@article{HenzeHomannRohnetal.2016, author = {Henze, Andrea and Homann, Thomas and Rohn, Isabelle and Aschner, Michael A. and Link, Christopher D. and Kleuser, Burkhard and Schweigert, Florian J. and Schwerdtle, Tanja and Bornhorst, Julia}, title = {Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin}, series = {Scientific reports}, volume = {6}, journal = {Scientific reports}, publisher = {Nature Publishing Group}, address = {London}, issn = {2045-2322}, doi = {10.1038/srep37346}, pages = {12}, year = {2016}, abstract = {The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.}, language = {en} } @misc{HenzeHomannRohnetal.2016, author = {Henze, Andrea and Homann, Thomas and Rohn, Isabelle and Aschner, Michael A. and Link, Christopher D. and Kleuser, Burkhard and Schweigert, Florian J. and Schwerdtle, Tanja and Bornhorst, Julia}, title = {Caenorhabditis elegans as a model system to study post-translational modifications of human transthyretin}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-103674}, pages = {12}, year = {2016}, abstract = {The visceral protein transthyretin (TTR) is frequently affected by oxidative post-translational protein modifications (PTPMs) in various diseases. Thus, better insight into structure-function relationships due to oxidative PTPMs of TTR should contribute to the understanding of pathophysiologic mechanisms. While the in vivo analysis of TTR in mammalian models is complex, time- and resource-consuming, transgenic Caenorhabditis elegans expressing hTTR provide an optimal model for the in vivo identification and characterization of drug-mediated oxidative PTPMs of hTTR by means of matrix assisted laser desorption/ionization - time of flight - mass spectrometry (MALDI-TOF-MS). Herein, we demonstrated that hTTR is expressed in all developmental stages of Caenorhabditis elegans, enabling the analysis of hTTR metabolism during the whole life-cycle. The suitability of the applied model was verified by exposing worms to D-penicillamine and menadione. Both drugs induced substantial changes in the oxidative PTPM pattern of hTTR. Additionally, for the first time a covalent binding of both drugs with hTTR was identified and verified by molecular modelling.}, language = {en} } @article{BhuvaneshMachatschekLysyakovaetal.2019, author = {Bhuvanesh, Thanga and Machatschek, Rainhard Gabriel and Lysyakova, Liudmila and Kratz, Karl and Schulz, Burkhard and Ma, Nan and Lendlein, Andreas}, title = {Collagen type-IV Langmuir and Langmuir-Schafer layers as model biointerfaces to direct stem cell adhesion}, series = {Biomedical materials : materials for tissue engineering and regenerative medicine}, volume = {14}, journal = {Biomedical materials : materials for tissue engineering and regenerative medicine}, number = {2}, publisher = {Inst. of Physics Publ.}, address = {Bristol}, issn = {1748-6041}, doi = {10.1088/1748-605X/aaf464}, pages = {17}, year = {2019}, abstract = {In biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement Membranes (BM), a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network-like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air-water (A-W) interface. Transferring these layers onto cell culture substrates using the Langmuir-Schafer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A-W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mN m(-1). Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mN m(-1) onto poly(ethylene terephthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the areal Col-IV density by LS transfer at 10, 15 and 20 mN m(-1) on PET is shown. The LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.