711 research outputs found
Charakterisierung der Bindung humaner Proteine an G-Quadruplex-DNA und -RNA
No full textThe doctoral project presented in this work was dedicated to the characterization of the binding properties of nuclear proteins in relation to so-called G-quadruplex nucleic acid structures. The G-quadruplex is a special secondary structure of nucleic acids in which four guanine molecules within a sequence come together to form a planar tetrad. Different numbers of such G-tetrads combine to form different types of this unusual secondary structure. The binding of the proteins was analyzed with regard to G-quadruplex structures (Myc22-DNA, Myc22-RNA, CEB25-L111, CEB25-L191) and non-G-quadruplex structures (guanine-rich ssDNA and dsDNA). Both the deoxyribooligonucleotide and the ribooligonucleotide of the Myc22 sequence already used by Rauser (Valerie Rauser, PhD thesis RWTH, 2019) were used here. Analogously to the method described by Rauser for generating monomeric structures of the G-quadruplex (G4) Pu27 DNA or Myc22 DNA under basic conditions, a method for generating monomeric Myc22 RNA structures was first established at the beginning of the project. The success of each treatment approach was verified by analytical methods such as size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). The pretreated oligonucleotides, folded into monomeric G4 structures, were then used for binding studies with certain predominantly nuclear proteins - starting from proteins that were identified by Rauser in 2019 as promising G4 binding candidates through proteomic analyzes of ovarian cancer cell lysates. As preliminary work for these binding studies, some of the potential G4-binding proteins (EED, hnRNPU and YBX1), each equipped with a His tag, were overexpressed in E. coli cells and purified using affinity chromatography (Ni-NTA column) and gel filtration. The characterization of the protein binding properties to the various DNA and RNA structures was carried out using biochemical and biophysical methods in order to enable the most accurate possible verification of the results. The pulldown assay and the EMSA (electrophoretic mobility shift assay) were used as biochemical methods. The pulldown assay is a type of affinity chromatographic isolation method in which proteins can be immobilized by their binding to oligonucleotide-coated magnetic particles. The protein-DNA complexes formed during the binding reaction can be dissociated after repeated washing of the samples with a sodium dodecyl sulfate solution (sodium dodecyl sulfate, SDS) and the fixed proteins can be identified and quantified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). In EMSA, a binding reaction with protein and fluorophore-labeled (Cy5) oligonucleotide is set up and incubated. The entire binding mixture is then applied to a native polyacrylamide gel, which typically reveals the eponymous shift of the oligonucleotide bands compared to the G4-protein complex bands. The FID assay (fluorescent intercalator displacement assay, FIDA) and MST (microscale thermophoresis) were used as biophysical methods. For the FIDA, the unmodified oligonucleotide is first incubated with a fluorophore (here acridine orange or thiazole orange) and this mixture is then gradually titrated with increasing concentrations of protein. Here, the successive decrease in fluorescence due to the displacement of the fluorophore by the added protein is recorded. In principle, microscale thermophoresis is also based on the titration of a Cy5-modified oligonucleotide of constant concentration with different protein concentrations. The evaluation is carried out by detecting the change in fluorescence or by quantifying the G4-protein complex formed as a function of the protein concentration used. After carrying out the various binding assays, the respective results for each protein to be characterized were analyzed, compared and classified according to G4 selectivity. It then finally became apparent whether the potential G4-binding proteins from previous proteomic analyzes are in fact G4 binders or whether there are deviations. In the end, a clearly recognizable G4 selectivity could only be seen for some of the proteins examined. Some of the other candidates showed an increased guanine selectivity, some showed a complete lack of selectivity in relation to the nucleic acid structures used
Isolierung und Identifikation von G-Quadruplex-DNA-bindenden Proteinen
