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[{"key": "dc.contributor.advisor", "value": "Nymark, Soile", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Fadjukov, Julia", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Hurskainen, Elina", "language": "", "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2021-01-04T07:24:32Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2021-01-04T07:24:32Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2020", "language": "", "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/73527", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Verkkokalvon pigmenttiepiteeli (eng. retinal pigment epithelium eli RPE) on v\u00e4ltt\u00e4m\u00e4t\u00f6n kudos n\u00e4k\u00f6kyvylle. RPE toimii esteen\u00e4 verenkierron ja verkkokalvon v\u00e4lill\u00e4, huoltaa valoreseptorisoluja mm. kuljettamalla ravinteita ja absorboimalla haitallista, siroavaa valoa, sek\u00e4 avustaa n\u00e4k\u00f6aistimuksessa. RPE:n toiminnan heikentyminen saattaa johtaa verkkokalvon rappeumaan sek\u00e4 lopulta n\u00e4k\u00f6kyvyn menett\u00e4miseen. Ionikanavien rooli on merkitt\u00e4v\u00e4 verkkokalvon pigmenttiepiteelin normaalille toiminalle. T\u00e4ss\u00e4 ty\u00f6ss\u00e4 tutkittiin RPE:n j\u00e4nniteherkki\u00e4 natriumkanavia (eng. voltage gated sodium channels eli NaV); tavoitteena oli vahvistaa erilaisten j\u00e4nniteherkkien natriumkanavien alayksik\u00f6iden esiintyminen verkkokalvon pigmenttiepiteeliss\u00e4 kuvantamisen keinoin sek\u00e4 erist\u00e4m\u00e4ll\u00e4 niit\u00e4 RPE-soluista ja todentaa ne Western blot -menetelm\u00e4ll\u00e4. Aluksi kaikki yhdeks\u00e4n NaV \u2013alayksikk\u00f6\u00e4 v\u00e4rj\u00e4ttiin sopivilla vasta-aineilla ja kuvattiin konfokaalimikroskoopilla. Ne alayksik\u00f6t, jotka havaittiin mikroskoopilla, oli m\u00e4\u00e4r\u00e4 erist\u00e4\u00e4 RPE-soluista ja edelleen tutkia Western blot \u2013menetelm\u00e4n avulla. Ainoastaan NaV1.4, NaV1.5, NaV1.6 ja NaV1.8 voitiin luotettavasti havaita vasta-aineleimatuista solun\u00e4ytteist\u00e4 mikroskoopilla. N\u00e4m\u00e4 em. j\u00e4nniteherk\u00e4n natriumkanavan alayksik\u00f6t valittiin siis jatkok\u00e4sittelyihin. Ennen varsinaista erist\u00e4mist\u00e4 ja NaV-alayksik\u00f6iden olemassaolon vahvistamista, sopivin kalvoproteiinien eristysmenetelm\u00e4 sek\u00e4 t\u00e4h\u00e4n tarkoitukseen soveltuva Western blot \u2013protokolla oli testattava ja optimoitava. NaV-proteiinien\neristyksess\u00e4 paras saanto saatiin radioimmunosaostuksen m\u00e4\u00e4rityspuskuri (RIPA), proteaasi-inhibiittoreiden ja etyleenidiamiinitetraetikkahapon (EDTA) yhdistelm\u00e4ll\u00e4. Eristetyt kalvoproteiinit eroteltiin toisista proteiineista SDS- geelielektroforeesin avulla, siirrettiin s\u00e4hk\u00f6virran avulla nitroselluloosakalvolle ja lopuksi v\u00e4rj\u00e4ttiin sopivilla vasta-aineilla. Tutkimuksissa varmistui edelleen, ett\u00e4 RPE solut selv\u00e4sti ilment\u00e4v\u00e4t NaV1.4, NaV1.5 ja NaV1.6 alayksik\u00f6it\u00e4. Tulokset viittasivat vahvasti siihen, ett\u00e4 RPE ilment\u00e4isi my\u00f6s alayksikk\u00f6\u00e4 1.8, mutta t\u00e4m\u00e4n kohdalla Western blot \u2013tulokset eiv\u00e4t olleet yht\u00e4 selkeit\u00e4 kuin muilla alayksik\u00f6ill\u00e4. NaV1.8 luotettava todentaminen vaatii siis viel\u00e4 ty\u00f6t\u00e4 ja mahdollisesti protokollien uudelleen optimointia.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "The retinal pigment epithelium (RPE) is an important tissue for vision. It functions as a barrier between retina and blood circulation but also takes care of the light sensing cells of the retina cells and aids with vision. The loss of different functions in RPE has been linked with retinal dysfunction and degeneration which can finally lead to the loss of vision. Ion channels have been discovered to be very important for the normal function of RPE and for visual health. The aim of this study was to verify the presence of different voltage gated sodium channel subunits (NaV) in RPE. All nine NaV subunits (NaV1.1-1.9) were assayed with suitable fluorescent antibodies from RPE cells and the results were examined with confocal microscopy. Only four NaV subunits, NaV1.4, NaV1.5, NaV1.6 and NaV1.8, were visibly abundant in the RPE cells. These NaV subunits needed to be extracted and immunoblotted. Before the verifications, the protocols for membrane protein extraction and immunoblotting were developed and optimised. The best extraction results were obtained with a combination of radioimmunoprecipitation assay buffer (RIPA), protease inhibitor cocktail and ethylenediaminetetraacetic acid (EDTA). The extracted sodium channel subunits were separated from other proteins with SDS gel electrophoresis, transferred into a nitrocellulose membrane and finally stained\nwith suitable antibodies. Taken together, the results verified the presence of NaV1.4, NaV1.5 and NaV1.6 subunits in RPE cells. The NaV1.8 was only slightly visible in the blots, so the existence could not be confirmed. However, all the results indicate that the RPE also expresses the NaV1.8 subunit, but this still requires some additional work.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by Paivi Vuorio (paelvuor@jyu.fi) on 2021-01-04T07:24:32Z\nNo. of bitstreams: 0", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2021-01-04T07:24:32Z (GMT). No. of bitstreams: 0\n Previous issue date: 2020", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "80", "language": "", "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.title", "value": "Verifying the presence of voltage gated sodium channels in retinal pigment epithelium", "language": "", "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-202101041008", "language": "", "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": "Jyv\u00e4skyl\u00e4n yliopisto", "language": "fi", "element": "contributor", "qualifier": "organization", "schema": "dc"}, {"key": "dc.contributor.organization", "value": "University of Jyv\u00e4skyl\u00e4", "language": "en", "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": "yvv.contractresearch.funding", "value": "0", "language": "", "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": null, "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": "", "element": "subject", "qualifier": "oppiainekoodi", "schema": "dc"}, {"key": "dc.subject.yso", "value": "verkkokalvon pigmenttiepiteeli", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "retinal pigment epithelium", "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": null, "element": "type", "qualifier": "okm", "schema": "dc"}]
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