Dokument: Development and engineering of optogenetic systems and reconstruction of light signaling pathways
| Titel: | Development and engineering of optogenetic systems and reconstruction of light signaling pathways | |||||||
| Weiterer Titel: | Entwicklung und Konstruktion von optogenetischen Systemen und Rekonstruktion von Licht-Signalwegen | |||||||
| URL für Lesezeichen: | https://docserv.uni-duesseldorf.de/servlets/DocumentServlet?id=52503 | |||||||
| URN (NBN): | urn:nbn:de:hbz:061-20200310-101052-0 | |||||||
| Kollektion: | Dissertationen | |||||||
| Sprache: | Englisch | |||||||
| Dokumententyp: | Wissenschaftliche Abschlussarbeiten » Dissertation | |||||||
| Medientyp: | Text | |||||||
| Autor: | Fischbach, Patrick [Autor] | |||||||
| Dateien: |
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| Beitragende: | Prof. Dr. Zurbriggen, Matias [Betreuer/Doktorvater] Prof. Dr. Möglich, Andreas [Gutachter] | |||||||
| Dewey Dezimal-Klassifikation: | 500 Naturwissenschaften und Mathematik » 570 Biowissenschaften; Biologie | |||||||
| Beschreibungen: | To reconstruct and understand complex regulatory networks and signaling pathways, the field of synthetic biology evolved from synthetic gene circuits to the construction of toggles switches
and complex molecular tools. Within those tools, new synthetic approaches such as the relatively new field of optogenetics arose and showed increased applicability in bacteria, plants and mammalian cells. Since the first optogenetic tool based on the light-dependent ion-channel channelrhodopsin was developed, various light-activatable systems to control and study cellular processes with a high spatiotemporal resolution were engineered. Those studies and the deciphering of complex pathways is particularly difficult in many organisms, because of genetic redundancy, interconnectivity and component number. To gain better understanding of light signaling pathways, a synthetic, optogenetic approach to screen, analyze and reconstruct complex plant signaling pathways in an orthogonal mammalian cell-based platform is one part of this work. Furthermore, an optogenetic tool for in vivo applications was generated since the control of gene expression in a non-invasive manner by light in in vivo situations with high precision and a deep tissue penetration still lags behind. A phytochrome B-based toggle switch was re-engineered for a highly customizable and easily exchangeable modular structure. This optimized toggle switch has been used i) to study the light-dependent interaction with truncated versions of PIF1,3 and 6 in detail, ii) as a screening platform for light-dependent interactions of various plant transcription factors and phytochromes in an orthogonal platform, iii) to engineer a novel red light-inducible phytochrome A-based split transcription factor system and iv) in a developed lentiviral delivery system in neuronal-like cells in view of future in vivo applications. Optogenetic tools based on different photoreceptors to target transcriptional inhibition, protein stability and mRNA levels simultaneously, were designed, characterized and combined for a complete light-induced protein knockout in mammalian cells. These approaches or switches were used for i) quantitative analysis, ii) control of programmed cell death and iii) control of cell cycle progression by endogenous protein knockout. The demonstration of the comprehensive applicability of optogenetic tools designed in this work exemplifies the possibilities and perspectives in various research fields and the importance of synthetic biology to answer today´s fundamental scientific questions. Furthermore, this work illustrates the transmission from the reconstruction of plant lightsignaling pathways to the design and construction of novel optogenetic tools based on this knowledge.Um regulatorische Netzwerke und Signalwege zu rekonstruieren und zu verstehen wurde im Laufe der Zeit im Forschungsfeld der Synthetischen Biologie das relativ neue Feld der Optogenetik etabliert. In diesem Forschungsfeld wurde im Rahmen dieser Arbeit an Phytochrom B basierten Schaltern gearbeitet um diesen zu für in vivo Applikationen zu optimieren, licht-abhängige Interaktionen zu studieren, einen neuen Phytochrom A basierten Schalter zu entwickeln und via viraler Partikel zu transduzieren. Weiter wurden verschiedene optogenetische Werkzeuge entwickelt um Transkription, mRNA und Protein Stabilität mit blauem Licht zu regulieren. Diese Werkzeuge wurden dann für quantitative Analysen, Kontrolle des programmierten Zelltods und reversible Zellzykluskontrolle benutzt. Diese Arbeit zeigt die Anwendbarkeit von optogenetischen Werkzeugen in verschiedenen Fragestellungen sowie die Übertragung von Resultaten der Rekonstruktion auf die Etablierung neuer optogenetischer Werkzeuge. | |||||||
| Lizenz: |  Urheberrechtsschutz | |||||||
| Fachbereich / Einrichtung: | Mathematisch- Naturwissenschaftliche Fakultät | |||||||
| Dokument erstellt am: | 10.03.2020 | |||||||
| Dateien geändert am: | 10.03.2020 | |||||||
| Promotionsantrag am: | 05.12.2019 | |||||||
| Datum der Promotion: | 17.02.2020 |
