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[{"key": "dc.contributor.advisor", "value": "Gilbert, Leona", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Rauham\u00e4ki, Sanna", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2015-06-25T13:40:43Z", "language": "", "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2015-06-25T13:40:43Z", "language": "", "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2013", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1476823", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/46419", "language": "", "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Virusinfektioita pidet\u00e4\u00e4n kasvavissa m\u00e4\u00e4rin suurimpana ymp\u00e4rist\u00f6per\u00e4isen\u00e4 syyn\u00e4, joka vaikuttaa autoimmuunisairauksien kehitykseen ja autoimmuniteetin muodostukseen. Esimerkiksi parvovirus B19 (B19V) on yhdistetty punahukkaan sek\u00e4 nivelreumaan. On todettu, ett\u00e4 B19V:n ei-rakenteellinen proteiini 1 (NS1) aiheuttaa apoptoosia soluissa, jotka eiv\u00e4t tavallisesti ole B19V infektion kohteena. Lis\u00e4ksi hiljattain on osoitettu, ett\u00e4 apoptoosin seurauksena muodostuvat apoptoottiset kappaleet kykenev\u00e4t esittelem\u00e4\u00e4n antigeenej\u00e4 niit\u00e4 esittelem\u00e4\u00e4n erikoistuneille soluille, kuten makrofageille tai dendriittisoluille. T\u00e4m\u00e4n tutkimuksen tarkoituksena on l\u00f6yt\u00e4\u00e4 B19V infektioon liittyvi\u00e4 autoantigeenej\u00e4 sek\u00e4 tarkastella NS1:n aiheuttamaa immuunivastetta. T\u00e4t\u00e4 varten NS1 on yhdistetty vihre\u00e4\u00e4n fluoresoivaan proteiiniin (EGFP) ja kloonattu bakulovirusvektoriin sytomegaloviruksen v\u00e4litt\u00f6m\u00e4n aikaisen promoottorin alle. T\u00e4m\u00e4n j\u00e4lkeen n\u00e4it\u00e4 yhdistelm\u00e4-bakuloviruksia lis\u00e4ttiin Sf9-hy\u00f6nteissolulinjan avulla. T\u00e4ll\u00e4 tavoin tuotettua virusta k\u00e4ytettiin ihmisen maksasta per\u00e4isin olevien HepG2-solujen transduktiossa. Kyseisen transduktion tehokkuus mitattiin virtaussytometrill\u00e4, hy\u00f6dynt\u00e4en EGFP-signaalia merkkin\u00e4 onnistuneesta transduktiosta. Transduktion tehokkuuden perusteella m\u00e4\u00e4ritettiin viruskannasta sellainen tilavuus, mik\u00e4 tuottaa 70 prosentin transduktion. T\u00e4t\u00e4 tilavuutta k\u00e4ytettiin apoptoottisten kappaleiden tuotannossa. Apoptoottiset kappaleet puhdistettiin ensin suodattamalla ja koottiin sitten pelleteiksi ultrasentrifugilla. Kootuista apoptoottisista kappaleista immunoleimattiin tumaper\u00e4iset antigeenit apolipoproteiini H (ApoH), histoni 4 (H4), histoni 2B (H2B), lysosomiin liittyv\u00e4 kalvoproteiini 2 (Lamp2), Ku80 ja Smith. Kaikki tumaper\u00e4iset antigeenit, poissulkien H2B, l\u00f6ydettiin apoptoottisista kappaleista yhdess\u00e4 NS1 kanssa. Tumaper\u00e4isten antigeenien l\u00f6ytyminen apoptoottisista kappaleista saattaa aiheuttaa h\u00e4iri\u00f6n immunitoleranssissa, mit\u00e4 tutkittiin seuraavaksi hy\u00f6dynt\u00e4en antigeenej\u00e4 esittelevi\u00e4 soluja. T\u00e4h\u00e4n tarkoitukseen valittiin akuutista monosyyttisesta leukemiasta per\u00e4isin olevat ihmisen monosyytit (THP-1), jotka erilaistettiin forboli-12-myristaatti-13-asetaatilla (PMA). Soluja viljeltiin PMA:a sis\u00e4lt\u00e4v\u00e4ll\u00e4 kasvatusalustalla ensin kolme p\u00e4iv\u00e4\u00e4 ja viljely\u00e4 jatkettiin viel\u00e4 erilaistumisen varmistamiseksi puhtaalla alustalla viiden p\u00e4iv\u00e4n ajan. Seuraavaksi THP-1 soluja stimuloitiiin apoptoottisilla kappaleilla ja niiden p\u00e4\u00e4tyminen solujen sis\u00e4\u00e4n varmistettiin konfokaalimikroskopialla. Koska erilaistetut THP-1 solut pystyiv\u00e4t ottamaan apoptoottisia kappaleita sis\u00e4\u00e4ns\u00e4, t\u00e4st\u00e4 johtuvaa sytokiiniprofiilia tarkasteltiin viel\u00e4 alustavasti tarkoitukseen sopivan kaupallisen tuotteen avulla. Mahdollisia immunologisia vaikutuksia tarkasteltaessa tultiin lopputulokseen, jossa ainoastaan yhden proteiinin, \u03b3-interferonin indusoima proteiini 10:n (IP-10), l\u00e4sn\u00e4olo erotti EGFP- ja EGFP-NS1-fuusioproteiinien avulla tuotetut n\u00e4ytteet kontrollin\u00e4ytteist\u00e4. Vaikka B19V:n infektiomekanismien aiheuttamat h\u00e4iri\u00f6t immunitoleranssiin vaativatkin viel\u00e4 lis\u00e4tutkimuksia, t\u00e4ss\u00e4 tutkimuksessa saadut tulokset tukevat alkuper\u00e4ist\u00e4 hypoteesia. B19V NS1:n avulla tuotetut apoptoottiset kappaleet, jotka ovat per\u00e4isin soluissa, joita B19V ei tavallisesti infektoi, sis\u00e4lt\u00e4v\u00e4t autoantigeenej\u00e4 ja n\u00e4m\u00e4 autoantigeenit voidaan esitell\u00e4 immuunij\u00e4rjestelm\u00e4lle antigeenej\u00e4 esittelevien solujen kautta.