}, language = {en} } @phdthesis{Wettstein2015, author = {Wettstein, Christoph}, title = {Cytochrome c-DNA and cytochrome c-enzyme interactions for the construction of analytical signal chains}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-78367}, school = {Universit{\"a}t Potsdam}, pages = {120}, year = {2015}, abstract = {Electron transfer (ET) reactions play a crucial role in the metabolic pathways of all organisms. In biotechnological approaches, the redox properties of the protein cytochrome c (cyt c), which acts as an electron shuttle in the respiratory chain, was utilized to engineer ET chains on electrode surfaces. With the help of the biopolymer DNA, the redox protein assembles into electro active multilayer (ML) systems, providing a biocompatible matrix for the entrapment of proteins. In this study the characteristics of the cyt c and DNA interaction were defined on the molecular level for the first time and the binding sites of DNA on cyt c were identified. Persistent cyt c/DNA complexes were formed in solution under the assembly conditions of ML architectures, i.e. pH 5.0 and low ionic strength. At pH 7.0, no agglomerates were formed, permitting the characterization of the NMR spectroscopy. Using transverse relaxation-optimized spectroscopy (TROSY)-heteronuclear single quantum coherence (HSQC) experiments, DNAs' binding sites on the protein were identified. In particular, negatively charged AA residues, which are known interaction sites in cyt c/protein binding were identified as the main contact points of cyt c and DNA. Moreover, the sophisticated task of arranging proteins on electrode surfaces to create functional ET chains was addressed. Therefore, two different enzyme types, the flavin dependent fructose dehydrogenase (FDH) and the pyrroloquinoline quinone dependent glucose dehydrogenase (PQQ-GDH), were tested as reaction partners of freely diffusing cyt c and cyt c immobilized on electrodes in mono- and MLs. The characterisation of the ET processes was performed by means of electrochemistry and the protein deposition was monitored by microgravimetric measurements. FDH and PQQ-GDH were found to be generally suitable for combination with the cyt c/DNA ML system, since both enzymes interact with cyt c in solution and in the immobilized state. The immobilization of FDH and cyt c was achieved with the enzyme on top of a cyt c monolayer electrode without the help of a polyelectrolyte. Combining FDH with the cyt c/DNA ML system did not succeed, yet. However, the basic conditions for this protein-protein interaction were defined. PQQ-GDH was successfully coupled with the ML system, demonstrating that that the cyt c/DNA ML system provides a suitable interface for enzymes and that the creation of signal chains, based on the idea of co-immobilized proteins is feasible. Future work may be directed to the investigation of cyt c/DNA interaction under the precise conditions of ML assembly. Therefore, solid state NMR or X-ray crystallography may be required. Based on the results of this study, the combination of FDH with the ML system should be addressed. Moreover, alternative types of enzymes may be tested as catalytic component of the ML assembly, aiming on the development of innovative biosensor applications.}, language = {en} } @misc{WessigBaderKlieretal.2016, author = {Wessig, Pablo and Bader, Denise and Klier, Dennis Tobias and Hettrich, Cornelia and Bier, Frank Fabian}, title = {Detecting carbohydrate-lectin interactions using a fluorescent probe based on DBD dyes}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-394382}, pages = {1235 -- 1238}, year = {2016}, abstract = {Herein we present an efficient synthesis of a biomimetic probe with modular construction that can be specifically bound by the mannose binding FimH protein - a surface adhesion protein of E. coli bacteria. The synthesis combines the new and interesting DBD dye with the carbohydrate ligand mannose via a Click reaction. We demonstrate the binding to E. coli bacteria over a large concentration range and also present some special characteristics of those molecules that are of particular interest for the application as a biosensor. In particular, the mix-and-measure ability and the very good photo-stability should be highlighted here.