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[{"key": "dc.contributor.advisor", "value": "Hyv\u00f6nen, Mervi", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.advisor", "value": "Takala, Heikki", "language": "", "element": "contributor", "qualifier": "advisor", "schema": "dc"}, {"key": "dc.contributor.author", "value": "Sutinen, Satu", "language": "", "element": "contributor", "qualifier": "author", "schema": "dc"}, {"key": "dc.date.accessioned", "value": "2024-12-20T06:50:54Z", "language": null, "element": "date", "qualifier": "accessioned", "schema": "dc"}, {"key": "dc.date.available", "value": "2024-12-20T06:50:54Z", "language": null, "element": "date", "qualifier": "available", "schema": "dc"}, {"key": "dc.date.issued", "value": "2024", "language": "", "element": "date", "qualifier": "issued", "schema": "dc"}, {"key": "dc.identifier.uri", "value": "https://jyx.jyu.fi/handle/123456789/99146", "language": null, "element": "identifier", "qualifier": "uri", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Optogenetiikka tutkii ja ohjaa soluja valoherkkien proteiinien, valoreseptorien avulla. Er\u00e4s valoreseptorityyppi on bakteerifytokromit, jotka aistivat punaista ja kaukopunaista valoa. Luonnossa bakteerifytokromit ovat usein valoherkki\u00e4 histidiinikinaaseja (HK) ja toimivat osana kaksikomponenttij\u00e4rjestelm\u00e4\u00e4, jonka avulla bakteerit aistivat ymp\u00e4rist\u00f6n signaaleja ja s\u00e4\u00e4telev\u00e4t soluvasteita, kuten geeniekspressiota. T\u00e4m\u00e4ntyyppisi\u00e4 valoreseptoreja ja signalointireittej\u00e4 voidaan hy\u00f6dynt\u00e4\u00e4 optogenetiikassa s\u00e4\u00e4telem\u00e4ll\u00e4 niiden avulla geeniekspressiota kohdeorganismin soluissa. T\u00e4ss\u00e4 Pro Gradu -ty\u00f6ss\u00e4 tuotettiin uusi, punaisella valolla aktivoituva optogeneettinen ty\u00f6kalu, mDERusk, geeniexpression ohjaamiseen nis\u00e4k\u00e4ssoluissa. mDERusk hy\u00f6dynt\u00e4\u00e4 bakteerifytokromin valoa aistivaa osaa kimeerisess\u00e4 HK komponentissa osana kaksikomponenttij\u00e4rjestelm\u00e4\u00e4, jolla s\u00e4\u00e4dell\u00e4\u00e4n kohdegeenin transkriptiota. Ty\u00f6kalu valmistettin k\u00e4\u00e4nt\u00e4m\u00e4ll\u00e4 olemassa olevan nis\u00e4k\u00e4ssolujen geeniekpressiota ohjaavaan optogeneettisen mREDusk ty\u00f6kalun toiminta pime\u00e4ss\u00e4 aktiivisesta punaisen valon avulla aktivoitavaksi, jotta saavutetaan tarkempi spatio-temporaalinen ohjailtavuus. Aktiivisuuden k\u00e4\u00e4ntyminen saatiin aikaan muokkaamalla valoa aistivan osan ja HKn v\u00e4list\u00e4 linkkerialuetta niin, ett\u00e4 kimeerisen HKn kinaasiaktiivisuus ja valovaste muuttuivat k\u00e4\u00e4nteisiksi. Mutatoitu ty\u00f6kalu kloonattiin Gibson assembly -menetelm\u00e4ll\u00e4 ja tuotettiin Escherichia coli -bakteereissa. Ty\u00f6kalun toimintaa tutkittiin HEK293 -soluissa kvantifioimalla luminesoivan reportterigeenin ilmentyminen indusoivan punaisen valon alla ja pime\u00e4ss\u00e4 inkuboiduissa soluissa, joissa ilmentyminen inhiboituu. Luminesenssimittauksen tulokset osoittivat, ett\u00e4 valosta riippuvaisen kinaasiaktiivisuuden k\u00e4\u00e4nt\u00e4minen onnistui ja punaisen valon alla havaittiin korkeampi reportteriproteiinin tuottuminen. Optimoimalla ty\u00f6kalun toimintaa edelleen, t\u00e4m\u00e4n tyyppisi\u00e4 ty\u00f6kaluja voidaan k\u00e4ytt\u00e4\u00e4 esimerkiksi uuden tyyppisten biol\u00e4\u00e4ketieteellisten sovellusten kehitt\u00e4miseen.", "language": "fi", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.abstract", "value": "Optogenetics studies and control living cells with light-sensitive proteins, photoreceptors. One photoreceptor class is the red and far-red light-sensing bacterial phytochromes (BphPs). In nature, BphPs are often light-sensitive histidine kinases (HKs) that are part of a two-component system (TCS). TCS is common signalling pathway that bacteria use to sense environmental cues and transduce the signals into cellular responses, such as gene expression. For optogenetic purposes, these types of photoreceptors and signalling pathways can be used to build optogenetic circuits to control events in different types of cells in the host organism. Here a novel, red light-activated optogenetic tool, mDERusk, was created for controlling gene expression in mammalian cells. mDERusk utilises photosensory module of BphP in chimaeric light-sensitive HK component of TCS to regulate target gene transcription. The strategy to construct mDERusk was to invert the light response of an existing optogenetic tool mREDusk from dark to red light activated for improved spatio-temporal regulation over the expression. The inversion was done with a modification to the linker region between the BphP photosensory and HK effector modules, which inverts the light-dependent kinase activity and the photoresponse of the chimaeric HK component. The mutated tool was cloned with the Gibson assembly method and produced in Escherichia coli -bacteria. The function of mDERusk was studied in HEK293 cells by quantifying the expression of luminescent reporter gene in inducing red light or inhibiting dark conditions. Luminescence assays showed successful inversion of the light-dependent kinase activity and upregulation of reporter protein expression with red light. With further optimization, these types of red light-sensitive optogenetic tools could be used e.g. in developing novel biomedical applications.", "language": "en", "element": "description", "qualifier": "abstract", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Submitted by Paivi Vuorio (paelvuor@jyu.fi) on 2024-12-20T06:50:54Z\nNo. of bitstreams: 0", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.description.provenance", "value": "Made available in DSpace on 2024-12-20T06:50:54Z (GMT). No. of bitstreams: 0\n Previous issue date: 2024", "language": "en", "element": "description", "qualifier": "provenance", "schema": "dc"}, {"key": "dc.format.extent", "value": "80", "language": "", "element": "format", "qualifier": "extent", "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": "light receptor", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "bacterial phytochrome", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.subject.other", "value": "two-component system", "language": "", "element": "subject", "qualifier": "other", "schema": "dc"}, {"key": "dc.title", "value": "Designing and testing red light-activated optogenetic tool for controlling gene expression in mammalian cells", "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-202412207950", "language": null, "element": "identifier", "qualifier": "urn", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Master\u2019s thesis", "language": "en", "element": "type", "qualifier": "ontasot", "schema": "dc"}, {"key": "dc.type.ontasot", "value": "Pro gradu -tutkielma", "language": "fi", "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": "restrictedAccess", "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": "geeniekspressio", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "synteettinen biologia", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "optogenetiikka", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "gene expression", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "synthetic biology", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.subject.yso", "value": "optogenetics", "language": null, "element": "subject", "qualifier": "yso", "schema": "dc"}, {"key": "dc.rights.url", "value": "https://rightsstatements.org/page/InC/1.0/", "language": null, "element": "rights", "qualifier": "url", "schema": "dc"}, {"key": "dc.rights.accessrights", "value": "The author has not given permission to make the work publicly available electronically. Therefore the material can be read only at the archival workstation at Jyv\u00e4skyl\u00e4 University Library (https://kirjasto.jyu.fi/collections/archival-workstation).", "language": "en", "element": "rights", "qualifier": "accessrights", "schema": "dc"}, {"key": "dc.rights.accessrights", "value": "Tekij\u00e4 ei ole antanut lupaa avoimeen julkaisuun, joten aineisto on luettavissa vain Jyv\u00e4skyl\u00e4n yliopiston kirjaston arkistoty\u00f6semalta. Ks. https://kirjasto.jyu.fi/kokoelmat/arkistotyoasema..", "language": "fi", "element": "rights", "qualifier": "accessrights", "schema": "dc"}]
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