No full textIn this work, progress was made in the subject of protein isolation and identification, which bind to the biologic relevant G-quadruplex forming DNA sequence derived from the promotor of the onkogen c-myc. Development of a new methodology enabled the transformation of the polymorphic structure of the guanine-rich sequence Pu27 of the c-myc promotor to a monomeric G4 structure. Multimers were denaturized by treatment with 150 mM NaOH at room temperature. Renaturization in presence of excessive amount of K+¬-buffer lead to a selective formation of the desired parallel oriented G4-monomer. The successful application of this method could be confirmed by SEC-analyses, gel electrophoresis, CD spectroscopy and UV-melting curves for the Pu27 sequence as well as related DNA sequences. The monomerized and biotinylized G4 DNA was immobilized on streptavidin coated magnetic beads for the isolation of the G4 binding proteins from cell lysate. Beside Pu27 and its derivative Myc22, single- and double-stranded DNA was tested as control sequence. Furthermore, the displaceability of the proteins was evaluated by treatment with the G4-binding ligand PhenDC3. Analysis via SDS-PAGE showed a selective binding of many proteins to the Pu27 and Myc22 sequence, which showed no affinity for the single- and double-stranded control sequence. On the other hand, those DNA structures enabled the isolation of non-G4-binding DNA. Other proteins could be found in all samples. Many of the G4-binding proteins could be displaced by PhenDC3. For identification of the isolated proteins, the generated samples were analyzed via mass spectrometry and assigned based on a human database in collaboration with the Institute Curie in Paris, France. Analysis of the protein affinity to G4-sequence in comparison to the control sequence enabled the selection of the relevant target proteins. In a second step, the promising proteins examined regarding the displaceability with PhenDC3. Proteins with a good binding property to the examined G4-sequence as well as a good displaceability with PhenDC3 are defined as target protein. 28 target proteins meeting those criteria could be identified via this method. Next, the affinity of purified target proteins to different DNA structures was examined. Within this study, binding of identified target proteins to G4 DNA was verified and the proteins YBX1 and hnRNP U were identified as promising candidates. Alltogether, a biologic relevant, monomeric G4-structure derived from the polymorphic c-myc promotor was formed in vitro. This G4 could be utilized for the isolation of proteins from humane cancer cells. Those target proteins have been identified and their binding selectivity was validated, which enables further investigations of their biologic function
MALDI-MS tissue imaging identification of biliverdin reductase B overexpression in prostate cancer
No full textAbstract not availableJohannes Dominikus Pallua, Georg Schaefer, Christof Seifarth, Michael Becker, Stephan Meding, Sandra Rauser, Axel Walch, Michael Handler, Michael Netzer, Marina Popovscaia, Melanie Osl, Christian Baumgartner, Herbert Lindner, Leopold Kremser, Bettina Sarg, Georg Bartsch, Christian W. Huck, Günther K. Bonn, Helmut Klocke
Quantitative Formation of Monomeric G-Quadruplex DNA from Multimeric Structures of c-Myc Promoter Sequence
No full textGeneration of virus-specific T-cells for adoptive transfer
No full textDie HCMV-Infektion stellt für Patienten nach allogener Stammzelltransplantation nach wie vor eine lebensbedrohliche Erkrankung dar. Da die medikamentöse Behandlung der HCMV-Infektion mit schweren Nebenwirkungen verbunden ist und zudem immer häufiger von Virustatika-resistenten HCMV-Stämmen berichtet wird, stellt die adoptive Immuntherapie eine wichtige alternative Therapieoption dar.
Die gängigen Protokolle zur Generierung HCMV-spezifischer T-Zellen verwenden vermehrungsfähige Viren oder allogene Zellkulturzusätze, die Expansion der T-Zellen dauert meist mehrere Wochen bis Monate.
In der vorliegenden Arbeit wurde ein Protokoll entwickelt, das die Generierung CMV-spezifischer CD4+ und CD8+ T-Zelllinien aus einer 500 ml Vollblutspende ermöglicht, unter Verwendung von ausschließlich autologen Zell- und Serumkomponenten. Nach Stimulation von Spenderlymphozyten mit verschiedenen HLA-restringierten Peptiden sowie CMV-Antigen und Anreicherung der IFN-g produzierenden Zellen mittels IFN-g secretion assay, konnte in der anschließenden Kultur innerhalb von 10 Tagen eine für einen Transfer ausreichende Menge an T-Zellen gewonnen werden. Die generierten T-Zellen waren auch nach der Expansion hochspezifisch und weiterhin vermehrungsfähig. Die CMV-spezifische Immunantwort wurde noch erweitert, indem ein neues HLA-A*0201 Peptid aus dem HCMV/IE-Antigen definiert wurde, gegen das sich ebenfalls spezifische T-Zellen generieren liessen. Im letzten Teil der Arbeit konnte gezeigt werden, dass sich das erarbeitete Protokoll auch zur Generierung von EBV-spezifischen T-Zellen einsetzten lässt. Der Transfer solcher T-Zellen stellt eine wichtige Therapieoption für Patienten mit EBV-positivem Hodgkin-Lymphom dar.
