Lehrstuhl für Biologisch-Chemische Mikrostrukturtechnik
Permanent URI for this collection
Browse
Recent Submissions
Item Untersuchung der Kaskadenreaktion von Glukose-Oxidase und Meerrettich-Peroxidase in räumlich definierten Anordnungen auf der Nano- und Mikrometerskala(2017) Müller, Joachim; Niemeyer, Christof M.; Rauh, DanielIn dieser Arbeit wurden zwei Strategien zum Aufbau von kompartimentierten GOX/HRP-Reaktionskaskaden untersucht. Die erste Strategie bestand in der Assemblierung der Enzyme im Nanometermaßstab durch Methoden der DNA Nanotechnologie. Hierdurch sollten künstliche Multienzymkomplexe aufgebaut werden, die auf eine mögliche Beschleunigung der Gesamtreaktion durch räumliche Nähe, dem sogenannten „Channeling“, untersucht wurden. Bei der zweiten Strategie wurden die Enzyme in einen Festbettreaktor im Mikrometermaßstab angeordnet, um in der Mikrofluidik die Kinetik der gekoppelten Reaktionen nach Michalis Menten zu bestimmen.Item Synthese von supramolekularen Nucleinsäure-Protein-Nanostrukturen(2014-04-24) Schmitte, Holger; Niemeyer, Christof M.; Brakmann, SusanneItem Imaging EphA3 receptor tyrosine kinase substrate interaction(2012-12-04) Hou, Jian; Bastiaens, Philippe I.; Winter, RolandReceptor tyrosine kinases (RTKs) function as key signaling molecules to influence several physiological processes such as cell growth, survival and migration. Crystal structure and biochemical evidence suggests that the kinase activity of some RTKs, like the Eph RTK family, is affected by the phosphorylation of several key tyrosine residues in the activation loop (AL) of the tyrosine kinase domain and in the conserved juxtamembrane segment (JMS). However, in living cells, little is known about this regulatory mechanism in which tyrosine phosphorylation affects the catalytic activity and substrate accessibility of RTKs in both space and time. In this thesis, we used the EphA3 RTK as an example to investigate the JMS auto- inhibition involved in the Eph RTK activity regulatory mechanism. To achieve this, a generic cellular EphA3 RTK substrate interaction-imaging assay was developed that is based on Förster Resonance Energy Transfer as measured by Fluorescence Lifetime Imaging Microscopy (FRET-FLIM). A high affinity EphA3 peptide substrate was applied to increase the probability of kinase-substrate complexes forming. Using this assay, it was possible to quantitatively investigate the formation of steady-state enzyme- substrate (ES) complexes between the purified EphA3 catalytic domain fused to mCitrine and the Cy3.5 labeled peptide substrate. A kinase/phosphatase reaction cycle could be reconstituted that gave rise to stable ES-intermediates as measured by FRET- FLIM. By using this ES imaging approach, EphA3 RTK (WT)-substrate interactions could be observed in EphA3-mCitrine expressing quiescent Cos-7 cells that had been microinjected with the Cy3.5-substrate. This suggests that a population of EphA3 receptors exists with a temporarily relieved JMS auto-repression in the absence of ephrin-A5 ligands. Stimulation with soluble ephrin-A5 ligands led to a detectable increase in ES-intermediates, that pointed to an autocatalytic phosphorylation mechanism of JMS auto-repression relieve. To further study the role of JMS tyrosine phosphorylation on active site accessibility, the ES imaging approach was applied to analyze the active site accessibility of JMS regulation deficient mutants (MTs). Different substrate accessibilities were observed for these JMS regulation-deficient mutants (MTs), which revealed a possible function of JMS tyrosine phosphorylation in the regulation of the EphA3 RTK active site accessibility in living cells. In this work, a generic living cell compatible FRET-FLIM approach has been developed that allows direct observation of kinase-substrate interactions and helps to elucidate conformational dynamics of EphA3 RTK in living cells. In principle, this assay could also be used to quantitatively address conformational dynamics of other RTKs in living cells.Item Chemistry and biology of natural product derived protease inhibitors(2012-07-19) Stolze, Sara Christina; Waldmann, Herbert; Kaiser, MarkusIm Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden. Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwickelt. Die Festphasenstrategie ermöglicht die Synthese maßgeschneiderter Ahp-Cyclodepsipeptide; sie beinhaltet eine Veresterung an der festen Phase, sowie eine Festphasen-Macrolaktamisierung und etabliert ein neues Protokoll zur Generierung eines Aldehyds bei Abspaltung von der festen Phase. Zur Überprüfung der neu-entwickelten Strategie wurde der Chymotrypsin-Inhibitor Symplocamide A erfolgreich an der festen Phase synthetisiert und im Anschluss auf seine biologische Aktivität hin untersucht. Mit Symplocamide A als der Leitstruktur wurden vereinfachte Analoga von Ahp-Cyclodepsipeptiden synthetisiert, um zu überprüfen ob diese Analoga, wie die Ahp-Cyclodepsipeptide auch, die sogenannte kanonische Konformation einnehmen. Die kanonische Konformation wurde zuerst in proteinogenen Serinprotease-Inhibitoren beobachtet und studiert. Kürzlich wurden auch peptidische Analoga dieser Inhibitoren untersucht und ein Zusammenhang mit den Ahp-Cyclodepsipeptiden erkannt, welche ebenfalls durch die Einnahme der kanonischen Konformation inhibieren. Im Rahmen dieser Dissertation war es möglich zu zeigen, dass die Ahp-Einheit unter Retention der biologischen Aktivität durch kommerziell erhältliche Aminosäuren ersetzt werden kann. Mittels Struktur-Wirkungsuntersuchungen konnten zudem die kritischen Determinanten zur Einnahme der kanonischen Konformation bestimmt, sowie ein Einblick in die molekulare Grundlage einer effizienten Inhibition gewonnen werden. Für den Naturstoff Symplostatin 4 wurde eine konvergente Lösungssynthese entwickelt, die einen flexiblen Zugang zu modifizierten Derivaten ermöglichte. Speziell die Synthese von Sonden für die Untersuchung von Symplostatin 4 mittels aktivitätsbasiertem Proteinprofiling war durch eine 1,3-dipolare Huisgen-Cycloaddition von propargyl-modifizierten Spezies mit Azid-modifizierten Reportermolekülen vereinfacht möglich. Durch aktivitätsbasiertes Profiling konnten mit den Cystein-Proteasen RD21A und RD21B die Zielenzyme von Symplostatin 4 in der Modellpflanze Arabidopsis thaliana identifiziert werden. Desweiteren wurde die anti-Malaria-Aktivität, die für das chemisch identische Gallinamide A publiziert worden war, für Symplostatin 4 und seine Derivate studiert. Im Rahmen von Untersuchungen mit dem Malariaerreger Plasmodium falciparum konnte festgestellt werden, dass Symplostatin 4 ein nanomolarer Inhibitor der Cystein-Proteasen Falcipain 2 und 3 ist, die für den Hemoglobinverdau zuständig sind und als wichtige Zielenzyme einer alternativen Malaria-Chemotherapie betrachtet werden.Item Isolation, Charakterisierung und hepatische Differenzierung von mesenchymalen Stammzellen aus adipösem Gewebe und ihr möglicher Einsatz bei klinischen Fragestellungen(2011-11-09) Seeliger, Claudine; Hengstler, Jan Georg; Niemeyer, ChristofHepatocyte transplantation is considered to be an alternative to orthotopic liver transplantation. Cells can be used to bridge patients waiting for a donor organ, decrease mortality in acute liver failure, and to support metabolic liver diseases. The limited availability of primary human hepatocytes for such applications has lead to the generation of alternative hepatocyte-like cells from various adult stem or precursor cells. The aim of this study was to generate hepatocyte-like cells from adipose-derived mesenchymal stem cells (Ad-MSCs) for clinical applications, which are available “off the shelf”. Characteristic CDmarker could be detected in the isolated Ad-MSCs. Furthermore cells showed a decrease of proliferation and telomere length over the passages. Epigenetic changes in hepatocyte-like cells were induced by 5-azacytidine, which in combination with other supplements leads to significantly improved metabolic and enzymatic activities compared to non-treated cells. Cells with sufficient hepatic features were generated with a 4-step protocol: 5-azacytidine (Step1), epidermal growth factor (Step 2), fibroblast growth factor-4, dexamethasone, insulintransferrin- sodium-selenite, nicotinamide (Step 3) and hepatocyte growth factor, dexamethasone, insulin-transferrin-sodium-selenite and nicotinamide (Step 4). Morphological changes were registered through the differentiation. Hepatocyte-like cells are more compact and hexagonal compared to the fibroblast-like Ad-MSCs. Generated differentiated cells had higher phase I (CYP1A1/2, CYP2E1, CYP2B6, CYP3A4) and phase II activities compared to the undifferentiated cells. A strong expression of CYP3A7 and a weak expression of CYP3A4, as well as the important detoxification markers, á-fetoprotein and albumin, could also be detected