Universitätspublikationen
783 search hits
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"... ergab sich bald ein merkwürdiges Hindernis ..." : zur Aktualität von Ludwig Edingers neurowissenschaftlichem Projekt
(2005)
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Gerald Kreft
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"A system for the intracellular generation of triple helix-forming oligonucleotides (TFOs) and the sequence-specific inhibition of human MCP-1 gene expression"
(2005)
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Kordula Kautz
- Chemokines play a key role in the cellular infiltration of inflamed tissue. They are released by a wide variety of cell types during the initial phase of host response to injury, allergens, antigens, or invading microorganisms, and selectively attract leukocytes to inflammatory foci, inducing both migration and activation. Monocyte chemoattractant protein-1 (MCP-1), a member of the CC chemokine superfamily, functions in attracting monocytes, T lymphocytes, and basophils to sites of inflammation. MCP-1 is produced by monocytes, fibroblasts, vascular endothelial cells and smooth muscle cells in response to various stimuli such as tumour necrosis factor-a (TNF-a), interferon-g (IFN-g), and interleukin-1b (IL-1b). It also plays an important role in the pathogenesis of chronic inflammation, and overexpression of MCP-1 has been implicated in diseases including glomerulonephritis and rheumatoid arthritis. Oligonucleotide-directed triple helix formation offers a means to target specific sequences in DNA and interfere with gene expression at the transcriptional level. Triple helix-forming oligonucleotides (TFOs) bind to homopurine/homopyrimidine sequences, forming a stable, sequence-specific complex with the duplex DNA. Purine-rich sequences are frequent in gene regulatory regions and TFOs directed to promoter sequences have been shown to prevent binding of transcription factors and inhibit transcription initiation and elongation. Exogenous TFOs that bind homopurine/ homopyrimidine DNA sequences and form triple-helices can be rationally designed, while the intracellular delivery of single-stranded RNA TFOs has not been studied in detail before. In this study, expression vectors were constructed which directed transcription of either a 19 nt triplex-forming pyrimidine CU-TFO sequence targeting the human MCP-1 or two different 19 nt GU- or CA-control sequences, respectively, together with the vector encoded hygromycin resistance mRNA as one fusion transcript. HEK 293 cells were stable transfected with these vectors and several TFO and control cell lines were generated. Functional relevant triplex formation of a TFO with a corresponding 19 bp GC-rich AP-1/SP-1 site of the human MCP-1 promoter was shown. Binding of synthetic 19 nt CUTFO to the MCP-1 promoter duplex was verified by triplex blotting at pH 6.7. Underlining binding specificity, control sequences, including the GU- and CA-sequence, a TFO containing one single mismatch and a MCP-1 promoter duplex containing two mismatches, did not participate in triplex formation. Establishing a magnetic capture technique with streptavidin microbeads it was verified that at pH 7.0 the 19 nt TFO embedded in a 1.1 kb fusion transcript binds to a plasmid encoded MCP-1 promoter target duplex three times stronger than the controls. Finally, cell culture experiments revealed 76 ± 10.2% inhibition of MCP-1 protein secretion in TNF-a stimulated CU-TFO harboring cell lines and up to 88% after TNF-a and IFN-g costimulation in comparison to controls. Expression of interleukin-8 (IL-8) as one TNF-a inducible control gene was not affected by CU-TFO, demonstrating both highly specific and effective chemokine gene repression. Furthermore, another chemokine target, regulated upon activation normal T cell expressed and secreted (RANTES), which plays an essential role in inflammation by recruiting T lymphocytes, macrophages and eosinophils to inflammatory sites, was analysed using the triplex approach. A 28 nt TFO was designed targeting the murine RANTES gene promoter, and gel mobility shift assays demonstrated that the phosphodiester TFO formed a sequencespecific triplex with the double-stranded target DNA with a Kd of 2.5 x 10-7 M. It was analysed whether RANTES expression could be inhibited at the transcriptional level testing the TFO in two different cell lines, T helper-1 lymphocytes and brain microvascular endothelial cells (bend3 cells). Although there was a sequence-specific binding of the TFO detectable in the gel shift assays, there was no inhibitory effect of the exogenously added and phosphorothioate stabilised TFO on endogenous RANTES gene expression visible. Additionally, the small interfering RNA (siRNA) approach was tested as another strategy to inhibit expression of the pro-inflammatory chemokines MCP-1 and RANTES. Two different methods were pursuit, describing transient transfection with vector derived and synthetic siRNA. The vector pSUPER containing the siRNA coding sequence was used to suppress endogenous MCP-1 in HEK 293 cells. An empty vector without RNA sequence served as a control. Inhibition due to the siRNA was measured in stimulated and unstimulated cells. In TNF-a stimulated cells MCP-1 protein synthesis was decreased by 35 ± 11% after siRNA transfection. Using a synthetic double-stranded siRNA, the TNF-a induced MCP-1 protein secretion could be successfully inhibited about 62.3 ± 10.3% in HEK 293 cells, indicating that the siRNA is functional in these cells to suppress chemokine expression. The siRNA approach targeting murine RANTES in Th1 cells and b-end3 cells revealed no inhibition of endogenous gene expression. Gene therapy approaches rely on efficient transfer of genes to the desired target cells. A wide variety of viral and nonviral vectors have been developed and evaluated for their efficiency of transduction, sustained expression of the transgene, and safety. Among them, lentiviruses have been widely used for gene therapy applications. In order to improve the delivery of TFOs or siRNAs into the target cells, cloning of the lentiviral transfer vector SEW, the production of lentiviral particles by transient transfection were performed with the aim to generate lentiviral vector-derived TFOs in further experiments. Here, Th1 cells were transduced with infectious lentiviral particles and transduction efficacy was measured. Transduction efficacy higher than 82% could be achieved using the lentiviral vector SEW, opening optimal possibilities for the TFO or siRNA approach.
