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Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking AnalysisDeighan, Clayton J. January 2015 (has links)
No description available.
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Characterization of the Immune Stimulated Release of Extracellular Vesicles from Murine CellsNorrie, Andrew 31 March 2021 (has links)
Introduction: Viruses, extracellular vesicles (EVs) and endogenous retroviruses (ERVs) are types of sub-micron particles which are known to be released from a vast range of cell types, across many species. There are many medically relevant sub-micron particles which can enter healthy cells and enable the intercellular delivery of functional host-derived and foreign products, through their enclosed lipid layers. While multiple particle subsets have been identified, many of the properties, behaviors and biochemical functions have not been fully described and have yet to be characterized.
Materials and Methods: CD4⁺ naïve T-cells were isolated from female C57BL6/N mice and stimulated with varying concentrations of PMA/I. In addition to concentration, the length of PMA/I activation was assessed. Supernatants and cells were harvested, filtered, and stained to be subsequently analyzed by Nanoscale Flow Cytometry, Nanoparticle Tracking Analysis and Flow Cytometry. Particle populations were quantified and sorted by size, by NTA. Labelling dye CFSE was used in conjunction with fluorescently conjugated CD81 and CD9 antibodies to separate EVs, including exosomes, from background signal. Naïve T-cell purity, viability and levels of activation were assessed by flow cytometry using CD3, CD4 and CD62L antibodies and viability staining.
Results: Increasing PMA concentration led to a global increase in particles by T-cells and a specific increase in smaller particle production and were demonstrated to be significant by Welch’s T-test, when compared to non-activated and DMSO controls (p<0.0001). In addition to concentration, activation length also correlated with increases in total particle counts and a specific increase in the secretion of smaller particles in comparison to non-activated and DMSO controls (p<0.0001). Labelling techniques by NFC revealed an increased presence of CFSE-CD81 positive and CFSE-CD9 positive particles secreted by T-cells, treated for 24 hours, compared to the 0- and 12-hour timepoints.
Conclusion: This work demonstrates preliminary steps and outlines methods to begin assessing discrete particle populations and subsets secreted by murine naïve T-cells. Being able to identify patterns of particle secretions by naïve T-cells, especially under immune-stimulated conditions, may be the solution to uncovering the necessary information on EV physiology, that is required to understand the roles EVs play in pathology and how these conserved pathways may lead conditions to become exacerbated. This knowledge is essential to uncovering the roles EVs play in pathophysiology, and in the development of novel rapid diagnostic tests, to screen for cancers, infections, autoimmune disorders, and numerous other pathological conditions.
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The potential of exosomes as a tool to guide human pluripotent stem cells to insulin producing cells.Mohamed, Idil January 2024 (has links)
The ability of human pluripotent stem cells (hPSCs) to differentiate into different types of cells has been regarded as a significant discovery in the development of cell replacement therapy for type 1 diabetic patients. MicroRNAs can be transported to recipient cells via vesicles, so-called exosomes. Exosomal microRNA differ from those of the parent cells suggesting that cells possess an active selecting mechanism of exosomes and their contents. Therefore, microRNAs may directly or indirectly regulate the expression of pancreatic islet-specific transcription factors to control the differentiation and maturation of pancreatic islet cells. In this study, the dynamic expression of exosomal and intracellular microRNAs from human pancreatic islets were analyzed and were compared with that in stem cell-derived islet-like clusters. The study also aimed to analyze the expression levels of intracellular microRNA in human pancreatic islets and stem cell-derived islet-like clusters compared to exosomal microRNA extracted from human islet media and stem cell media, respectively. The primary method of exosome extraction was ultracentrifugation, followed by microRNA isolation using a kit. The exosomes were then characterized with NTA, and the isolated microRNAs were detected using RT-qPCR. The results showed that the expression of microRNAs was generally low in human islets compared to isolated exosomes. The microRNA expression levels in stem cell-derived islet-like clusters and their respective isolated exosomes were also analyzed and it showed that let-7a, miR-375 and miR-26a were more abundant in exosomes. The results can contribute to the generation of more functional stem cell-derived islet-like cell clusters prepared from hPSCs to some extent. However, continued research in this area is required.