at the mRNA-level. Importantly, urea metabolism (basal, NH4-stimulated, NH4 and ornithine-stimulated) was comparable to freshly isolated human hepatocytes and, unlike cryopreserved human hepatocytes, this activity was maintained after six months of cryopreservation. These findings suggest that these cells may be suitable for clinic application, especially for treatment of urea cycle disorders.Item Entwicklung eines in vitro Systems zur Untersuchung von substanzinduzierten Genexpressionsänderungen bei primären Hepatozyten der Ratte(2011-06-29) Schug, Markus; Hengstler, Jan Georg; Kiemer, Alexandra Kathrin; Niemeyer, ChristofDevelopment of an in vitro system for the detection of substance induced gene expression alterations in primary rat hepatocytes Following the new European legislation for chemicals (REACH) a large amount of chemicals need to be evaluated from the toxicological perspective. With commonly used techniques, the current testing capacities will be overloaded. Therefore, there is a requirement for faster and more reliable test systems for toxicology. In the last couple of years there has been a lot of progress in the field of toxicogenomics. After exposing animals to toxic substances, it is possible to detect the mode of action based on the substance induced gene expression alterations. This kind of analysis requires animal experiments and is not feasible for high throughput testing. An alternative test method would be the investigation of cultured hepatocytes. Approx. 600 million hepatocytes can be isolated from one rat liver, which would be sufficient for roughly 100 six well plates. At the beginning of this thesis the majority of groups working in this field saw no feasible option to perform gene expression analysis of cultured hepatocytes. This is based on the fact, that cultured hepatocytes undergo a (“spontaneous”) change of more then 1000 genes caused only by the culture conditions. Nonetheless, in this thesis an in vitro system based on primary rat hepatocytes has been established which is able to detect similar substance induced gene expression changes as observed in the in vivo rat model. To achieve this, three commonly used culture systems for the cultivation of primary cells were investigated: (1) the classical collagen monolayer culture, where hepatocytes are cultured on a thin film of collagen, (2) the collagen sandwich culture, where hepatocytes are cultivated between two layers of a collagen gel and (3) Matrigel®-culture, a commercially available smooth extracellular matrix in which the hepatocytes are embedded. The hepatocytes were incubated with methapyrilene as a test substance in a concentration of 100 and 200 μM and the gene expression of four markergenes (Abat, Gsk3b, Myd116 and Sult1a1) was measured. These genes were already known as characteristically up- (Gsk3b, Myd116) and downregulated (Abat, Sult1a1) under the influence of methapyrilene. It could be shown that these characteristic deregulations were the most pronounced and the most reproducible in the collagen sandwich culture. Therefore, the collagen sandwich was used in subsequent experiments. Besides the choice of the collagen sandwich culture as the optimal culture system, it was discovered that there is an advantage to cultivate both control and treated cells on the same six well dish. The variability on the same plate was less then the plate-to-plate variability. When developing a new in vitro system, the choice of the correct concentration of test substances is one critical aspect. This concentration should be in the range of a concentration which can also be obtained in vivo. The results, using the optimized system, show a deregulation at a level of 0.39 μM and a clear deregulation at a level of 6.25 μM methapyrilene. It is known from the literature, that an i.p.-injection of 0.7 mg/kg BW methapyrilene results in a plasma level of approx. 