TY  - THES
A1  - Schäfer, Ingo
T1  - Fremdgenexpression in humanen Mitochondrien
T1  - Artificial gene expression in human mitochondria
N2  - Bei einer Vielzahl neuromuskulärer und neurodegenerativer Erkrankungen spielen Fehlfunktionen der Mitochondrien eine wichtige Rolle. Da die Proteine der Atmungsketten-komplexe sowohl durch die mitochondriale DNA als auch durch das Kerngenom codiert werden, können Mutationen in beiden Genomen die Auslöser dieser Erkrankungen darstellen. Veränderungen der mitochondrialen DNA lassen sich - im Gegensatz zum Kerngenom - bisher nicht korrigieren, weshalb bei einem großen Teil der Erkrankungen nur die Symptome und nicht die Auslöser behandelt werden können. Das grundlegende Problem stellt dabei der Transport der DNA in die Mitochondrien dar. Ziel dieser Arbeit war es, mit Hilfe von physikalischen Transfektionsmethoden exogene DNA in die Mitochondrien menschlicher Kulturzellen einzubringen. Dazu wurden unterschiedliche Vektoren hergestellt, die in Mitochondrien das an die Mitochondrien angepasste grün fluoreszierende mtEGFP exprimieren sollen. Die Expressionsfähigkeit und Prozessierung dieser Konstrukte konnte in in-vitro-Assays mit einem Mitochondrienextrakt nachgewiesen werden. Bei Transfektionsversuchen mit der Gene Gun gelang es erstmals, exogene Plasmid-DNA in die Mitochondrien menschlicher Zellen einzubringen. Das durch die transfizierten Vektoren exprimierte mtEGFP konnte am Fluoreszenzmikroskop eindeutig in den Mitochondrien der Zellen lokalisiert werden. Eine Transfektion mit Hilfe magnetischer Partikel erwies sich jedoch nicht als zielführend, da die die Partikel eine Eigenfluoreszenz aufwiesen, die eine Detektion der mtEGFP-Expression verhinderten. Eine wichtige Voraussetzung für die Transfektion von Mitochondrien durch mechanische Methoden wie die Mikroinjektion ist die reversible Induktion von Megamitochondrien, da sie erst in diesem Zustand penetriert werden können. Durch eine Ansäuerung des Kulturmediums mit Natriumacetat bzw. Essigsäure konnten Mitochondrien erzeugt werden, die beinahe die Größe des Zellkerns aufwiesen und somit ideale Bedingungen für die Mikroinjektion darstellen. Bei den anschließenden Mikroinjektionsversuchen mit den hergestellten mitochondrialen Expressionsvektoren wurden wiederum Zellen mit eindeutig grün fluoreszierenden Mitochondrien gefunden. Zusammenfassend wurden im Rahmen dieser Arbeit erstmalig menschliche Mitochondrien mit exogener DNA transfiziert. Dies stellt einen grundlegenden Schritt für die Entwicklung neuer Therapieformen bei mitochondrialen Myopathien dar. Zuvor müssen die Transfektionsmethoden jedoch noch weiter optimiert werden, um eine höhere Transfektionseffizienz zu erreichen.
N2  - Mitochondrial dysfunctions play an important role in a variety of neuromuscular and neurodegenerative diseases. As the proteins of the respiratory chain complexes are encoded by the mitochondrial DNA as well as the nuclear genome, mutations in both could trigger solely the diseases. Up to now, changes of the mitochondrial DNA could not be corrected, hence, therapies were designed to decrease symptoms in patients. In this context, the limiting factor to cure these disease relies on the DNA transport into the mitochondria. The aim of this work was to insert exogenous DNA into the mitochondria of human cultured cells by physical transfection methods. A variety of different vectors was constructed to express the mitochondrially adapted green fluorescent mtEGFP within mitochondria. The ability of these constructs to be expressed and processed was proved by in vitro assays using a mitochrondrial extract. In the transfection experiments using the gene gun, we succeeded for the first time to introduce exogenous plasmid DNA into human mitochondria. The mtEGFP expressed by the transfected vectors could definitely be localized to the mitochondria of the cells. Transfections using magnetic particles as mediator could not be used, because the particles exhibit an autofluorescence which prevents a detection of the mtEGFP expression. An essential prerequisite for the transfection of mitochondria by mechanical methods like microinjection is the reversible induction of megamitochondria, since they could not be penetrated as small regular organelles. Acidification of the culture medium with sodium acetate or acetic acid led to mitochondria exhibiting almost the size of the nucleus, thus giving ideal conditions for microinjection. In the applied microinjection experiments using the mitochondrial expression vectors, cells displayed mitochondria with distinct green fluorescence. In summary, human mitochondria were transfected successfully for the first time with mitochondrial expression vectors. This is a fundamental step in the development of new therapies targeting mitochondrial myopathies. However, the transfection methods have to be optimized to achieve higher transfection efficiencies.
KW  - Mitochondrium
KW  - Heterologe Genexpression
KW  - Mitochondrien
KW  - mitochondria
KW  - gene expression
KW  - Transfektion
KW  - Mensch
KW  - Genexpression
Y1  - 2011
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-85202
ER  - 
TY  - JOUR
A1  - Le Provost, Gaëtane
A1  - Thiele, Jan
A1  - Westphal, Catrin
A1  - Penone, Caterina
A1  - Allan, Eric
A1  - Neyret, Margot
A1  - van der Plas, Fons
A1  - Ayasse, Manfred
A1  - Bardgett, Richard D.
A1  - Birkhofer, Klaus
A1  - Boch, Steffen
A1  - Bonkowski, Michael
A1  - Buscot, Francois
A1  - Feldhaar, Heike
A1  - Gaulton, Rachel
A1  - Goldmann, Kezia
A1  - Gossner, Martin M.
A1  - Klaus, Valentin H.
A1  - Kleinebecker, Till
A1  - Krauss, Jochen
A1  - Renner, Swen
A1  - Scherreiks, Pascal
A1  - Sikorski, Johannes
A1  - Baulechner, Dennis
A1  - Blüthgen, Nico
A1  - Bolliger, Ralph
A1  - Börschig, Carmen
A1  - Busch, Verena
A1  - Chisté, Melanie
A1  - Fiore-Donno, Anna Maria
A1  - Fischer, Markus
A1  - Arndt, Hartmut
A1  - Hoelzel, Norbert
A1  - John, Katharina
A1  - Jung, Kirsten
A1  - Lange, Markus
A1  - Marzini, Carlo
A1  - Overmann, Jörg
A1  - Paŝalić, Esther
A1  - Perović, David J.
A1  - Prati, Daniel
A1  - Schäfer, Deborah
A1  - Schöning, Ingo
A1  - Schrumpf, Marion
A1  - Sonnemann, Ilja
A1  - Steffan-Dewenter, Ingolf
A1  - Tschapka, Marco
A1  - Türke, Manfred
A1  - Vogt, Juliane
A1  - Wehner, Katja
A1  - Weiner, Christiane
A1  - Weisser, Wolfgang
A1  - Wells, Konstans
A1  - Werner, Michael
A1  - Wolters, Volkmar
A1  - Wubet, Tesfaye
A1  - Wurst, Susanne
A1  - Zaitsev, Andrey S.
A1  - Manning, Peter
T1  - Contrasting responses of above- and belowground diversity to multiple components of land-use intensity
JF  - Nature Communications
N2  - Land-use intensification is a major driver of biodiversity loss. However, understanding how different components of land use drive biodiversity loss requires the investigation of multiple trophic levels across spatial scales. Using data from 150 agricultural grasslands in central Europe, we assess the influence of multiple components of local- and landscape-level land use on more than 4,000 above- and belowground taxa, spanning 20 trophic groups. Plot-level land-use intensity is strongly and negatively associated with aboveground trophic groups, but positively or not associated with belowground trophic groups. Meanwhile, both above- and belowground trophic groups respond to landscape-level land use, but to different drivers: aboveground diversity of grasslands is promoted by diverse surrounding land-cover, while belowground diversity is positively related to a high permanent forest cover in the surrounding landscape. These results highlight a role of landscape-level land use in shaping belowground communities, and suggest that revised agroecosystem management strategies are needed to conserve whole-ecosystem biodiversity.
KW  - biodiversity
KW  - community ecology
KW  - grassland ecology
Y1  - 2021
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-371552
VL  - 12
ER  - 
TY  - JOUR
A1  - Steimle, Alex
A1  - Menz, Sarah
A1  - Bender, Annika
A1  - Ball, Brianna
A1  - Weber, Alexander N. R.
A1  - Hagemann, Thomas
A1  - Lange, Anna
A1  - Maerz, Jan K.
A1  - Perusel, Raphael
A1  - Michaelis, Lena
A1  - Schäfer, Andrea
A1  - Yao, Hans
A1  - Löw, Hanna-Christine
A1  - Beier, Sina
A1  - Mebrhatu, Mehari Tesfazgi
A1  - Gronbach, Kerstin
A1  - Wagner, Samuel
A1  - Voehringer, David
A1  - Schaller, Martin
A1  - Fehrenbacher, Birgit
A1  - Autenrieth, Ingo B.
A1  - Oelschlaeger, Tobias A.
A1  - Frick, Julia-Stefanie
T1  - Flagellin hypervariable region determinessymbiotic properties of commensalEscherichia coli strains
JF  - PLoS Biology
N2  - Escherichia coli represents a classical intestinal gram-negative commensal. Despite this commensalism, different E. coli strains can mediate disparate immunogenic properties in a given host. Symbiotic E. coli strains such as E. coli Nissle 1917 (EcN) are attributed beneficial properties, e.g., promotion of intestinal homeostasis. Therefore, we aimed to identify molecular features derived from symbiotic bacteria that might help to develop innovative therapeutic alternatives for the treatment of intestinal immune disorders. This study was performed using the dextran sodium sulphate (DSS)-induced colitis mouse model, which is routinely used to evaluate potential therapeutics for the treatment of Inflammatory Bowel Diseases (IBDs). We focused on the analysis of flagellin structures of different E. coli strains. EcN flagellin was found to harbor a substantially longer hypervariable region (HVR) compared to other commensal E. coli strains, and this longer HVR mediated symbiotic properties through stronger activation of Toll-like receptor (TLR)5, thereby resulting in interleukin (IL)-22–mediated protection of mice against DSS-induced colitis. Furthermore, using bone-marrow–chimeric mice (BMCM), CD11c+ cells of the colonic lamina propria (LP) were identified as the main mediators of these flagellin-induced symbiotic effects. We propose flagellin from symbiotic E. coli strains as a potential therapeutic to restore intestinal immune homeostasis, e.g., for the treatment of IBD patients.
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-239501
VL  - 17
ER  -