}, language = {en} } @article{LauxErmilovaPannwitzetal.2018, author = {Laux, Eva-Maria and Ermilova, Elena and Pannwitz, Daniel and Gibbons, Jessica and H{\"o}lzel, Ralph and Bier, Frank Fabian}, title = {Dielectric Spectroscopy of Biomolecules up to 110 GHz}, series = {Frequenz}, volume = {72}, journal = {Frequenz}, number = {3-4}, publisher = {De Gruyter}, address = {Berlin}, issn = {0016-1136}, doi = {10.1515/freq-2018-0010}, pages = {135 -- 140}, year = {2018}, abstract = {Radio-frequency fields in the GHz range are increasingly applied in biotechnology and medicine. In order to fully exploit both their potential and their risks detailed information about the dielectric properties of biological material is needed. For this purpose a measuring system is presented that allows the acquisition of complex dielectric spectra over 4 frequency decade up to 110 GHz. Routines for calibration and for data evaluation according to physicochemical interaction models have been developed. The frequency dependent permittivity and dielectric loss of some proteins and nucleic acids, the main classes of biomolecules, and of their sub-units have been determined. Dielectric spectra are presented for the amino acid alanine, the proteins lysozyme and haemoglobin, the nucleotides AMP and ATP, and for the plasmid pET-21, which has been produced by bacterial culture. Characterisation of a variety of biomolecules is envisaged, as is the application to studies on protein structure and function.}, language = {en} } @misc{KuekenSommerYanevaRoderetal.2018, author = {K{\"u}ken, Anika and Sommer, Frederik and Yaneva-Roder, Liliya and Mackinder, Luke C.M. and H{\"o}hne, Melanie and Geimer, Stefan and Jonikas, Martin C. and Schroda, Michael and Stitt, Mark and Nikoloski, Zoran and Mettler-Altmann, Tabea}, title = {Effects of microcompartmentation on flux distribution and metabolic pools in Chlamydomonas reinhardtii chloroplasts}, series = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam : Mathematisch-Naturwissenschaftliche Reihe}, number = {1122}, issn = {1866-8372}, doi = {10.25932/publishup-44635}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-446358}, pages = {25}, year = {2018}, abstract = {Cells and organelles are not homogeneous but include microcompartments that alter the spatiotemporal characteristics of cellular processes. The effects of microcompartmentation on metabolic pathways are however difficult to study experimentally. The pyrenoid is a microcompartment that is essential for a carbon concentrating mechanism (CCM) that improves the photosynthetic performance of eukaryotic algae. Using Chlamydomonas reinhardtii, we obtained experimental data on photosynthesis, metabolites, and proteins in CCM-induced and CCM-suppressed cells. We then employed a computational strategy to estimate how fluxes through the Calvin-Benson cycle are compartmented between the pyrenoid and the stroma. Our model predicts that ribulose-1,5-bisphosphate (RuBP), the substrate of Rubisco, and 3-phosphoglycerate (3PGA), its product, diffuse in and out of the pyrenoid, respectively, with higher fluxes in CCM-induced cells. It also indicates that there is no major diffusional barrier to metabolic flux between the pyrenoid and stroma. Our computational approach represents a stepping stone to understanding microcompartmentalized CCM in other organisms.