0.5 μM 10 minutes after injection. This led to the conclusion that this test system is able to detect a substance induced effect on gene expression, even at a level close to in vivo relevant concentrations. This was confirmed by measuring additional genes (Bax, Cdkn1, Gsta2, Hsf1, Mdm2 und Nqo1). A concentration dependent effect could also be shown for aflatoxin B1, piperonylbutoxide and 2-nitrofluorene as additional liver-carcinogens. Based on these encouraging results, a larger study was performed to investigate if the substance induced gene expression changes in the in vivo rat liver and in vitro primary rat hepatocytes are comparable. Two genotoxic (aflatoxin B1, 2-nitrofluorene) and two non-genotoxic (methapyrilene, piperonylbutoxide) compounds were used as test substances. Two gene groups were analyzed: one gene group associated with stress response, DNA-repair and metabolism (gene group 1: Abcb1, Ugt1a6, Gsta5, Gsta2, Hsf1, Nqo1, Apex1, Bax, Cdkn1a, Gadd45a, Mt1a, Sds, Mdm2, Myc) and one gene group which is active during cell proliferation (gene group 2: Mcm6, Cdc2, Hdc, 3-Pgdh, Cdc20, Igfbp1, Top2a, Map3k1, Atf3). The in vitro experiments were performed related to this thesis. The results from the in vivo experiments were provided by a cooperation-partner. Interestingly, there was a very good correlation between the in vivoand the in vitro-results when analysing gene group 1 (p< 0.001). For gene group 2 no significant correlation could be found (p=0.286). This could be explained by the fact that stress-response, DNArepair and metabolism are similar processes both in vitro and in vivo. The missing correlation of proliferation-associated genes (gene group 2) could be due to the fact, that there is no replacement proliferation in vitro. Replacement proliferation in organs means that damaged cells are replaced by the proliferation of surrounding healthy cells. With the current state of the art in culturing primary hepatocytes, the effect of replacement-proliferation can not be seen in vitro. The investigation of gene groups, whose substance induced gene expression alterations correlate between in vitro and in vivo systems, will facilitate the development of toxicological test-systems by focusing only on the relevant genes. At present, the use of primary rat hepatocytes is very often criticized. One reason is that hepatocytes produce artificial or false positive results in vitro. For example, the genes Gsk3b and Myd116 were altered by the influence of methapyrilene in vitro only, but not in rat liver in vivo. If this argumentation is correct, a further use of cultivated rat hepatocytes in toxicogenomics would not make any sense. This was the basis for investigating the apparently occurring in vitro/in vivo discrepancy. The result of these investigations showed that this discrepancy is simply based on the different pharmacokinetics in vitro and in vivo. Methapyrilene has a half-life of 2.8 h. The previous studies focused on 24 h as the earliest time point for sampling, therefore the substance was already completely excreted and the gene expression change had already returned to basal level. By investigation of earlier timepoints, it could be shown, that an induction of Gsk3b and Myd116 also occurred in vivo . Due to this fact, the argument of a discrepancy between in vitro and in vivo is no longer valid. In summary, it has been shown that the culture system described in this thesis, is able to detect substanceinduced gene expression alterations at in vivo relevant concentrations. These changes in gene expression correlate well with the in vivo situation concerning the group of stress-associated genes.Item Oncogene induced senescence via the tyrosine kinase receptor ERBB2(2010-01-12T15:29:56Z) Maccoux, Lindsey; Hengstler, Jan G.; Niemeyer, ChristophItem Untersuchung der subzellulären Lokalisation der Rezeptortyrosinkinase HER-3(2008-12-11T09:43:38Z) Schormann, Wiebke; Hengstler, J. G.; Niemeyer, C. M.Receptors tyrosine kinases play a pivotal role in tumour development and progression, and its altered expression has been observed in breast cancer, ovarian cancer and various other malignant tumour diseases. For example, approximately 20-30 % of breast cancer patients show an overexpression of the human epidermal growth factor receptor 2, HER-2. Therefore to counterpart this overexpression of HER-2, a therapeutic monoclonal humanized antibody (Herceptin®) directed against the extracellular domain of HER-2 was developed and is currently approved for clinical therapy. Receptors are normally localized to the cell membrane where they are activated by ligandbinding to trigger an intracellular signalling network. In the last decade several nuclear receptors were identified, but the function of many of these receptors remains unclear. To determine the subcellular distribution of the HER-2 and HER-3 an immunhistochemical analysis was conducted in ovarian and breast cancer tissues. In ovarian cancer