04 Fakultät Energie-, Verfahrens- und Biotechnik
Permanent URI for this collectionhttps://elib.uni-stuttgart.de/handle/11682/5
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Item Open Access Precision 3D‐printed cell scaffolds mimicking native tissue composition and mechanics(2020) Erben, Amelie; Hörning, Marcel; Hartmann, Bastian; Becke, Tanja; Eisler, Stephan A.; Southan, Alexander; Cranz, Séverine; Hayden, Oliver; Kneidinger, Nikolaus; Königshoff, Melanie; Lindner, Michael; Tovar, Günter E. M.; Burgstaller, Gerald; Clausen‐Schaumann, Hauke; Sudhop, Stefanie; Heymann, MichaelCellular dynamics are modeled by the 3D architecture and mechanics of the extracellular matrix (ECM) and vice versa. These bidirectional cell‐ECM interactions are the basis for all vital tissues, many of which have been investigated in 2D environments over the last decades. Experimental approaches to mimic in vivo cell niches in 3D with the highest biological conformity and resolution can enable new insights into these cell‐ECM interactions including proliferation, differentiation, migration, and invasion assays. Here, two‐photon stereolithography is adopted to print up to mm‐sized high‐precision 3D cell scaffolds at micrometer resolution with defined mechanical properties from protein‐based resins, such as bovine serum albumin or gelatin methacryloyl. By modifying the manufacturing process including two‐pass printing or post‐print crosslinking, high precision scaffolds with varying Young's moduli ranging from 7‐300 kPa are printed and quantified through atomic force microscopy. The impact of varying scaffold topographies on the dynamics of colonizing cells is observed using mouse myoblast cells and a 3D‐lung microtissue replica colonized with primary human lung fibroblast. This approach will allow for a systematic investigation of single‐cell and tissue dynamics in response to defined mechanical and bio‐molecular cues and is ultimately scalable to full organs.Item Open Access Insights into the structural and functional properties of the eukaryotic porin Tom40(2012) Gessmann, Dennis; Nußberger, Stephan (Prof. Dr.)Tom40 forms the preprotein conducting channel in the outer membrane of mitochondria enabling transport of up to 1500 different preproteins through an optimized pore environment. Moreover, Tom40 exhibits a voltage-dependent gating mechanism in terms of an ‘electrical switch’ making this eukaryotic beta-barrel a promising target for nanopore based applications. In this work, new bioinformatics methods were developed and verified through practical approaches to shed light on the structural elements of Tom40 facilitating its particular function in mitochondria. Based on these results, Tom40 proteins were designed with modified and optimized structural properties. TmSIP, a physical interaction model developed for TM beta-barrel proteins, was used to identify weakly stable regions in the TM domain of Tom40 from mammals and fungi. Three unfavorable beta-strands were determined for human Tom40A. Via CD and Trp-fluorescence spectroscopy it was shown that substitution of key amino acid residues in theses strands resulted in an improved resistance of the protein to chemical and thermal perturbations. Further, the mutated form of hTom40A was strictly found in its monomeric state. Equal improvements were gained for the apparent stability of Tom40 from Aspergillus fumigatus. Tom40 was isolated and purified in its native state from Neurospora crassa mitochondria. Time-limited proteolysis of native NcTom40 coupled to mass spectrometry revealed comparable protease-accessibility to VDAC isoform 1 from mammals suggesting a similar fold. Thus, a homology model of NcTom40 was developed on the basis of the solved mouse VDAC-1 crystal structure. It was found that Tom40 forms a 19-stranded beta-barrel with an N-terminal alpha-helix inside the pore. Further, a conserved ‘polar slide’ in the pore interior is possibly involved in preprotein translocation and a second conserved domain, termed ‘helix anchor region’, in arresting the helix inside the Tom40 pore. Based on the homology model of NcTom40, the structure and function of the N-terminal domain of Tom40 was addressed. Examination of the model structure revealed two different domains for the N-terminus, the inner-barrel and outer-barrel N-terminus. In vivo investigations showed that both parts prevent a heat-induced dysfunction of Tom40 in N. crassa mitochondria independently. By applying CD spectroscopy the predicted N-terminal alpha-helix could be allocated to the inner-barrel N-terminus. Further, in combination with Trp-fluorescence spectroscopy it was found that the N-terminal alpha-helix unfolds independently from the Tom40 