Die Spezifität und mögliche Alloreaktivität der generierten T-Zellen wurde zwar ex vivo mit verschiedenen Methoden bestimmt, erst in einer klinischen Studie kann aber letztlich die Wirksamkeit und Unbedenklichkeit der generierten und transferierten CD4+ und CD8+ CMV-spezifischen T-Zellen getestet werden. Das entwickelte Protokoll lässt sich dabei leicht an die erforderlichen GMP-Bedingungen anpassen, alle benötigten Reagenzien stehen in geeigneter Qualität zur Verfügung und auch entsprechende Reinräume sind vorhanden.Human cytomegalovirus (HCMV) infection still accounts for high morbidity and mortality in patients undergoing allogeneic stem cell transplantation. Because of an increasing number of reports of HCMV-isolates resistent to current standard antiviral therapy as well as the toxicity of these antiviral agents, the interest in adoptive immunotherapy as an alternative means for treatment and prevention of HCMV disease was promoted.
Current protocols for generating HCMV-specific T-cells utilize live virus or allogeneic cellular and serum components and are very time consuming for ex vivo expansion.
In the present thesis, a clinical scale protocol was developed for generation of combined CD4+ and CD8+ CMV-specific T-cells by utilizing autologous cellular and serum components derived from one single 500 mL blood draw. Donor lymphocytes were stimulated simultaneously with CMV-specific MHC-I peptides and CMV-antigen. Activated T-cells were isolated with the IFN-g secretion assay and could be expanded within 10 days to numbers sufficient for adoptive transfer. The generated T-cells were highly specific after isolation by efficiently lysing HCMV-infected targets and expansion and further proliferated upon restimulation with CMV-antigen. CMV-specific immune response could be further expanded by defining a new HLA-A*0201 peptide from HCMV/IE and generating specific T-cells against this epitope. The last part of this thesis shows, that the developed protocol can also be translated to EBV-specific T-cells specific for the EBV-proteins expressed by Hodgkin and Non-Hodgkin. Transfer of such T-cells can be an important therapy-option for patients with EBV-positive Hodgkin-lymphoma.
Important for transferring the developed protocol to a clinical trial, the alloreactivity against third party against was evaluated, demonstrating a vast reduction in mounting such a response. Nevertheless, clinical efficacy as well toxicity of generated CD4+ and CD8+ CMV-specific T-cells will be evaluated in a clinical trial
Understanding uncertainty as a key interdisciplinary problem in earth system science
No full textThe emerging field of Earth system science integrates strongly disciplinary sciences with a multitude of new interdisciplinary fields in the joined attempt to understand the interactions and properties of the Earth system as a whole. Cross-disciplinary language problems are a natural consequence as every discipline brings with it a multitude of definitions, historical connotations, and interpretations of scientific language. In an attempt to help remedy this situation for Earth system science, the Young Earth System Science community (YESS) has worked in recent years on creating a description of what is meant when we talk of uncertainty in the Earth system sciences. The difference in methodology and quality of what different scientists mean when they refer to uncertainty is large, and in a series of workshops in 2012 and 2014 and an international conference in 2013, YESS has tried to bridge the disciplinary gaps a little. We believe that a consistent uncertainty language treatment within our field of research is a first step to a concrete representation of uncertainty in all spheres of scientific communication, from the disciplinary communication to the communication with stakeholders and the public. © 2017 by the American Geophysical Union
MALDI imaging mass spectrometry on formalin-fixed paraffin-embedded tissues.
No full textTissue samples have been routinely used for decades to distinguish healthy from diseased tissue in histopathological characterization. While nucleic acid-based methodologies have been successfully in use for many years, protein-based techniques, in contrast, are at a very early stage (with the exception of immunohistochemistry). In this chapter, a method for the analysis of FFPE tissues by MALDI imaging mass spectrometry is described
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