HER-3 appear to have prognostic relevance in the patient’s survival. Patients with high HER-3 expression have a poorer prognosis than patients with low HER-3 expression. Additionally, breast cancer patients have a significant association between HER-2 expression and overall survival. Interestingly, 43.2 % of breast cancer patients showed a nuclear distribution of HER-3 with little or no HER-3 in cytoplasm. Reverse is also true where patients with cytoplasmic HER-3 had little or none localized to the nucleus. Thus, there appears to be "make-or-break" situation in the tumour samples but neither the mechanism nor the driving force behind the phenomenon is currently known. A further noteworthy observation is that cultured tumour cells have noticeably lower nuclear HER-3 expression (3-9%) compared to the patients’ specimens, providing a potential explanation as to why this observation has been neglected in the past. To investigate this phenomenon in more detail, cultured tumour cells were injected subcutaneously in nude mice and the nuclear HER-3 expression was analysed. Immunohistochemical analysis revealed high expression of HER-3 in the newly formed tumour tissue leading to the assumption that the nuclear HER-3 expression might be associated with the three dimensional feature of solid tumours. A main characteristic of solid tumours is the altered morphology which causes an undersupply of nutrients, oxygen and energy within the tumour. To determine whether challenging cells ( with nutrient depletion or drug treatment) altered HER-3 localization, different cell lines were depleted of ATP, glucose or treated with the drug cisplatin and the expression of HER-3 analysed. ATP depletion was carried out using Oligomycin B and 2 deoxyglucose, resulting in an accumulation of HER-3 in the nucleus to all cells examined. In contrast, the response to glucose depletion was cell line-dependent. Under glucose depletion the cervical cancer cell line HeLa showed an elevated nuclear HER-3 expression, whereas the breast cancer cell line MCF-7 did not show increased nuclear HER-3 localization. Dittmann et al. showed translocation of human epidermal growth factor (EGFR) into the nucleus after cisplatin exposure. Based on this observation, HER-3 localization was also analyzed after cisplatin exposure in MCF-7 cells. The result showed a clear nuclear translocation of HER-3. Although this effect was not comparable to the depletion of ATP and glucose, this finding provided further evidence that subcellular distribution of HER-3 is influenced by genotoxic stress. An additional factor which was found to influence the cellular distribution of HER-3 was confluency. A subconfluent growing cell culture shows higher amount of nuclear HER-3 compared to cell culture with a cell density of 100 %. Therefore an association between proliferation and the nuclear HER-3 expression may exist. Two main factors are linked to the signalling of HER-3: the dimerization partner of HER-3 (HER-2) and the ligand (HRGb1). Both factors were investigated to determine if they influence the subcellular distribution of HER-3. A Doxycycline-inducible HER-2 overexpressing cell line showed no increase in nuclear HER-3 expression with increased expression of HER-2. Furthermore, stimulation with HRGb1 did not have any effect on the nuclear HER-3 expression. Overall, the present results suggest that the translocation of HER-3 to the nucleus may be an important step in its signalling process. Depleting cells of ATP or glucose, or treating with a genotoxic chemical such as cisplatin, resulted in nuclear HER-3 expression. The current study is also the first to show that HER-3 nuclear location in tumour samples are more abundant compared to cell lines, tools commonly used to study receptor signalling. Although the functional role of nuclear HER-3 remains unclear, this work provides some key initial factors that can be used to further elucidate its function. Additionally, the significant increase in nuclear HER-3 expression in solid tumours may be in some way linked to resistance of such tumours to irradiation and chemotherapy.Item Luminescent semiconductor quantum dots(2008-06-11T08:41:48Z) Lu, Hua-Chang; Niemeyer, C. M.; Becker, C.Item Water compatible molecularly imprinted polymers for the recognition of biologically active compounds in aqueous media(2005-11-22T13:39:02Z) Manesiotis, Panagiotis; Sellergren, Börje; Niemeyer, M.