beta-barrel, but is not necessary for pore stability or integrity. However, a conserved amino acid residue, Ile47 of NcTom40, in the inner-barrel N-terminus is essential for the structural integrity of the N-terminal alpha-helix. In conclusion, these results may offer a basis for future works on TM beta-barrel proteins with the aim to alter the structural properties in the absence of a high atomic resolution structure or an established knowledge of the biochemical and biophysical properties.Item Open Access Anti-adhesive surfaces inspired by bee mandible surfaces(2023) Saccardi, Leonie; Schiebl, Jonas; Balluff, Franz; Christ, Ulrich; Gorb, Stanislav N.; Kovalev, Alexander; Schwarz, OliverPropolis, a naturally sticky substance used by bees to secure their hives and protect the colony from pathogens, presents a fascinating challenge. Despite its adhesive nature, honeybees adeptly handle propolis with their mandibles. Previous research has shown a combination of an anti-adhesive fluid layer and scale-like microstructures on the inner surface of bee mandibles. Our aim was to deepen our understanding of how surface energy and microstructure influence the reduction in adhesion for challenging substances like propolis. To achieve this, we devised surfaces inspired by the intricate microstructure of bee mandibles, employing diverse techniques including roughening steel surfaces, creating lacquer structures using Bénard cells, and moulding resin surfaces with hexagonal patterns. These approaches generated patterns that mimicked the bee mandible structure to varying degrees. Subsequently, we assessed the adhesion of propolis on these bioinspired structured substrates. Our findings revealed that on rough steel and resin surfaces structured with hexagonal dimples, propolis adhesion was significantly reduced by over 40% compared to unstructured control surfaces. However, in the case of the lacquer surface patterned with Bénard cells, we did not observe a significant reduction in adhesion.Item Open Access Light-addressable actuator-sensor platform for monitoring and manipulation of pH gradients in microfluidics : a case study with the enzyme penicillinase(2021) Welden, Rene; Jablonski, Melanie; Wege, Christina; Keusgen, Michael; Wagner, Patrick Hermann; Wagner, Torsten; Schöning, Michael J.The feasibility of light-addressed detection and manipulation of pH gradients inside an electrochemical microfluidic cell was studied. Local pH changes, induced by a light-addressable electrode (LAE), were detected using a light-addressable potentiometric sensor (LAPS) with different measurement modes representing an actuator-sensor system. Biosensor functionality was examined depending on locally induced pH gradients with the help of the model enzyme penicillinase, which had been immobilized in the microfluidic channel. The surface morphology of the LAE and enzyme-functionalized LAPS was studied by scanning electron microscopy. Furthermore, the penicillin sensitivity of the LAPS inside the microfluidic channel was determined with regard to the analyte’s pH influence on the enzymatic reaction rate. In a final experiment, the LAE-controlled pH inhibition of the enzyme activity was monitored by the LAPS.Item Open Access Flux calculation for primary metabolism reveals changes in allocation of nitrogen to different amino acid families when photorespiratory activity changes(2024) Friedrichs, Nils; Shokouhi, Danial; Heyer, Arnd G.Photorespiration, caused by oxygenation of the enzyme Rubisco, is considered a wasteful process, because it reduces photosynthetic carbon gain, but it also supplies amino acids and is involved in amelioration of stress. Here, we show that a sudden increase in photorespiratory activity not only reduced carbon acquisition and production of sugars and starch, but also affected diurnal dynamics of amino acids not obviously involved in the process. Flux calculations based on diurnal metabolite profiles suggest that export of proline from leaves increases, while aspartate family members accumulate. An immense increase is observed for turnover in the cyclic reaction of glutamine synthetase/glutamine-oxoglutarate aminotransferase (GS/GOGAT), probably because of increased production of ammonium in photorespiration. The hpr1-1 mutant, defective in peroxisomal hydroxypyruvate reductase, shows substantial alterations in flux, leading to a shift from the oxoglutarate to the aspartate family of amino acids. This is coupled to a massive export of asparagine, which may serve in exchange for serine between shoot and root.Item Open Access A systems biology approach to analyse leaf carbohydrate metabolism in Arabidopsis