}, language = {en} } @phdthesis{Buller2013, author = {Buller, Jens}, title = {Entwicklung neuer stimuli-sensitiver Hydrogelfilme als Plattform f{\"u}r die Biosensorik}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-66261}, school = {Universit{\"a}t Potsdam}, year = {2013}, abstract = {Diese Arbeit befasst sich mit der Synthese und der Charakterisierung von thermoresponsiven Polymeren und ihrer Immobilisierung auf festen Oberfl{\"a}chen als nanoskalige d{\"u}nne Schichten. Dabei wurden thermoresponsive Polymere vom Typ der unteren kritischen Entmischungstemperatur (engl.: lower critical solution temperature, LCST) verwendet. Sie sind bei niedrigeren Temperaturen im L{\"o}sungsmittel gut und nach Erw{\"a}rmen oberhalb einer bestimmten kritischen Temperatur nicht mehr l{\"o}slich; d. h. sie weisen bei einer bestimmten Temperatur einen Phasen{\"u}bergang auf. Als Basismaterial wurden verschiedene thermoresponsive und biokompatible Polymere basierend auf Diethylenglykolmethylethermethacrylat (MEO2MA) und Oligo(ethylenglykol)methylethermethacrylat (OEGMA475, Mn = 475 g/ mol) {\"u}ber frei radikalische Copolymerisation synthetisiert. Der thermoresponsive Phasen{\"u}bergang der Copolymere wurde in w{\"a}ssriger L{\"o}sung und in gequollenen vernetzten d{\"u}nnen Schichten beobachtet. Außerdem wurde untersucht, inwiefern eine selektive Proteinbindung an geeignete funktionalisierte Copolymere die Phasen{\"u}bergangstemperatur beeinflusst. Die thermoresponsiven Copolymere wurden {\"u}ber photovernetzbare Gruppen auf festen Oberfl{\"a}chen immobilisiert. Die n{\"o}tigen lichtempfindlichen Vernetzereinheiten wurden mittels des polymerisierbaren Benzophenonderivates 2 (4 Benzoylphenoxy)ethylmethacrylat (BPEM) in das Copolymer integriert. D{\"u}nne Filme der Copolymere mit ca. 100 nm Schichtdicke wurden {\"u}ber Rotationsbeschichtung auf Siliziumwafer aufgeschleudert und anschließend durch Bestrahlung mit UV Licht vernetzt und auf der Oberfl{\"a}che immobilisiert. Die Filme sind stabiler je gr{\"o}ßer der Vernetzeranteil und je gr{\"o}ßer die Molmasse der Copolymere ist. Bei einem Waschprozess nach der Vernetzung wird beispielsweise aus einem Film mit moderater Molmasse und geringem Vernetzeranteil mehr unvernetztes Copolymer ausgewaschen als bei einem h{\"o}hermolekularen Copolymer mit hohem Vernetzeranteil. Die Quellbarkeit der Polymerschichten wurde mit Ellipsometrie untersucht. Sie ist gr{\"o}ßer je geringer der Vernetzeranteil in den Copolymeren ist. Schichten aus thermoresponsiven OEG Copolymeren zeigen einen Volumenphasen{\"u}bergang vom Typ der LCST. Der thermoresponsive Kollaps der Schichten ist komplett reversibel, die Kollapstemperatur kann {\"u}ber die Zusammensetzung der Copolymere eingestellt werden. F{\"u}r einen Vergleich dieser Eigenschaften mit dem gut charakterisierten und derzeit wohl am h{\"a}ufigsten untersuchten thermoresponsiven Polymer Poly(N-isopropylacrylamid) (PNIPAM) wurden zus{\"a}tzlich photovernetzte Schichten aus PNIPAM hergestellt und ebenfalls ellipsometrisch vermessen. Im Vergleich zu PNIPAM verl{\"a}uft der Phasen{\"u}bergang der Schichten aus den Copolymeren mit Oligo(ethylenglykol)-seitenketten (OEG Copolymere) {\"u}ber einen gr{\"o}ßeren Temperaturbereich. Mit Licht einer Wellenl{\"a}nge > 300 nm wurden die photosensitiven Benzophenongruppen selektiv angeregt. Bei der Verwendung kleinerer Wellenl{\"a}ngen vernetzten die Copolymerschichten auch ohne die Anwesenheit der lichtempfindlichen Benzophenongruppen. Dieser Effekt ließ sich zur kontrollierten Immobilisierung und Vernetzung der OEG Copolymere einsetzen. Als weitere Methode zur Immobilisierung der Copolymere wurde die Anbindung {\"u}ber Amidbindungen untersucht. Dazu wurden OEG Copolymere mit dem carboxylgruppenhaltigen 2 Succinyloxyethylmethacrylat (MES) auf mit 3 Aminopropyldimethylethoxysilan (APDMSi) silanisierte Siliziumwafer rotationsbeschichtet, und mit dem oligomeren α, ω Diamin Jeffamin® ED 900 vernetzt. Die Vernetzungsreaktion erfolgte ohne weitere Zus{\"a}tze durch Erhitzen der Proben. Die Hydrogelschichten waren anschließend stabil und zeigten neben thermoresponsivem auch pH responsives Verhalten. Um