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[{"key": "dc.contributor.advisor", "value": "Karttunen, Jenni", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Mattila, Sari", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Heiskanen, Mette", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2017-06-20T07:24:48Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2017-06-20T07:24:48Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2017", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1703947", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/54592", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Eksosomit ovat halkaisijaltaan 40-100 nm kokoisia solunulkoisia vesikkeleit\u00e4, joita solut eritt\u00e4v\u00e4t solunsis\u00e4isen multivesikulaarisen rakkulan yhdistyess\u00e4 solukalvoon. Eksosomit ovat t\u00e4rke\u00e4ss\u00e4 roolissa solujen v\u00e4lisess\u00e4 viestinn\u00e4ss\u00e4, sill\u00e4 ne kuljettavat monenlaisia biomolekyylej\u00e4, kuten useita eri RNA-tyyppej\u00e4, lipidej\u00e4 ja proteiineja. Aivoissa eksosomit liittyv\u00e4t neurodegeneratiivisiin sairauksiin, kuten Parkisonin tautiin, Alzheimerin tautiin ja aivovammaan. Eksosomit kykenev\u00e4t l\u00e4p\u00e4isem\u00e4\u00e4n veri-aivoesteen, ja tutkijat ovatkin esitt\u00e4neet paljon kiinnostusta eksosomien mahdollisuuksiin uutena hoitokeinona. Lis\u00e4ksi eksosomien kuljettamat molekyylit sis\u00e4lt\u00e4v\u00e4t tietoa eksosomit eritt\u00e4neen solun toiminnoista, mink\u00e4 ansiosta ne voisivat mahdollisesti toimia biomarkkereina eri sairauksille. Eksosomeja voidaan puhdistaa usealla tavalla, esimerkiksi saostamalla, ultrafuugaamalla ja pylv\u00e4skromatografialla. Puhdistuksen onnistumisen toteamiseksi suositellaan eksosomien karakterisointia ISEV:n (International Society for Extracellular Vesicles) asettamien suuntaviivojen mukaisesti.\n\nT\u00e4m\u00e4n tutkimuksen tavoitteet olivat 1) Western blot -menetelm\u00e4n pystytt\u00e4minen rotan plasman\u00e4ytteille 2) Western blot menetelm\u00e4n pystytt\u00e4minen eksosomien karakterisointia varten 3) saostusmenetelm\u00e4n tehokkuuden arviointi eksosomipuhdistuksessa rotan plasmasta. Western blot -menetelm\u00e4n pystytt\u00e4miseen sis\u00e4ltyi protokollan ja karakterisoinnissa k\u00e4ytett\u00e4vien vasta-aineiden optimointi. Vasta-aineilla tunnistettavat proteiinit olivat Alix (vesikkelimarkkeri), kalneksiini (solukontaminaatiomarkkeri) ja albumiini (plasmaproteiinien kontaminaatiomarkkeri). Saostusmenetelm\u00e4n tehokkuus arvioitiin usealla menetelm\u00e4ll\u00e4 (NTA, SDS-PAGE, Western blot).\n\nAnti-Alix, anti-kalneksiini ja anti-albumiini vasta-aineiden optimointi onnistui, mutta eksosomimarkkeriproteiini CD63:n ja flotilliini-1:n tunnistavia vasta-aineita ei saatu toimimaan. Anti-CD63 ja anti-flotilliini-1 eiv\u00e4t luultavasti toimineet ollenkaan, sill\u00e4 niill\u00e4 ei saatu signaalia edes positiivikontrollista. Tutkimuksessa huomattiin, ett\u00e4 sekundaarivasta-aineet reagoivat vahvasti rotan plasmassa esiintyvien immunoglobuliinien kanssa, mik\u00e4 aiheutti ylim\u00e4\u00e4r\u00e4isi\u00e4 vy\u00f6hykkeit\u00e4 Western blotissa. Eksosomien karakterisointi paljasti, ett\u00e4 eristystuote sis\u00e4lsi eksosomeja, mutta my\u00f6s paljon albumiinia. NTA:n tulokset n\u00e4yttiv\u00e4t, ett\u00e4 suurempien partikkelien osuus kasvoi, kun taas 60 nanometri\u00e4 pienempien partikkelien (proteiinit ja lipoproteiinit) osuus pieneni selke\u00e4sti eristysprosessin aikana. Arvioinnin muut tulokset osoittivat, ett\u00e4 saostusmenetelm\u00e4 onnistui poistamaan noin 60 % plasmaproteiineista, joista albumiini on kaikista yleisin. Tutkimuksessa onnistuttiin pystytt\u00e4m\u00e4\u00e4n Western blot -menetelm\u00e4 rotan plasman\u00e4ytteiden karakterisoinnille. Lis\u00e4ksi havaittiin, ett\u00e4 saostusmenetelm\u00e4 ennemmin rikasti kuin puhdisti eksosomeja, sill\u00e4 eksosomien ohella saostui paljon plasman proteiineja. Tulevaisuudessa olisi kannattavaa yritt\u00e4\u00e4 parantaa eksosomien eristyst\u00e4 kokeilemalla jotakin toista puhdistusmenetelm\u00e4\u00e4, esimerkiksi pylv\u00e4skromatografiaa.