thaliana(2011) Henkel, Sebastian; Nägele, Thomas; Hörmiller, Imke; Sauter, Thomas; Sawodny, Oliver; Ederer, Michael; Heyer, Arnd G.Plant carbohydrate metabolism comprises numerous metabolite interconversions, some of which form cycles of metabolite degradation and re-synthesis and are thus referred to as futile cycles. In this study, we present a systems biology approach to analyse any possible regulatory principle that operates such futile cycles based on experimental data for sucrose (Scr) cycling in photosynthetically active leaves of the model plant Arabidopsis thaliana. Kinetic parameters of enzymatic steps in Scr cycling were identified by fitting model simulations to experimental data. A statistical analysis of the kinetic parameters and calculated flux rates allowed for estimation of the variability and supported the predictability of the model. A principal component analysis of the parameter results revealed the identifiability of the model parameters. We investigated the stability properties of Scr cycling and found that feedback inhibition of enzymes catalysing metabolite interconversions at different steps of the cycle have differential influence on stability. Applying this observation to futile cycling of Scr in leaf cells points to the enzyme hexokinase as an important regulator, while the step of Scr degradation by invertases appears subordinate.Item Open Access Field-effect sensors for virus detection : from Ebola to SARS-CoV-2 and plant viral enhancers(2020) Poghossian, Arshak; Jablonski, Melanie; Molinnus, Denise; Wege, Christina; Schöning, Michael J.Coronavirus disease 2019 (COVID-19) is a novel human infectious disease provoked by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Currently, no specific vaccines or drugs against COVID-19 are available. Therefore, early diagnosis and treatment are essential in order to slow the virus spread and to contain the disease outbreak. Hence, new diagnostic tests and devices for virus detection in clinical samples that are faster, more accurate and reliable, easier and cost-efficient than existing ones are needed. Due to the small sizes, fast response time, label-free operation without the need for expensive and time-consuming labeling steps, the possibility of real-time and multiplexed measurements, robustness and portability (point-of-care and on-site testing), biosensors based on semiconductor field-effect devices (FEDs) are one of the most attractive platforms for an electrical detection of charged biomolecules and bioparticles by their intrinsic charge. In this review, recent advances and key developments in the field of label-free detection of viruses (including plant viruses) with various types of FEDs are presented. In recent years, however, certain plant viruses have also attracted additional interest for biosensor layouts: Their repetitive protein subunits arranged at nanometric spacing can be employed for coupling functional molecules. If used as adapters on sensor chip surfaces, they allow an efficient immobilization of analyte-specific recognition and detector elements such as antibodies and enzymes at highest surface densities. The display on plant viral bionanoparticles may also lead to long-time stabilization of sensor molecules upon repeated uses and has the potential to increase sensor performance substantially, compared to conventional layouts. This has been demonstrated in different proof-of-concept biosensor devices. Therefore, richly available plant viral particles, non-pathogenic for animals or humans, might gain novel importance if applied in receptor layers of FEDs. These perspectives are explained and discussed with regard to future detection strategies for COVID-19 and related viral diseases.Item Open Access Regulation of mitochondrial dynamics in Parkinson’s disease : is 2-methoxyestradiol a missing piece?(2021) Bastian, Paulina; Dulski, Jaroslaw; Roszmann, Anna; Jacewicz, Dagmara; Kuban-Jankowska, Alicja; Slawek, Jaroslaw; Wozniak, Michal; Gorska-Ponikowska, MagdalenaMitochondria, as “power house of the cell”, are crucial players in cell pathophysiology. Beyond adenosine triphosphate (ATP) production, they take part in a generation of reactive oxygen species (ROS), regulation of cell signaling and cell death. Dysregulation of mitochondrial dynamics may lead to cancers and neurodegeneration; however, the fusion/fission cycle allows mitochondria to adapt to metabolic needs of the cell. There are multiple data suggesting that disturbed mitochondrial homeostasis can lead to Parkinson’s disease (PD) development. 