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "There are several factors, of which viral infections are increasingly regarded as the greatest environmental cause of autoimmunity, influencing the development of an autoimmune disease. For example human parvovirus B19 (B19V) has been previously connected to systemic lupus erythematosus and rheumatic arthritis. It has also been shown that nonstructural protein 1 (NS1) of B19V induces apoptosis in cells not normally permissive for B19V infections. In addition, it has been recently demonstrated that apoptotic bodies resulting from apoptosis can present antigens to antigen presenting cells (APCs) such as macrophages or dendritic cells. The aim of this study is to detect self antigens related to B19V infections and study the immune response caused by NS1. For that, the NS1 has been fused with enhanced green fluorescent protein (EGFP) and incorporated into baculovirus vector under cytomegalovirus immediate early promoter. These recombinant baculovirus particles were amplified in Sf9 insect cell line. The resulting virus was then used to transduce HepG2 human hepatocytic cells. The transduction efficiency of the virus was measured with flow cytometry, employing the presence of the EGFP as a sign of transduction. Based on the transduction efficiency, a volume providing 70 % transduction efficiency of the virus stock was used to produce apoptotic bodies. These apoptotic bodies were first purified by filtering and then pelleted by ultracentrifugation. Collected apoptotic bodies were immunolabeled using nuclear antigens apolipoprotein H (ApoH), histone 4 (H4), histone 2B (H2B), lysosomal-associated membrane protein 2 (Lamp2), Ku80 and Smith. The selected nuclear antigens as well as NS1 were detected from the apoptotic bodies, except for H2B. The presence of nuclear antigens in the apoptotic bodies is a potential method for immune tolerance disruption which was then addressed by utilizing APCs. The APCs of choice were acute monocytic leukemia derived human monocytes (THP-1) due to their close resemblance to peripheral blood mononuclear cells. THP-1 cells were differentiated using phorbol-12-myristate-13-acetate (PMA). The cells were incubated in medium containing PMA for three days and then five more days in fresh medium to ensure proper differentiation. Differentiated THP-1 cells (dTHP-1) were next fed with purified apoptotic bodies. Internalization of B19V NS1 induced apoptotic bodies was observed using confocal microscopy. Since the APCs were capable of internalizing apoptotic bodies, the following cytokine profile was tentatively studied. The examination of possible immunological effects derived to a conclusion that only the presence of interferon \u03b3 inducible protein 10 (IP-10) separated the EGFP or EGFP-NS1 fusion protein containing samples from the control conditions. Although the underlying mechanisms leading into immune tolerance disruption by B19V infection mechanisms warrants further investigation, the data here supports the hypothesis that B19V NS1 induced apoptotic bodies from nonpermissive cells contain self-antigens which could be introduced to the immune system though APCs.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Sanna Rauham\u00e4ki (samarauh) on 2015-06-25 13:40:41.864115. Form: Pro gradu -lomake (https://kirjasto.jyu.fi/julkaisut/julkaisulomakkeet/pro-gradu-lomake). 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"dc.subject.other", "value": "immune tolerance", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "apoptotic bodies", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "nuclear antigens", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "self-antigens", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Immune tolerance disruption by human parvovirus B19 viral infection mechanisms", "language": null, "element": "title", "qualifier": null, "schema": "dc"}, {"key": "dc.type", "value": "master thesis", "language": null, "element": "type", "qualifier": null, "schema": "dc"}, {"key": "dc.identifier.urn", "value": "URN:NBN:fi:jyu-201506252452", "language": null, "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": 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