Les microparticules (MP) sont des vésicules de la membrane plasmique émises après stress cellulaire. Nous avons étudié le rôle des MPs leucocytaires extraites de la rate de rats comme marqueur du vieillissement et effecteurs de la senescence et de la dysfonction endothéliales induites par les fortes concentrations de glucose (HG). L’émission basale de MP augmente avec l’âge qui favorise leur génération en réponse au LPS ou au PMA/ionophore A23187 (MPLPS, MPPMA/I). Les MP de rats âgés mais pas de jeunes induisent la sénescence de cellules endothéliales primaires d’artères coronaires (AC) de porc. MPLPS ou MPPMA/I de rats jeunes, mais pas MPCTL (cellules non traitées) réduisent la relaxation dépendante de l’endothélium d’anneaux d’AC en réponse à la bradykinine avec sous-expression de eNOS, surexpression de COX2, ICAM-1, VCAM-1. HG favorise l’émission des MP de rate. Dans les AC en HG, la vasoconstriction en réponse au U46619l est diminuée de manière dépendante du SGLT1/2 et de l’EDHF. / Microparticles (MP) are plasma membrane vesicles shed from stimulated cells. We investigated whether leukocyte MP extracted from rat spleen are reliable markers of aging and effectors of high glucose (HG)-induced endothelial senescence and dysfunction. Data indicate that ageing enhances MP shedding from spleen cells of middle-age and aged rats and raises MP release in response to LPS, or to PMA and ionophore A23187. Of note, MP from aged but not young rats induced senescence of porcine coronary artery primary endothelial cells. In young rats, MPLPS, MPPMA/I but not from resting cells (MPCTL) reduced the endothelial-dependent relaxation of coronary artery rings (CAR) in response to bradykinin with down-regulation of eNOS, up-regulation of COX-2, ICAM-1, VCAM-1. HG enhanced early and late MP release from spleen cells. Prolonged exposure to HG potentiated endothelial dysfunction in CAR and altered vasoconstriction in response to U46619l in a SGLT1/2 and EDHF dependent manner.

A L. (L.) amazonensis é capaz de evadir a resposta imune do macrófago hospedeiro induzindo a expressão de CD200 na célula. Porém, ainda não se sabe como ocorre este mecanismo. O objetivo deste trabalho foi avaliar a participação dos TLRs na indução de CD200 em macrófagos infectados por L. (L.) amazonensis. Os resultados mostraram que a indução de CD200 por L. (L.) amazonensis é dependente de TLR9 e das proteínas adaptadoras MyD88 e TRIF. Além disso, observamos que CD200 pode ser induzida pelo DNA do parasito, assim como por vesículas extracelulares (VEs) contendo DNA liberadas por ele. Os resultados in vivo mostraram que a ausência de TLR9 não altera o tamanho da lesão e nem a expressão de CD200 nos macrófagos presentes. Contudo, a carga parasitária foi maior nos camundongos selvagens. A partir dos resultados obtidos podemos concluir que a L. (L.) amazonensis induz CD200 de maneira dependente da via de TLRs e que esta indução pode ser estimulada pelo DNA do parasito. / L. (L.) amazonensis evades the immune response of host macrophage inducing the CD200 expression in the cell. However, it is not yet known how this mechanism occurs. The objective of this work was to evaluate the participation of TLRs in CD200 induction in infected macrophages by L. (L.) amazonensis. The results showed that the CD200 induction by the parasite is dependent on TLR9 and the adaptor proteins MyD88 and TRIF. In addition, we observed that the CD200 can be induced by the parasite DNA, as well as by extracellular vesicles (EVs) containing DNA released by it. In vivo results showed that the absence of TLR9 does not alter the lesion size nor the CD200 expression in macrophages present in the lesion. However, the parasite load was higher in wild type mice. Therefore, we can conclude that the CD200 induction by L. (L.) amazonensis amastigotes is TLR dependent and this can be stimulated by the parasite DNA.