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Impact of psychotomimetic molecules on glutamatergic N-Methyl-D-Aspartate receptors surface trafficking / Impact de molécules psychotomimétiques sur la diffusion de surface des récepteurs glutamatergiques de type N-Methyl-D-AspartateJezequel, Julie 18 November 2016 (has links)
Les récepteurs glutamatergiques de type N-Méthyl-D-Aspartate (RNMDA) jouent un rôle majeur dans de nombreux processus physiologiques, et leur implication dans la physiopathologie de certains troubles neuropsychiatriques tels que la schizophrénie est suggérée par un robuste faisceau de données cliniques et précliniques. Cependant, les mécanismes cellulaires et moléculaires conduisant à une telle dérégulation des RNMDA restent inexpliqués. La diffusion membranaire, mécanisme de contrôle spatial et temporel de la distribution des RNMDA à la surface des neurones, constitue un puissant régulateur de la transmission synaptique. Mon projet de thèse repose ainsi sur l’hypothèse originale qu’une altération de la diffusion de surface des RNMDA jouerait un rôle central dans l’émergence de troubles psychotiques. Afin d‘explorer cette piste, j’ai étudié l’impact de molécules aux propriétés psychomimétiques (i.e induisant un état psychotique) sur la diffusion de surface des RNMDA. Les résultats obtenus au cours de ma thèse démontrent que des molécules psychomimétiques, aux modes d’action distincts (antagonistes du RNMDA et autoanticorps anti-RNMDA), perturbent la diffusion membranaire ainsi que la localisation synaptique des RNMDA, conduisant à terme à des défauts de transmission glutamatergique. Mon travail de thèse propose donc qu’un défaut de diffusion membranaire des RNMDA conduirait à des altérations fonctionnelles pouvant contribuer à l’émergence de troubles psychotiques. L’ensemble de mon travail apporte ainsi un regard nouveau sur la mécanistique des troubles psychotiques et ouvre la voie à de nouvelles pistes thérapeutiques. / Glutamatergic N-Methyl-D-Aspartate receptors (NMDAR) play a key role in many physiological processes, and their implication in the pathophysiology of several neuropsychiatric disorders is now well established. Multiple lines of evidence converge towards a dysregulation of the NMDAR in psychotic disorders such as schizophrenia (SCZ). However, the molecular and cellular deficits underlying NMDAR dysfunction remain misunderstood. By tightly controlling NMDAR synaptic localization, surface trafficking represents a powerful regulator of synaptic transmission. Could an alteration of NMDAR surface trafficking underlie NMDAR dysfunction and contribute to the emergence of psychotic disorders? To tackle this question, my PhD project aimed at investigating the impact of different psychotomimetic molecules on NMDAR surface trafficking. In the first part of my project, I explored the impact of NMDAR autoantibodies (NMDAR-Ab) from SCZ and healthy subjects. My results revealed that NMDAR-Ab from SCZ patients rapidly disturb NMDAR synaptic trafficking and distribution, through a loss of NMDAR-EphrinB2 receptor interaction, eventually preventing the induction of synaptic plasticity. In the second part of my PhD project, I showed that psychotomimetic NMDAR antagonists also alter NMDAR synaptic mobility and localization. Downregulation of PSD proteins expression prevented NMDAR antagonists-induced deficits, suggesting that such alterations ensue from modifications of NMDAR intracellular interactions. Taken together, these results demonstrate that psychotomimetic molecules profoundly impact NMDAR surface trafficking, supporting a pathogenic role of this unsuspected process in the emergence of psychotic symptoms.
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Determination of titanium dioxide nanoparticles in personal care products / Determination of titanium dioxide nanoparticles in personal care productsKošík, Juraj January 2016 (has links)
Předkládaná diplomová práce se zabývá extrakcí nanočástic oxidu titaničitého z produktů osobní péče, konkrétně opalovacích krémů a následnou charakterizací těchto částic. Počet komerčně dostupných produktů s obsahem nanočástic TiO2 se neustále zvyšuje a to se sebou přináší potřebu vyhodnotit potenciální osud a nepřímou expozici TiO2 nanošástic o různých velikostí a tvarů a zkoumat jejich celý životní cyklus. Bylo zkoumáno použití ultrafiltrace a ultracentrifugace jako extrakční metody. Dvě metody pro extrakci TiO2 nanočástic byly vyvinuty a aplikovány na vzorky opalovacích krémů. Extrahované částice mohou být použity pro ekotoxikologické studie, případně experimenty v mesokosmu. Velikost částic byla stanovena pomocí metody dynamického rozptylu světla a transmisní elektronové mikroskopie.
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In Situ and Ex Situ Study of Nanoparticles Stability and Transformation in Simulated Aquatic Natural Media / Situ och Ex Situ studie av Nanopartiklars Stabilitet och Transformation i Simulerade Akvatiska MiljöerNguyen, Dinh, Samuelsson, Jonathan, Grönvall, Vilma January 2022 (has links)
Nanopartiklar är partiklar med storlekar i området 1 till 100 nm, och som uppvisar nanospecifika egenskaper. Egenskaperna kan variera helt mellan olika typer av nanopartiklar, även bland partiklarna och deras respektive material i bulk format på grund av deras unika storlekar, former och strukturer. I denna studie har vi analyserat storlekarna och den kolloidal stabiliteten på tre nanopartiklar, Co, Y2O3, CeO2 i färskvatten och färskvatten med naturligt organiskt material med hjälp av sonifikation, NTA, AAS och ICP analys. Nanopartiklarna lät vara utsatta i lösningarna i upp till six timmar, där analys med hand av NTA skedde efter den nollte, första och sjätte timmen. Partiklarna uppvisade olika egenskaper, och alla tre partiklar varierade i storlek under experimentet. Kobalt hade tendensen att minska i storlek i bägge lösningar, medan storlekarna på yttrium- och cerium oxid tenderade att variera. För att främja vår förståelse av nanopartiklar, behövs fler studier för att få en full förståelse för de unika egenskaperna hos dessa partiklar. / Nanoparticles are particles with sizes in the range of 1 to 100 nm, which also have nanospecific properties. Their properties vary wildly between different types of nanoparticles, even among nanoparticles and their respective particles in bulk format heeding to their highly individual shapes, sizes and structures. In this study we analyzed the sizes and colloidal stability of three different nanoparticles, Co, Y2O3 and CeO2 in freshwater and freshwater with natural organic matter solutions using sonication, NTA-, AAS-, and ICP analysis. The nanoparticles were exposed to the solutions for up to six hours, with analysis being performed at the zero, first and sixth hour. The particles indeed showed different properties, as all three particles varied in size throughout the experiment. Cobalt had the tendency to decrease in size as time progressed in both solutions, while the mean size of yttrium- and cerium oxide varied. To further our understanding of nanoparticles, more studies need to be performed to properly understand the individual properties of these nanoparticles.
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