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[{"key": "dc.contributor.advisor", "value": "Ihalainen, Janne", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Ihalainen, Teemu", "language": null, "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Takkinen, Heini", "language": null, "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2016-02-17T18:30:04Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2016-02-17T18:30:04Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2016", "language": null, "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.other", "value": "oai:jykdok.linneanet.fi:1522485", "language": null, "element": "identifier", "qualifier": "other", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/48816", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Fytokromit ovat bakteerien, levien, kasvien ja sienten punaista valoa aistivia proteiineja. Keskeinen piirre fytokromien toiminnassa on valon vaikutuksesta tapahtuva siirtym\u00e4 punaista valoa absorboivan (Pr) ja kaukopunaista valoa absorboivan (Pfr) tilan v\u00e4lill\u00e4. Deinococcus radiodurans -bakteerifytokromin valosensoriosa (CBD-PHY) on dimeerinen proteiini, joka koostuu C-terminaalisesta kromoforia sitovasta domeenista (CBD) ja fytokromiin liittyv\u00e4st\u00e4 domeenista (PHY). Viimeaikaiset CBD-PHY:n kristalli- ja liuosrakenteet ovat osoittaneet, ett\u00e4 PHY-domeenien v\u00e4linen et\u00e4isyys kasvaa, kun proteiini siirtyy Pr-tilasta Pfr-tilaan absorboituaan punaista valoa.\n\nT\u00e4ss\u00e4 ty\u00f6ss\u00e4 suunniteltiin F\u00f6rsterin resonanssienergiansiirtoon (FRET) perustuvat mittaukset vaihtoehtoiseksi, systeemi\u00e4 h\u00e4iritsem\u00e4tt\u00f6m\u00e4ksi ja kustannustehokkaaksi tavaksi mitata PHY-domeenien v\u00e4limatka Pr- ja Pfr-tiloissa. CBD-PHY -proteiinia, jonka PHY-domeenien k\u00e4rkiin oli lis\u00e4tty kysteiini-insertiot (E373/CC/G374), ja jonka yksi pintakysteiini oli korvattu seriinill\u00e4 (C93S), tuotettiin BL21 (DE3) -kannan E. coli -soluissa ja puhdistettiin korkean erotuskyvyn nestekromatografialla. Ty\u00f6ss\u00e4 kehitettiin malemidipohjainen leimausmenetelm\u00e4 FRET donori (D) \u2013 akseptori (A) -parin (Alexa Fluor 488 \u2013 Alexa Fluor 546) kiinnitt\u00e4miseksi inserttikysteiineihin. Leimattujen proteiinin\u00e4ytteiden steady-state -emissiospektrit, fluoresenssin elinajat sek\u00e4 steady-state- ja aikaerotteiset anisotropiat mitattiin FRET-tehokkuuksien ja niit\u00e4 vastaavien D-A-et\u00e4isyyksien m\u00e4\u00e4ritt\u00e4miseksi Pr- ja Pfr-tiloissa.\n\nDA-leimatun CBD-PHY:n Pr- ja Pfr-tilojen emissiospektreiss\u00e4 n\u00e4htiin FRET:iin viittaava intensiteetin muutos sek\u00e4 D:n ett\u00e4 A:n emissiossa. Aikaerotteisista tuloksista FRET-signaalia ei yll\u00e4tt\u00e4en havaittu. T\u00e4m\u00e4n lis\u00e4ksi eri mittaustekniikat eiv\u00e4t paljastaneet eroa Pr:n ja Pfr:n v\u00e4lill\u00e4, eiv\u00e4tk\u00e4 emissiospektreist\u00e4 m\u00e4\u00e4ritetyt D-A -et\u00e4isyydet vastanneet aiemmista liuosrakenteista arvioituja et\u00e4isyyksi\u00e4. Odottamattomille tuloksille voidaan esitt\u00e4\u00e4 useita mahdollisia syit\u00e4. On ilmeist\u00e4, ett\u00e4 ty\u00f6ss\u00e4 suunniteltu leimausprotokolla vaatii edelleen kehitt\u00e4mist\u00e4. Toisaalta on my\u00f6s mahdollista, ett\u00e4 systeemi on todellisuudessa ennakkok\u00e4sityksi\u00e4 monimutkaisempi esimerkiksi v\u00e4riainemolekyylien sammutuksen tai v\u00e4riaineiden ja CBD-domeeneihin sitoutuneiden biliverdiini-molekyylien v\u00e4lisen FRET:in vuoksi.