Les vésicules extracellulaires (EVs) participent à la communication intercellulaire. Dans la littérature actuelle, elles sont divisées en deux classes principales selon leur origine intracellulaire. En premier lieu, les exosomes sont formés à l'intérieur des endosomes multivésiculaires et sont libérés lors de la fusion de ces compartiments avec la membrane plasmique (MP). La taille des exosomes est contrôlée au cours de leur biogenèse et varie de 50 à 150 nm. Deuxièmement, les EVs sont formées par bourgeonnement direct et sécrétion à partir de la MP. Ces EVs sont plus hétérogènes et leur taille varie de 50 à 1000 nm. Malgré le fait que la nature hétérogène de EVs soit clairement documentée dans la littérature, la composition en protéines et les mécanismes exacts de la biogenèse des différentes EVs restent un sujet de débat en cours. Le but principal de ce travail était de redéfinir autant de sous-types différents d’EVs que possible, en trouvant des marqueurs protéiques spécifiques, et d'étudier les outils possibles pour affecter spécifiquement leur sécrétion. Dans ce projet, nous avons mis en place plusieurs outils utiles pour la caractérisation d’EVs. Tout d'abord, mes principaux efforts ont été concentrés sur la mise en place de plusieurs protocoles d'isolation et d'analyse d’EVs. Cela a conduit à la production d'une cartographie des protéines vésiculaires, qui si elle est appliquée pour caractériser les EVs, permettra de mieux les identifier par leur composition. Deuxièmement, j'ai étudié la façon dont la sécrétion de ces sous-populations d’EVs peut être modulée par l'inhibition de quelques protéines de la famille RAB et par certaines drogues. Enfin, grâce à une collaboration établie au sein de l'unité, j'ai eu l'occasion de participer à une comparaison des propriétés fonctionnelles entre les EVs et les virus sécrétés simultanément par les cellules infectées. Mes résultats confirment l'hypothèse selon laquelle l'origine intracellulaire des EVs sera reflétée dans leur composition. Les résultats présentés confirment la coexistence de plusieurs classes d'EVs et donnent un aperçu sur les moyens de les caractériser dans une préparation d’EVs donnée. En outre, nous fournissons un exemple de l'application de notre ensemble de protéines dans les études portant sur la biogenèse des EVs. / Extracellular vesicles (EVs) are participating in intercellular communication. Classically, in the current literature, they are divided into two main classes depending on their intracellular origin. Firstly, exosomes are formed within multivesicular endosomes and released upon fusion of these compartments with plasma membrane. The size of exosomes is controlled during their biogenesis and ranges from 50 to 150 nm. Secondly, EVs are formed by direct budding and pinching off from the plasma membrane. These EVs are more heterogeneous and their size varies from 50 to 1000 nm. Despite the fact that a heterogeneous nature of EVs is clearly documented in the literature, the exact protein content and biogenesis mechanisms of different EVs remain a matter of on-going debate. The principal goal of this work was to re-define as many different subtypes of EVs as possible, by finding specific protein markers, and investigate possible tools to affect specifically their secretion. In this project, we set up several tools useful for EV characterization. Firstly, my main efforts were concentrated on establishment of several protocols to isolate and analyse EVs. This led to the foundation of a vesicle protein cartography, which if applied to characterize EVs, will allow better understanding of the composition of the studied EVs. Secondly, I investigated how secretion of these EV subpopulations might be modulated by inhibition of a few RAB proteins and by some drugs. Finally, thanks to a collaboration established within the unit, I had the opportunity to participate in a comparison of the functional properties between EVs and viruses secreted simultaneously by infected cells. My results confirmed the hypothesis that the intracellular origin of EVs will be reflected in their composition. The results presented in this study point at the coexistence of several EV classes and provide insights on how to demonstrate their presence in a given EV preparation. In addition, we provide an example of the application of our set of proteins in studies addressing EV biogenesis.