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Exosomes are 40\u2013100 nm membrane-enclosed extracellular vesicles (EVs) secreted outside the cell by the fusion of multivesicular bodies with the plasma membrane. Exosomes play an important role in intercellular signaling by carrying different biomolecules, such as several types of RNA (mRNA, miRNA and other non-coding RNA), lipids and proteins. In the brain, exosomes are involved in neurodegenerative diseases, such as Parkinson\u2019s disease, Alzheimer\u2019s disease and traumatic brain injury (TBI). Exosomes can pass the blood-brain barrier, and there has been a growing interest in the therapeutic potential of exosomes. Also, the contents of exosomes carry information about the ongoing processes in their cell of origin, which makes the exosome cargo, for example miRNAs, potential disease biomarkers. To claim the presence of exosomes in the isolate, they are recommended to be characterized according to the guidelines of International Society for Extracellular Vesicles (ISEV).\n\nThe aims of this study were 1) to set up a Western blot method for samples from rat plasma 2) to set up a Western blot method for characterization of exosomes according to the ISEV guidelines 3) evaluate the efficiency of the precipitation method in exosome isolation from rat plasma. The set-up of the Western blot method included optimization of the protocol and the antibodies, which were then used in exosome characterization for detection of Alix (EV marker), calnexin (cell contamination marker) and albumin (plasma protein contamination marker). Efficiency of the exosome precipitation was analysed by nanoparticle tracking analysis (NTA), SDS-PAGE and Western blot.\n\nIt was succeeded to optimize the anti-Alix, anti-calnexin and anti-albumin primary antibodies. However, finding working conditions for antibodies targeting other exosome marker proteins (CD63 and flotillin-1) failed, possibly because of unfunctional antibodies. In addition, the secondary antibodies were observed to strongly cross-react with the immunoglobulins naturally present in rat plasma, which caused unwanted bands in Western blot. Characterization confirmed the presence of exosomes, however, also the vast amount of contaminating plasma proteins in the exosome isolate. NTA results indicated that the share of larger particles increased during the isolation process, whereas the share of <60 nm particles (proteins and lipoproteins) clearly decreased. The results of the evaluation of the precipitation method efficiency by SDS-PAGE and Western blot indicated that the method removed ~60 % of the contaminating plasma proteins, of which albumin is the most abundant. In conclusion, it was succeeded to establish a Western blot characterization method for rat plasma samples. It was also concluded that the precipitation method enriched exosomes rather than purified them, since a lot of plasma proteins were coisolated with exosomes. To improve the isolation of exosomes from plasma in future studies, use of alternative isolation methods i.e. size-exclusion chromatography should be investigated.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Mette Heiskanen (memaheis) on 2017-06-20 07:24:47.527123. Form: Pro gradu -lomake (https://kirjasto.jyu.fi/julkaisut/julkaisulomakkeet/pro-gradu-lomake). JyX data: [jyx_publishing-allowed (fi) =True]", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by jyx lomake-julkaisija (jyx-julkaisija.group@korppi.jyu.fi) on 2017-06-20T07:24:48Z\nNo. of bitstreams: 2\nURN:NBN:fi:jyu-201706202963.pdf: 918675 bytes, checksum: 6a388d448ca85bdaa3f2664ef024c104 (MD5)\nlicense.html: 4906 bytes, checksum: 91233a03ef4ebc806284caebca0469f3 (MD5)", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2017-06-20T07:24:48Z (GMT). No. of bitstreams: 2\nURN:NBN:fi:jyu-201706202963.pdf: 918675 bytes, checksum: 6a388d448ca85bdaa3f2664ef024c104 (MD5)\nlicense.html: 4906 bytes, checksum: 91233a03ef4ebc806284caebca0469f3 (MD5)\n Previous issue date: 2017", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "1 verkkoaineisto (39 sivua)", "language": null, "element": "format", "qualifier": "extent", "schema": "dc"}, {"key": "dc.format.mimetype", "value": "application/pdf", "language": null, "element": "format", "qualifier": "mimetype", "schema": "dc"}, {"key": "dc.language.iso", "value": "eng", "language": null, "element": "language", "qualifier": "iso", "schema": "dc"}, {"key": "dc.rights", "value": "In Copyright", "language": "en", "element": "rights", "qualifier": null, "schema": "dc"}, {"key": "dc.subject.other", "value": "solunulkoiset vesikkelit", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "eksosomit", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "karakterisointi", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "Western blot", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Western blot characterization of exosomes isolated from rat plasma and evaluation of the efficiency of a precipitation method in exosome isolation", "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-201706202963", "language": null, "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Pro gradu -tutkielma", "language": "fi", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Master\u2019s thesis", "language": "en", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Matemaattis-luonnontieteellinen tiedekunta", "language": "fi", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.faculty", "value": "Faculty of Sciences", "language": "en", "element": "contributor", "qualifier": "faculty", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Bio- ja ymp\u00e4rist\u00f6tieteiden laitos", "language": "fi", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.department", "value": "Department of Biological and Environmental Science", "language": "en", "element": "contributor", "qualifier": "department", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "University of Jyv\u00e4skyl\u00e4", "language": "en", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Solu- ja molekyylibiologia", "language": "fi", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.subject.discipline", "value": "Cell and molecular biology", "language": "en", "element": "subject", "qualifier": "discipline", "schema": "dc"}, {"key": "dc.date.updated", "value": "2017-06-20T07:24:48Z", "language": null, "element": "date", "qualifier": "updated", "schema": "dc"}, {"key": "yvv.contractresearch.funding", "value": "0", "language": null, "element": "contractresearch", "qualifier": "funding", "schema": "yvv"}, {"key": "dc.type.coar", "value": "http://purl.org/coar/resource_type/c_bdcc", "language": null, "element": "type", "qualifier": "coar", "schema": "dc"}, {"key": "dc.rights.accesslevel", "value": "openAccess", "language": "fi", "element": "rights", "qualifier": "accesslevel", "schema": "dc"}, {"key": "dc.type.publication", "value": "masterThesis", "language": null, "element": "type", "qualifier": "publication", "schema": "dc"}, {"key": "dc.subject.oppiainekoodi", "value": "4013", "language": null, "element": "subject", "qualifier": "oppiainekoodi", "schema": "dc"}, {"key": "dc.subject.yso", "value": "saostus", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "plasma (neste)", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.format.content", "value": "fulltext", "language": null, "element": "format", "qualifier": "content", "schema": "dc"}, {"key": "dc.rights.url", "value": "https://rightsstatements.org/page/InC/1.0/", "language": null, "element": "rights", "qualifier": "url", "schema": "dc"}, {"key": "dc.type.okm", "value": "G2", "language": 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