, many of miRNAs altered with age, return to levels comparable to young. In addition, we showed that at the skeletal muscle level, aging and resistance training affect exosome biogenesis and miRNA expressions. In conclusion, we provide evidence that aging significantly alters circulatory exosomes and miRNA and show that resistance training normalizes the miRNA profile to levels seen in exosomes derived from young plasma. How exosomes and their molecular signals change with aging and how exercise affects them gives us an insight on how exercise elicits multi-systemic benefits against aging and sarcopenia.
15

EXTRACELLULAR VESICLES IN THE VASCULATURE: NOVEL MEANS OF COMMUNICATION DURING VASCULAR INSULT

Boyer, Michael, 0000-0001-7080-8767 January 2020 (has links)
Endothelial dysfunction, present in most cardiovascular disease, results in up-regulation of inflammatory adhesion molecules/cytokines, increases in vascular permeability, and decreased vasoprotective factors leading to vascular dysfunction. A novel means of communication between almost all cells are small vesicles containing biologically active proteins, nucleic acids, and lipids known as extracellular vesicles. Despite the advances in cardiovascular biology, the role of extracellular vesicles between endothelial cells and cells of vascular wall are underexplored. Therefore, we hypothesized that endothelial activation results in the release of pro-inflammatory vesicles that initiate inflammatory remodeling of vascular smooth muscle cells of the aorta. Extracellular vesicles were released from both endothelial cells and vascular smooth muscle cells with characteristic size, shape, and content. However, serum-free collection in endothelial cells resulted in endothelial activation of cell in culture and resulted in altered function in vascular smooth muscle cells, characterized by increased monocyte adhesion, altered protein synthesis/signal transduction, and signs of pro-senescent features. These effects were not recapitulated in any combination of endothelial-vascular smooth muscle cell extracellular vesicle communication. Unbiased mass spectroscopy of vascular smooth muscle cell treated with serum-free endothelial vesicles identified several proteins significantly up- regulated, including high mobility group box 1 and 2. Pharmacologic and genetic inhibition of these molecules significantly attenuated NF-kB activation, VCAM-1 expression, and monocyte adhesion. In summation, we suggest a new axis through which endothelial activation releases vesicles that skew the function of vascular smooth muscle cells to phenotype characterized by inflammatory properties through up-regulation of high mobility group box proteins 1 and 2. This highlights the importance of extracellular vesicles as a novel communication method between cells of the vasculature and how alterations in the host cell function may change the function of these vesicles. / Biomedical Sciences
16

Analysis of extracellular RNA in cerebrospinal fluid

Saugstad, Julie A., Lusardi, Theresa A., Van Keuren-Jensen, Kendall R., Phillips, Jay I., Lind, Babett, Harrington, Christina A., McFarland, Trevor J., Courtright, Amanda L., Reiman, Rebecca A., Yeri, Ashish S., Kalani, M. Yashar S., Adelson, P. David, Arango, Jorge, Nolan, John P., Duggan, Erika, Messer, Karen, Akers, Johnny C., Galasko, Douglas R., Quinn, Joseph F., Carter, Bob S., Hochberg, Fred H. 24 May 2017 (has links)
We examined the extracellular vesicle (EV) and RNA composition of pooled normal cerebrospinal fluid (CSF) samples and CSF from five major neurological disorders: Alzheimer's disease (AD), Parkinson's disease (PD), low-grade glioma (LGG), glioblastoma multiforme (GBM), and subarachnoid haemorrhage (SAH), representing neurodegenerative disease, cancer, and severe acute brain injury. We evaluated: (I) size and quantity of EVs by nanoparticle tracking analysis (NTA) and vesicle flow cytometry (VFC), (II) RNA yield and purity using four RNA isolation kits, (III) replication of RNA yields within and between laboratories, and (IV) composition of total and EV RNAs by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and RNA sequencing (RNASeq). The CSF contained similar to 106 EVs/mu L by NTA and VFC. Brain tumour and SAH CSF contained more EVs and RNA relative to normal, AD, and PD. RT-qPCR and RNASeq identified disease-related populations of microRNAs and messenger RNAs (mRNAs) relative to normal CSF, in both total and EV fractions. This work presents relevant measures selected to inform the design of subsequent replicative CSF studies. The range of neurological diseases highlights variations in total and EV RNA content due to disease or collection site, revealing critical considerations guiding the selection of appropriate approaches and controls for CSF studies.
17

Functional characterization of npcRNAs - Intercellular trafficking of generegulatory components via exosomes

Böker, Kai Oliver 15 December 2016 (has links)
No description available.
18

A Rapid Lipid-based Approach for Normalization of Quantum Dot-detected Biomarker Expression on Extracellular Vesicles in Complex Biological Samples

January 2019 (has links)
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
19

Development of Capillary Electrophoresis-Based Methods for Analysis of Extracellular Vesicles Isolated from Cancer Cell Lines and Human Saliva

Ren, Lixuan 16 September 2020 (has links)
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.
20

Tiny but mighty: mesenchymal stem cell-derived extracellular vesicles as a therapeutic in a monkey model of cortical injury

Go, Veronica 17 February 2021 (has links)
Cortical injury, such as that following stroke, is one of the leading causes of long-term disabilities world-wide. While some neuroprotective agents given within hours of stroke can reduce damage, there are currently no neurorestorative therapeutics that can enhance long-term recovery. To address this, we tested Mesenchymal Stem Cell (MSC) derived Extracellular Vesicles (EVs) as a treatment for cortical injury in rhesus monkeys (Macaca mulatta). Monkeys treated with EVs 24 hours after injury and again at 14 days after injury recovered more completely and more rapidly than monkeys given a vehicle control. However, the cellular changes associated with enhanced recovery remained unknown. In this dissertation, it was hypothesized that EVs modulated cells within the brain to enhance recovery after cortical injury. To explore this hypothesis, three specific aims were tested. Aim 1: To determine the effects of EVs on microglial reactivity. Since EVs in this study were derived from MSCs, it was hypothesized that they would have an immunomodulatory effect. Using immunohistochemistry, image analyses, and 3-D reconstruction, we showed that microglia shifted from reactive, damaging phenotypes towards homeostatic, surveilling functions in EV-treated monkeys. These effects correlated with reduced time to recovery, suggesting that reduced microglial reactivity enhanced recovery. Aim 2: To assess the effects of EVs on myelination. Because MSCs have regenerative effects, it was hypothesized that these MSC-derived EVs would improve neurorestoration. Using immunohistochemistry, qRT-PCR, Spectral Confocal Reflectance microscopy, and ELISA, we assessed myelination after cortical injury with and without EV treatment. EVs limited oligodendrocyte damage and increased densities of mature oligodendrocytes to enhance myelin maintenance. These effects correlated with improved recovery, suggesting the importance of myelination in recovery after cortical injury. Aim 3: To assess the neuroprotective role of EVs on infarct volumes. While it was hypothesized that EVs would reduce the densities of inflammatory cells (astrocytes, macrophages/microglia, T-cells), hemosiderin accumulation, and infarct volume, we found that EVs did not alter these endpoints. Collectively, our results suggest that EVs modulated microglia and oligodendrocytes to promote neurorestoration. Overall, these findings demonstrate the therapeutic potential of EVs for neurorestoration after cortical injury.

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