Submitted by Angela Maria de Oliveira (amolivei@uepg.br) on 2018-11-27T13:38:37Z No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Felipe Biscaia.pdf: 705544 bytes, checksum: d29d4058c2971e1453c934610a2e4d2b (MD5) / Made available in DSpace on 2018-11-27T13:38:37Z (GMT). No. of bitstreams: 2 license_rdf: 811 bytes, checksum: e39d27027a6cc9cb039ad269a5db8e34 (MD5) Felipe Biscaia.pdf: 705544 bytes, checksum: d29d4058c2971e1453c934610a2e4d2b (MD5) Previous issue date: 2018-08-22 / Câncer é um conjunto de doenças causadas por uma lesão gênica associada a uma expansão clonal com crescimento desproporcional. O melanoma é o câncer de pele com menor perspectiva de sobrevida e com alta capacidade de metástase. O câncer de mama é um dos mais abundantes entre as mulheres, com alta incidência de morte. Estudos relatam que todas as células do corpo humano são capazes de secretar microvesículas e, que os tecidos tumorais secretam micropartículas com mais abundancia. Essas micelas desempenham um papel importante na comunicação celular. Assim, a comunicação entre as células tumorais possui uma função essencial para o desenvolvimento do câncer. Neste trabalho, as linhagens de melanoma B16F10 e adenocarcinoma 4T1 foram cultivadas e submetidas ao tratamento dos fármacos Dexametasona e tamoxifeno, bem como microvesículas extraídas dos sobrenadante de B16F10 tratado com dexametasona. Como resultados, observamos redução da viabilidade celular de ambas as linhagens ocasionada pelos três tratamentos isolados, assim como pelos tratamentos combinados do compostos utilizados. Os fármacos e as microvesículas desencadearam efeitos citotóxicos sobre as linhagens tumorais, mostrando-se potenciais alternativas para tratamento. / Cancer is a set of diseases caused by a simple gene injury associated with a clonal expansion with disproportionate growth. Melanoma is a skin cancer with a lower survival prospect and high metastatic capacity. Breast cancer is one of the most abundant among women, with a high incidence of death. Several studies report that all human cells are able to secrete microvesicles, and that tumor tissues secrete microparticles with high abundance. These micelles play an important role in cellular communication. Thus, communication between tumor cells has an essential function for the development of cancer. In this work, the B16F10 melanoma and 4T1 adenocarcinoma cell lines were cultured and treated with the drugs Dexamethasone and tamoxifen, as well as microvesicles extracted from the B16F10 supernatant treated with dexamethasone. The results shows a reduction in the cell viability of both strains caused by the three isolated treatments, as well as by the combined treatments of the compounds used. Drugs and microvesicles triggered cytotoxic effects on tumor lines, showing potential treatment alternatives.

No description available.

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.

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

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.

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.

La mucoviscidose est une maladie génétique due à des mutations du gène CFTR (Cystic Fibrosis Transmembrane conductance Regulator), conduisant à un défaut d'adressage de la protéine CFTR à la membrane apicale des cellules épithéliales, ou à un déficit de sa fonction de canal à ions chlorure. Ce travail a consisté à étudier les vésicules extracellulaires (EV), microvésicules (MV) et exosomes (Exo), comme vecteurs de la protéine CFTR et de son ARN messager. La preuve de concept du transfert de matériel biologique d'intérêt par l'intermédiaire d'EV, d'abord apportée sur un modèle de cellules animales (CHO), a été validée en cellules humaines. Les EV ont été isolées à partir de surnageant de Calu-3, cellules exprimant la protéine CFTR de manière endogène, et de A549 transduites par le vecteur adenoviral Ad5-GFP-CFTR, surexprimant la protéine de fusion GFP-CFTR. Les cellules cibles choisies, A549 et CF15, étaient déficientes en CFTR. Le transfert s'est révélé plus efficace en système homologue (A549/A549) qu'en système hétérologue (A549/CF15). Par ailleurs, l'utilisation d'inhibiteurs métaboliques suggère que les EV ne suivent pas une voie d'internalisation cellulaire unique, mais que plusieurs mécanismes sont mis en jeu, dont l'endocytose clathrine dépendante et la macropinocytose. Les deux types d'EV sont capables de rétablir la fonction canal associée au CFTR dans les cellules CF15 de façon dose-dépendante, mais avec un effet de seuil minimum. L'activité CFTR reste stable pendant 3 jours, et à un niveau encore détectable après 5 jours. Notre travail démontre l'intérêt potentiel des MV et Exo comme vecteurs de biothérapie de pathologies génétiques / Cystic fibrosis is a genetic disease in which its prognosis depends on the lung damage. It is caused by mutations in the cystic fibrosis transmembrane conductance regulator gene (CFTR), resulting in a dysfunctional CFTR protein normally located at the plasma membrane of epithelial cells. This thesis is a study of a novel therapeutic approach to use extracellular vesicles (EVs), microvesicles and exosomes, as transfer vectors for CFTR mRNA and protein to target cells. The proof of concept for the transfer of CFTR mRNA and protein was first done in the CHO hamster model. To validate this concept on human cells, we used human bronchial Calu-3 cells, which express the endogenous CFTR protein, and A549 lung epithelial cells transduced by the adenoviral vector Ad5-GFP-CFTR to overexpress the fusion exogenous protein GFP-CFTR. We show that EVs produced by these cells could transfer a new functionality to CF15 target cells carrying the CFTRdeltaF508 mutation and the transfer seems to be more efficient in a homologous cell system versus a heterologous system. Interestingly, the exosomes seem to be more efficient in CFTR transfer than the microvesicles. A study of the mechanism of EVs cellular uptake show that it is temperature dependent and that endocytosis and macropinocytosis are implicated. Collectively, this study demonstrates the potential application of EVs for CFTR transfer and functional correction of the genetic defect in human CF cells