zu untersuchen, ob die Phasen{\"u}bergangstemperatur durch eine Proteinbindung beeinflusst werden kann, wurde ein polymerisierbares Biotinderivat 2 Biotinyl-aminoethylmethacrylat (BAEMA) in das thermoresponsive Copolymer eingebaut. Der Einfluss des biotinbindenen Proteins Avidin auf das thermoresponsive Verhalten des Copolymers in L{\"o}sung wurde untersucht. Die spezifische Bindung von Avidin an das biotinylierte Copolymer verschob die {\"U}bergangstemperatur deutlich zu h{\"o}heren Temperaturen. Kontrollversuche zeigten, dass dieses Verhalten auf eine selektive Proteinbindung zur{\"u}ckzuf{\"u}hren ist. Thermoresponsive OEG Copolymere mit photovernetzbaren Gruppen aus BPEM und Biotingruppen aus BAEMA wurden {\"u}ber Rotationsbeschichtung auf Gold- und auf Siliziumoberfl{\"a}chen aufgetragen und durch UV Strahlung vernetzt. Die spezifische Bindung von Avidin an die Copolymerschicht wurde mit Oberfl{\"a}chenplasmonenresonanz und Ellipsometrie untersucht. Die Bindungskapazit{\"a}t der Schichten war umso gr{\"o}ßer, je kleiner der Vernetzeranteil, d. h. je gr{\"o}ßer die Maschenweite des Netzwerkes war. Die Quellbarkeit der Schichten wurde durch die Avidinbindung erh{\"o}ht. Bei hochgequollenen Systemen verursachte eine Mehrfachbindung des tetravalenten Avidins allerdings eine zus{\"a}tzliche Quervernetzung des Polymernetzwerkes. Dieser Effekt wirkt der erh{\"o}hten Quellbarkeit durch die Avidinbindung entgegen und l{\"a}sst die Polymernetzwerke schrumpfen.}, language = {de} } @misc{LaemkeBaeurle2017, author = {L{\"a}mke, J{\"o}rn and B{\"a}urle, Isabel}, title = {Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants}, series = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, journal = {Postprints der Universit{\"a}t Potsdam Mathematisch-Naturwissenschaftliche Reihe}, number = {792}, issn = {1866-8372}, doi = {10.25932/publishup-43623}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus4-436236}, pages = {11}, year = {2017}, abstract = {Plants frequently have to weather both biotic and abiotic stressors, and have evolved sophisticated adaptation and defense mechanisms. In recent years, chromatin modifications, nucleosome positioning, and DNA methylation have been recognized as important components in these adaptations. Given their potential epigenetic nature, such modifications may provide a mechanistic basis for a stress memory, enabling plants to respond more efficiently to recurring stress or even to prepare their offspring for potential future assaults. In this review, we discuss both the involvement of chromatin in stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.}, language = {en} } @article{LaemkeBaeurle2017, author = {L{\"a}mke, J{\"o}rn and B{\"a}urle, Isabel}, title = {Epigenetic and chromatin-based mechanisms in environmental stress adaptation and stress memory in plants}, series = {Genome biology : biology for the post-genomic era}, volume = {18}, journal = {Genome biology : biology for the post-genomic era}, publisher = {BioMed Central}, address = {London}, issn = {1474-760X}, doi = {10.1186/s13059-017-1263-6}, pages = {8685 -- 8693}, year = {2017}, abstract = {Plants frequently have to weather both biotic and abiotic stressors, and have evolved sophisticated adaptation and defense mechanisms. In recent years, chromatin modifications, nucleosome positioning, and DNA methylation have been recognized as important components in these adaptations. Given their potential epigenetic nature, such modifications may provide a mechanistic basis for a stress memory, enabling plants to respond more efficiently to recurring stress or even to prepare their offspring for potential future assaults. In this review, we discuss both the involvement of chromatin in stress responses and the current evidence on somatic, intergenerational, and transgenerational stress memory.}, language = {en} }