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"Also los und Mut! ... Die Werkstätte, unsere Werkstätte muß eingerichtet werden" : Hans Poelzigs Skizzenbücher und Briefe liefern neue Erkenntnisse über Werk und Ateliergemeinschaft mit Marlene Moeschke
(2005)
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Heike Hambrock
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"Auferstanden aus Ruinen ..." : Bechers Nationalismus - Brechts Kritik
(2005)
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Benjamin Ortmeyer
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"Der Schöpfer des modernen Frankfurt" : Wandel zur anerkannten Großstadt - Franz Adickes' Wirken als Oberbürgermeister - zum 90. Todestag
(2005)
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Gudrun Jäger
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"Die Entfernung ist schrecklich" : Freunde trotz erzwungener Distanz ; der Briefwechsel zwischen Alfred Schütz und Eric Voegelin
(2005)
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Thorsten Benkel
- Rezension zu: Alfred Schütz / Eric Voegelin : Eine Freundschaft, die ein Leben ausgehalten hat. Briefwechsel 1938 –1959. Hrsg. von Gerhard Wagner und Gilbert Weiss, UVK Verlagsgesellschaft, Konstanz, 2004, ISBN 389669-699-8, 610 Seiten, 128 Euro.
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"Disputation gegen die Frauen zum Beweis, dass sie keine Menschen sind" : ein Frankfurter Sammelband zum Geschlechterstreit am Beginn der europäischen Moderne
(2005)
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Andreas Kraß
- Rezension zu: Gisela Engel / Friederike Hassauer / Brita Rang / Heide Wunder (Hrsg.) : Geschlechterstreit am Beginn der europäischen Moderne, Die Querelle des Femmes, Kulturwissenschaftliche Gender Studies, Band 6, Ulrike Helmer Verlag, Königstein/Taunus, 2004, ISBN 3-89741-170-9, 353 Seiten, 34,95 Euro.
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"Durch Methodenkombination ein Neuro-Forschungszentrum erster Güte" : Stefan Kieß im Gespräch mit Prof. Dr. Helmuth Steinmetz
(2005)
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Stefan Kieß
Helmuth Steinmetz
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"Ein sozioökonomischer Blick auf Deutschland unter armutspolitischen Gesichtspunkten"
(2005)
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Christian Vas
- 1 Institutionelle Organisation und politischer Aufbau der Bundesrepublik Deutschland 2 Die Sozio-ökonomische Situation 2.1 Einkommensverteilung und Armut 2.1.1 Generelle Einkommensentwicklung der 90er Jahre 2.1.2 Ungleichheit der Einkommen 2.1.3 Armut und Armutsquoten 2.1.4 Verteilung der Armutsquoten auf bestimmte Personengruppen 2.1.5 Die Verweildauer von in Armut lebenden Menschen 2.2 Erwerbsarbeit und Erwerbslosigkeit 2.2.1 Strukturen der Erwerbsarbeit 2.2.2 Strukturen der Arbeitslosigkeit 2.3 Bildung und Ausbildung 2.4 Wohnsituation 2.5 Gesundheit und Armut 3 Demographische Situation 4 Strategien zur Bekämpfung von sozialer Ausgrenzung und Armut 4.1 Charakterisierung des deutschen Sozialstaats 4.2 Sozialpolitische Strategien zur Armutsbekämpfung 4.2.1 Der Armuts- und Reichtumsbericht der Bundesregierung 4.2.2 Der Nationale Aktionsplan zur Bekämpfung von sozialer Ausgrenzung und Armut 4.2.3 Forum Teilhabe und soziale Integration (FORTEIL) 4.2.4 Die Agenda 2010 als Teilumsetzung des Armutsberichtes und von NAP-incl
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"Eng ist die Welt, und das Gehirn ist weit" : vom Unfug des gefesselten Willens – Ansichten eines Arztes
(2005)
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Wolfgang Schlote