2-methoxyestradiol (2-ME), metabolite of 17β-estradiol (E2) and potential anticancer agent, was demonstrated to inhibit cell growth of hippocampal HT22 cells by means of nitric oxide synthase (NOS) production and oxidative stress at both pharmacologically and also physiologically relevant concentrations. Moreover, 2-ME was suggested to inhibit mitochondrial biogenesis and to be a dynamic regulator. This review is a comprehensive discussion, from both scientific and clinical point of view, about the influence of 2-ME on mitochondria and its plausible role as a modulator of neuron survival.Item Open Access Neurobiologische Untersuchungen zur Rolle von Dopamin bei risikoabhängigem Entscheidungsverhalten(2019) Mai, Bettina; Hauber, Wolfgang (apl. Prof. Dr.)Um das Überleben in einer sich ständig ändernden Umwelt zu sichern, müssen Menschen und Tiere tagtäglich eine Vielzahl von Kosten-Nutzen-abhängigen Entscheidungen fällen. Das Ziel dieses Entscheidungsprozesses ist es, eine situationsgerecht optimale Entscheidung zu treffen, bei der mit möglichst geringen Kosten der Nutzen maximiert wird. Der Präfrontalcortex (PFC) und der Nucleus Accumbens (Acb) sind die Schlüsselstrukturen eines neuralen Netzwerkes, welches eine wichtige Kategorie von Kosten-Nutzen-abhängigen Entscheidungen steuert, nämlich sogenannte risikoabhängige Entscheidungen. Beide Strukturen empfangen Dopamin-Signale aus dem Mittelhirn, die Informationen über Kosten und Nutzen kodieren. Das Ziel meiner Arbeit bestand darin, die noch unbekannte kausale Bedeutung von Dopamin-Signalen im PFC und im Acb bei risikoabhängigem Entscheidungsverhalten zu verstehen. Im Rahmen meiner Arbeit wurde das risikoabhängige Entscheidungsverhalten von Ratten beim Durchführen von Risikoaufgaben in Skinner-Boxen untersucht. Dabei hatte das Tier die Wahl zwischen zwei Hebeln. Bei dem „sicheren Hebel“ führte ein Hebeldruck sicher zu einer geringen Futterbelohnung. Bei dem „riskanten Hebel“ führte ein Hebeldruck entweder zu einer hohen oder gar keiner Futterbelohnung. Die Belohnungswahrscheinlichkeit, also die Wahrscheinlichkeit beim riskanten Hebel eine hohe Futterbelohnung zu erhalten, nahm entweder zwischen verschiedenen Sitzungen (konstante Risikoaufgabe) oder innerhalb einer Sitzung (absteigende Risikoaufgabe) ab. Aus meinen Messungen mit der konstanten Risikoaufgabe geht hervor, dass die permanente Inaktivierung der Dopamin-Innervation der orbitalen Subareale des PFC (OFC) sowie der Core- und Shell-Subareale des Acb (AcbC und AcbS) das risikoabhängige Entscheidungsverhalten nicht beeinträchtigt. Diese Befunde ließen sich in nachfolgenden Untersuchungen mit der absteigenden Risikoaufgabe erhärten: Tiere mit einer Blockade der Dopamin D1- und D2-Rezeptoraktivität des OFC beziehungsweise des Acb waren unverändert sensitiv gegenüber der abnehmenden Belohnungswahrscheinlichkeit. Allerdings war unter Blockade der D1- und D2-Rezeptoraktivität im AcbC die generelle Präferenz für die riskante Handlungsoption signifikant verringert. Dopamin-Signale im PFC und im Acb scheinen also nicht an der Erfassung des Risikos und dessen Veränderung beteiligt zu sein. Meine Daten sprechen vielmehr dafür, dass die Dopamin-Rezeptoraktivität im AcbC die generelle Präferenz für riskante Handlungsoptionen reguliert. Die dem risikoabhängigem Entscheidungsverhalten zugrunde liegende Neurobiologie ist komplex und die Datenlage teilweise uneinheitlich. Ein wesentlicher Grund dafür sind die in der Literatur verwendeten verschiedenartigen Risikoaufgaben, die auf unterschiedlichen Risikokonzepten basieren. Aus diesem Grund unterscheiden sich sowohl die erforderlichen kognitiven Leistungen als auch die beteiligten neuralen Schaltkreise in den verschiedenen Risikoaufgaben und die Effekte von dopaminergen Manipulationen sind teilweise inkonsistent. Meine Befunde sprechen dafür, dass Dopamin-Signale im AcbC die Präferenz von Tieren für Handlungsoptionen steigern, die trotz eines hohen Risikos mit hohem Nutzen verbunden sind. Bei den hier untersuchten risikoabhängigen Entscheidungen unterstützen die Dopamin-Signale im AcbC demnach nicht die Risikoerfassung an sich, sondern sie verstärken generell die Präferenz für kostenintensive Handlungsoptionen, die mit einer höheren, riskanten Belohnung verknüpft sind. Möglicherweise führt eine solche Dopamin-vermittelte Regulation von Handlungspräferenzen zu einer höheren biologischen Fitness und bildet damit einen Selektionsvorteil. Die Daten stehen im Einklang mit klinischen Befunden, wonach Suchtmittel und Medikamente, welche die Dopamin-Transmission erhöhen, riskante Entscheidungen des Menschen fördern. Meine Daten zeigen, dass der AcbC eine der Strukturen zu sein scheint, welche diese Wirkungen vermittelt.Item Open Access Local membrane curvature pins and guides excitable membrane waves in chemotactic and macropinocytic cells : biomedical insights from an innovative simple model(2021) Hörning, Marcel; