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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.
1

Neutrophil microvesicles restrict the phlogistic activation of macrophages

Rhys, Hefin Ioan January 2017 (has links)
Released in response to cellular activation, microvesicles are a major vector mechanism for the delivery of protein, nucleic acid and bioactive lipid payloads in local tissues and plasma. Large numbers of microvesicles (including those from neutrophils) are found within inflammatory sites, such as the rheumatoid synovium. Human neutrophil microvesicles promote tissue protection, and in some cases repair, by affecting function and phenotype of other inflammatory cells. Of these, tissue macrophages are central to the recovery of homeostasis after an inflammatory insult. The data herein indicate that microvesicles released by activated neutrophils impede lipopolysaccharide and interferon gamma-induced \M1-like" polarisation of macrophages via phosphatidylserine (PtdSer) exposure, and induce annexin A1-dependent release of transforming growth factor beta (TGFb). Macrophages treated with these vesicles stimulate the production of cartilage matrix from chondrocytes, and are unable to induce an inflammatory phenotype in fi broblasts. The efficacy of these vesicles is reproduced in two in vivo models of acute inflammation, zymosan-induced peritonits and K/BxN serum-transfer arthritis. Finally, the possibility of using both autologous, and cell-line-derived microvesicles as pharmacodynamic tools is explored. Microvesicles generated from neutrophils from patients with rheumatoid arthritis are found to be protective, and can outcompete the pro-inflammatory effects of both platelet microvesicles, and those isolated from synovial fluid of patients with rheumatoid arthritis. By building on the observation that anxA1 on microvesicles stimulates TGFb release in macrophages, a cell line was transfected to release anxA1+ microvesicles, and their e ects compared to those of their wild type counterparts.
2

Glycomic insights into microvesicle biogenesis

Batista, Bianca Stella 22 September 2011 (has links)
Cells can mediate intercellular communication by the secretion and uptake of microvesicles, nano-sized membranous particles that carry signaling molecules, antigens, lipids, mRNA and miRNA between cells. The biological function of these vesicles is dependent upon their composition and cellular origin which is regulated by mechanisms that are not well understood. Based on their molecular content, microvesicles may play a role in immune regulation, cancer progression, the spread of infectious agents and numerous other important normal and pathogenic processes. The proteomic content of microvesicles from diverse sources has been intensely studied. In contrast, little is known about their glycomic content. The glycosylation pattern of a protein or lipid plays a key role in determining its functional properties in several ways. Glycans can determine the trafficking of a protein to particular regions of the cell as well as the protein’s half life. In addition, the glycan-dervied oligomerization of glycolipids and glycoproteins is a known mechanism for the activation of receptors and recognition of ligands on the surface of the cell. Glycomic analysis may thus provide valuable insights into microvesicle function. I utilized lectin microarray technology to compare the glycosylation patterns of microvesicles derived from a variety of biological sources. When compared to cellular membranes, microvesicles were enriched in high mannose, polylactosamine, α2-6 sialic acid, and complex N-linked glycans but exclude terminal blood group A and B antigens. The polylactosamine signature in microvesicles from different cell lines derives from distinct glycoprotein cohorts. After treatment of Sk-Mel-5 cells with lactose to inhibit lectin-glycan interactions, secretion of microvesicle resident proteins was severely reduced. Taken together, this work provides evidence for a role of glycosylation in microvesicle-directed protein sorting. / text
3

Measurement and characterisation of microvesicles and nanovesicles in pregnancy and pre-eclampsia

Dragovic, Rebecca January 2011 (has links)
Excessive release of syncytiotrophoblast vesicles (STBM) from the placenta into the maternal circulation may cause the inflammatory response, endothelial dysfunction and activation of the coagulation system characteristic of pre-eclampsia (PE). Consequently, other cell types including platelets, leukocytes, red blood cells (RBC) and endothelium may be activated to release cellular vesicles which exacerbate the disease. This thesis aimed to develop methodology for enumerating and phenotyping STBM and the other vesicle types to determine whether they could be used as biomarkers for PE. In vitro derived STBM and vesicles from the other cells of the vascular compartment were examined to select a suitable panel of antibodies to analyse these same vesicle types in plasma samples from non-pregnant (NonP), normal pregnant (NormP) and PE women. Our flow cytometer was shown to detect microvesicles ≥290nm, hence smaller nanovesicles and exosomes could not be detected by this method. Therefore, a novel technique for analysing both microvesicles and nanovesicles, Nanoparticle Tracking Analysis (NTA), was explored and was found to be able to detect vesicles as small as 70nm. The origins of the vesicles that change in pregnancy are not yet known. Flow cytometry and NTA were used in parallel to determine the size, number and phenotype of STBM and other cellular vesicles in NonP, NormP and PE women. Flow cytometry showed that majority of vesicles were derived from platelets, followed by RBC vesicles, leukocyte vesicles and STBM. NTA showed that the total number of vesicles in plasma was significantly elevated in NormP and late-onset PE women compared to NonP controls, and the vesicles were smaller in size. Similarly, flow cytometry showed differences in the composition of vesicles between pregnant and non-pregnant women, demonstrating that pregnancy affects vesicle release. However, no differences were found between NormP and PE women. This was probably due to the majority of samples studied being from late rather than early-onset PE. Thus, although this is the most comprehensive analysis of circulating vesicles in pregnancy to date, their use as biomarkers for PE remains an open question.
4

Apoptosis-driven microenvironmental conditioning by microvesicles in non-Hodgkin lymphoma

Patience, Lauren Alexandra January 2017 (has links)
Plasma membrane derived microvesicles (MV) are nanoscale particles released from cells both constitutively and in response to stimuli including stress, apoptosis and oncogenic transformation. Due to their mechanism of biogenesis, the majority of MV expose phosphatidylserine (PS) on their surface and as such can be identified by staining with annexin V (AxV). First observed nearly 40 years ago as coagulant ‘dust’ originating from activated platelets, MV were initially studied for their role in thrombosis. In more recent years it has become apparent that MV release is increased in several diseases including cancer; this, in conjunction with their ability to carry cargo such as proteins and nucleic acid species, strongly implicates them in disease pathology. Given their small size it is considered likely that MV are able to travel to distal sites within the body allowing the widespread dissemination of effects otherwise not achievable by their parent cells. In the context of malignancy, the contribution of MV is especially important in that MV have been demonstrated to have roles in oncogenic transformation, promotion of tumour growth and increasing metastatic potential. Although clearly important in pathogenesis, their small size makes qualitative and quantitative analysis extremely difficult. Furthermore, the study of MV has been greatly hampered by a lack of standardised protocols for their isolation and as such the majority of studies have been in vitro. In line with this, the relevance of observed effects to in vivo systems is often questioned; given the high quantities of MV used in in vitro systems, the question of whether these concentrations bear any relevance in vivo remain to be answered. We hypothesise that the high rates of apoptosis observed in many tumours, most notably in the high grade B cell malignancy, Non-Hodgkin’s lymphoma (NHL), provides an environment whereby MV are continually released into the surrounding milieu allowing for an amplification of effects. As apoptosis has been previously implicated in promoting tumourigenesis we propose that this is extended to include MV released from apoptotic tumour cells (aMV). Given the numerous technical challenges involved in MV research, initial studies involved identifying the limitations of the instruments available for MV analysis. Preliminary experiments identified considerable resolution issues with the older style EPICS XL flow cytometer (Beckman Coulter) and so a newer flow cytometer, The Attune™ (Thermo Fisher), capable of higher resolution was utilised for the remainder of the project. Despite this improvement, flow cytometry was demonstrated to be less effective at quantifying MV than nanoparticle tracking analysis (NTA). As the fluorescent capacity of NTA is still in its infancy, it was used in concert with flow cytometry in order to quantify and phenotype MV as accurately as possible. As there is currently no concensus on an optimal method of MV isolation subsequent studies focused on determining a method of MV isolation that was appropriate for our experimental system. To this end, centrifugation, filtration and antibody coated magnetic bead-based methods were all tested and their limitations identified. In terms of bead-based isolation strategies, the generation of AxV, protein S, gla domain and gas 6 fusion proteins was attempted with the intention to conjugate to magnetic beads and provide a novel means to isolate aMV. Unfortunately this aspect of the project was ultimately abandoned due to time constraints and although commerically available antibody coated beads were tested for their ability to isolate MV, later co-culture experiments demonstrated that the beads had off target effects that were deleterious to cells. As a result, centrifugation and filtration methods were next researched and validated extensively. TEM analysis of MV morphology identified damage likely induced by the high-speed centrifugation of a fragile apoptotic cell population. As such, a protocol combining low speed centrifugation and filtration was designed and validated by several methods including TEM and staining with AxV. The surface levels of parent cell markers (CD19 and CD20) and apoptosis associated proteins were compared in aMV and vMV (MV released from viable tumour cells) and results demonstrated that B cell surface markers were off loaded into MV to a greater extent following apoptosis. Additional phenotypic studies extended previous work from the group demonstrating the presence of apoptotic cell associated molecular patterns (ACAMPs) capable of binding a panel of antibodies to LPS. Flow cytometry results confirmed the presence of ACAMPs on aMV and results from co-culture experiments with CD14 positive and negative cells suggested that unlike recognition of LPS, binding via ACAMPs was not CD14 dependent. The protein and nucleic acid content of MV was also studied and interestingly, results demonstrated significantly increased quantities of DNA and RNA in aMV compared to vMV. Furthermore, aMV were also shown to contain the matrix metalloproteinases, MMP2 and MMP12 alluding to a role for aMV in angiogenesis. The final stage of the project was focused on determining the roles of aMV in the tumour microenvironment and effects relating to cell growth, cell cycle and angiogenesis were studied and compared to vMV. Results showed that both aMV conditioned supernatant and aMV concentrated by the centrifugation were able to significantly increase the growth of the parent cell population. Further studies using DAPI staining to determine the cell cycle status of cells co-cultured with aMV demonstrated an increase in DNA synthesis and cell division upon incubation with aMV. An in vitro angiogenesis assay was designed to determine any pro-angiogenic capabilities of aMV given the earlier results demonstrating the presence of MMPs. These results provided some of the most interesting findings of the project and showed that aMV were able to increase the angiogenic potential of human endothelial cells (HUVECs); an effect that was shown to be greatly reduced following storage at either 4 or - 80°C. These results demonstrated that aMV possess factors capable of manipulating the tumour microenvironment to favour disease progression and that previously described pro-tumour functions of MV are increased as a result of apoptosis. These findings have implications both in terms of extending the previously described hallmarks of cancer and also when designing a course of therapy whereby in some instances the generation of large amounts of apoptosis may in fact serve to promote regeneration of the tumour cell population.
5

Rôle des exosomes comme nouvelle voie de communication entre les neurones / Role of exosomes as a novel way of interneuronal communication

Javalet, Charlotte 30 September 2016 (has links)
Les exosomes sont des vésicules d’origine endosomale sécrétées par les cellules dans leur environnement après fusion à la membrane plasmique des endosomes multivésiculés. Les exosomes représentent un nouveau mode de communication entre les cellules en permettant un transfert direct de protéines, de lipides et d’ARN. L’objectif de ma thèse était d’étudier le rôle des exosomes dans la communication entre les neurones. Précédemment, le laboratoire a montré que les neurones sécrètent des exosomes de manière régulée par l’activité synaptique. Nous avons observé que les exosomes neuronaux ne sont endocytés que par les neurones. Après avoir montré qu’ils ne contiennent que des ARN courts, nous avons réalisé un séquençage complet de leurs microARN et observé que ces microARN étaient sélectivement exportés dans les exosomes. Nos observations suggèrent que les microARN contenus dans les exosomes peuvent modifier la physiologie des neurones receveurs. Nos résultats renforcent l’hypothèse du rôle des exosomes dans la communication entre les neurones via le transfert de microARN. / Exosomes are vesicles of endocytic origin released by cells into their environment following fusion of multivesicular endosomes with the plasma membrane. Exosomes represent a novel mechanism of cell communication allowing direct transfer of proteins, lipids and RNA. The goal of my PhD thesis was to study that exosomes represent a novel way of interneuronal communication. Our team has previously reported that neurons release exosomes in a way tightly regulated by synaptic activity. We observed that exosomes released by neurons are only endocytosed by neurons. We found that exosomes contain only small RNA and did a deep sequencing of all their microRNA. MicroRNA are selectively exported into exosomes. It seems that exosomal microRNA can modify the physiology of receiving neurons. Our results strengthen the hypothesis of the role of exosomes in the interneuronal communication by the way of microARN transfert.
6

Particle Balances in Therapeutic Extracellular Vesicle Development and in depth Characterization of Fluorescence Nanoparticle Tracking Analysis

Deighan, Clayton J. January 2015 (has links)
No description available.
7

Characterization and miRNA analysis of cancer cell-secreted microvesicles

Guzman, Nicole Denise 27 June 2012 (has links)
No description available.
8

Prions and platelets: a possible role for cellular prion protein

Robertson, Catherine 28 April 2005 (has links)
Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion.
9

Cardiac hypertrophy : transcription patterns, hypertrophicprogression and extracellular signalling / Hjärthypertrofi : transkriptionsmönster, hypertrofisk progression och extracellulär signalering

Gennebäck, Nina January 2012 (has links)
Background: The aim of this thesis was to study transcription patterns and extracellular signalling of the hypertrophic heart to better understand the mechanisms initiating, controlling and maintaining cardiac hypertrophy. Cardiac hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophy of the myocardium is a state, independent of underlying disease, where the myocardium strives to compensate for an increased workload. This remodelling of the heart includes physiological changes induced by a changed gene expression, alteration of the extracellular matrix and diverse cell-to-cell signalling. Shedding microvesicles and exosomes are membrane released vesicles derived from the plasma membrane, which can mediate messages between cells and induce various cell-related processes in target cells. Methods and materials: Two different microarray studies on different materials were performed. In the first study, cardiac myectomies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls without cardiac disease were used. In the second study, myocardial tissue from 6 aorta ligated and 6 sham operated (controls) rats at three different time points (1, 6 and 42 days post-surgically) were analysed. To reveal differences in gene expression the materials were analyzed with Illumina whole genome microarray and multivariate data analysis (PCA and OPLS-DA). Cultured cardiomyocytes (HL-1) were incubated with and without growth factors (TGF-β2 or PDGF BB). Microvesicles and exosomes were collected and isolated after differential centrifugations and ultracentrifugations of the cell culture medium. The microvesicles and exosomes were characterized with dynamic light scattering (DLS), flow cytometry, western blot, electron microscopy and Illumina whole genome microarray. Results: The two different microarray studies revealed differentially expressed gene transcripts and groups of transcripts. When comparing HOCM patients to controls significant down-regulation of the MYH6 gene transcript and two immediate early genes (IEGs, EGR1 and FOS), as well as significant up-regulation of the ACE2, JAK2 and HDAC5 gene transcripts were found. In the rat model, 5 gene groups showed interesting clustering after multivariate data analysis (OPLS-DA) associated with the hypertrophic development: “Atherosclerosis”, “ECM and adhesion molecules”, “Fatty acid metabolism”, “Glucose metabolism” and “Mitochondria”. The shedding microvesicles were rounded vesicles, 40-300 nm in size and surrounded by a bilayered membrane. Chromosomal DNA sequences were identified in the microvesicles. The microvesicles could be taken up by fibroblasts resulting in an altered gene expression in the fibroblasts. The exosomes from cultured cardiomyocytes (incubated with TGF-β2 or PDGF BB) had an average diameter of 50-80 nm, similar to the unstimulated control exosomes. A large, for all cardiomyocyte derived exosomes, common pool of mRNA seems stable and a smaller pool varied in mRNA content according to treatment of the cardiomyocyte. Of the common mRNA about 14% were ribosomal, 14% were of unknown locus and 5% connected to the function of the mitochondria. Conclusions: The microarray studies showed that transcriptional regulation at a stable stage of the hypertrophic development is a balance of pro and anti hypertrophic mechanisms and that diverse gene groups are differently regulated at different time points in the hypertrophic progression. OPLS-DA is a very useful and powerful tool when analyzing gene expression data, especially in finding clusters of gene groups not seen with traditional statistics. The extracellular vesicle studies suggests that microvesicles and exosomes released from cardiomyocytes contain DNA and can be involved in events in target cells by facilitating an array of processes including gene expression changes. Different treatment of the cardiomyocyte influence the content of the exosome produced, indicating that the signal function of the exosome might vary according to the state of the cardiomyocyte. / Bakgrund: Syftet med den här avhandlingen var att studera transkriptions-mönster och extracellulär signalering vid hjärthypertrofi för att bättre förstå de mekanismer som startar, styr och underhåller tillväxten. Hjärthypertrofi, onormal tillväxt av hjärtmuskeln, är en riskfaktor för andra hjärt-kärlsjukdomar och dödlighet. Hypertrofi av hjärtmuskeln är ett tillstånd, oberoende av bakomliggande sjukdom, där hjärtmuskeln strävar efter att kompensera för ökad arbetsbelastning. Denna omställning av hjärtat innefattar fysiologiska förändringar orsakade av ett förändrat genuttryck, modifiering av miljön utanför cellen och ändrad cell-till-cell signalering. Mikrovesiklar och exosomer är små membranomslutna bubblor som frisätts från cellmembranet, ut i cellens omgivning. De kan förmedla budskap mellan celler och påverka olika processer i målceller. Metoder och material: Avhandlingen innefattar två olika microarraystudier på olika material. I den första studien användes hjärtbiopsier från 8 patienter med hypertrofisk obstruktiv kardiomyopati (HOCM) och 5 kontroller utan hjärtsjukdom. I det andra projektet användes hjärtvävnad från 6 aortaligerade och 6 skenopererade (kontroller) råttor vid tre olika tidpunkter (1, 6 och 42 dagar efter kirurgiskt ingrepp). För att påvisa skillnader i genuttryck analyserades proverna med Illumina helgenom microarray och multivariat dataanalys. Avhandlingens andra del innehåller två studier om mikrovesiklar och exosomer. Odlade hjärtmuskelceller (HL-1) stimulerades med tillväxt-faktorer (TGF-β2 eller PDGF BB) och ostimulerade celler användes som kontroll. Mikrovesiklar och exosomer renades fram med centrifugeringar och ultracentrifugering av cellodlingsmediet för att sedan karakteriseras med olika metoder för att studera storlek, ytmarkörer och innehåll. Illumina helgenom microarray användes för att studera microvesiklarnas och exosomernas mRNA innehåll. Resultat: I de två olika microarraystudierna hittades gentranskript och grupper av gentranskript som skiljde sig mellan kontroller och den hypertrofa hjärtvävnaden. När HOCM patientproverna jämfördes med kontroller hittades nedreglering av MYH6, EGR1 och FOS samt uppreglering av ACE2, JAK2 och HDAC5. Efter multivariat dataanalys av materialet från råtta, hittades 5 grupper av gentranskript med intressanta mönster som kunde kopplas till den hypertrofiska utvecklingen av hjärtmuskeln: "Ateroskleros", "ECM och adhesionsmolekyler", "Fettsyrametabolism", "Glukosmetabolis-men" och "Mitokondrien". Mikrovesiklarna hade en diameter på 40-300 nm och innehöll kromosomala DNA-sekvenser. När mikrovesiklarna överfördes till en annan celltyp (fibroblaster) resulterade det i ett förändrat genuttryck i fibroblasterna. Exosomer från hjärtmuskelcellerna som odlats med eller utan tillväxtfaktor hade en diameter på 50-80 nm. En stor pool av olika gentranskript var gemensam för alla exosomer oavsett stimulering eller ej. En mindre pool av gentranskript varierade i innehåll mellan de stimulerade och ostimulerade hjärtmuskelcellerna. I den gemensamma gentranskript poolen var ca 14 % ribosomala, ca 14 % var okända och ca 5 % var associerade till mitokondrien och dess funktion. Slutsats: Microarraystudierna visade att transkriptionsreglering i ett stabilt skede av hypertrofiutvecklingen är en balans mellan pro- och anti-hypertrofiska mekanismer och att olika gengrupper var olika reglerade vid olika tidpunkter i hjärtmuskeltillväxten. OPLS-DA är ett mycket användbart och kraftfullt verktyg när man analyserar genexpressionsdata, särskilt för att hitta grupper av gen-transkript som är svåra att upptäcka med traditionell statistik. Microvesikel- och exosomstudierna visade att mikrovesiklar och exosomer som frisätts från hjärtmuskelceller innehåller både DNA och RNA och kan vara inblandade i händelserna i målceller genom att underlätta en rad processer, inklusive ändringar av genuttryck. Olika stimulering av hjärtmuskelcellen kan påverka innehållet i exosomernas som produceras, vilket indikerar att exosomernas signalfunktion kan variera beroende på hjärtmuskelcellens tillstånd.
10

Prions and platelets: a possible role for cellular prion protein

Robertson, Catherine 28 April 2005 (has links)
Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion. / May 2005

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