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Phytochromes are red-light sensing proteins of bacteria, algae, plants, and fungi. A central feature of the function of phytochromes is a light-induced conversion between a red absorbing (Pr) state and a far-red absorbing state (Pfr). The photosensory unit (CBD-PHY) of Deinococcus radiodurans bacteriophytochrome is a dimeric protein consisting of a C-terminal chromophore binding (CBD) domain and a phytochrome-associated (PHY) domain. Recent crystal and solution structures of CBD-PHY have shown that the separation between the two PHY domains increases when the protein is converted from Pr to Pfr state as a result of red-light absorption.\n\nF\u00f6rster resonance energy transfer (FRET) based measurements were designed in this work to be used as an alternative, non-invasive, and cost-efficient way for probing the PHY domain separation in Pr and Pfr. CBD-PHY engineered with cysteine insertions at the ends of the PHY domains (E373/CC/G374) and one surface cysteine substituted with serine (C93S) was expressed in E. coli strain BL21 (DE3) cells and purified with high-pressure liquid chromatography. A maleimide-based labeling scheme was developed for attaching a FRET donor (D) \u2013 acceptor (A) pair (Alexa Fluor 488 \u2013 Alexa Fluor 546) to the cysteine insertions. Steady-state fluorescence emission spectra, fluorescence decays, and steady-state and time-resolved fluorescence anisotropies were measured for determining FRET efficiencies and corresponding D-A distances in Pr and Pfr.\n\nEmission spectrum of DA-labeled CBD-PHY in Pr and Pfr revealed FRET-associated intensity changes in both D and A emission. Surprisingly, FRET signal was absent from time-resolved results. Furthermore, none of the measurement techniques revealed a difference between Pr and Pfr, and there was discrepancy between D-A distances determined from steady-state results and those evaluated from earlier solution structures. Multiple explanations can be proposed for the unexpected results. It is evident that further development of protein labeling protocols is still needed. There is also a possibility that the system is more complicated than what was initially expected because of processes such as quenching of the dyes or FRET between the dyes and biliverdin molecules bound to the CBD-domains.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted using Plone Publishing form by Heini Takkinen (hetakkin) on 2016-02-17 18:30:00.954123. 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 2016-02-17T18:30:03Z\nNo. of bitstreams: 2\nURN:NBN:fi:jyu-201602171599.pdf: 3090387 bytes, checksum: 528d97f790b4753c2cf63ea19412e85f (MD5)\nlicense.html: 4898 bytes, checksum: e04b152f4239123526b354bf882e0ab8 (MD5)", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2016-02-17T18:30:04Z (GMT). No. of bitstreams: 2\nURN:NBN:fi:jyu-201602171599.pdf: 3090387 bytes, checksum: 528d97f790b4753c2cf63ea19412e85f (MD5)\nlicense.html: 4898 bytes, checksum: e04b152f4239123526b354bf882e0ab8 (MD5)\n Previous issue date: 2016", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "1 verkkoaineisto (58 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": "fytokromi", "language": null, "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Fluorescence labeling of the photosensory unit of Deinococcus radiodurans bacteriophytochrome for F\u00f6rster resonance energy transfer studies", "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-201602171599", "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": "2016-02-17T18:30:05Z", "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": "fluoresenssi", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "proteiinit", "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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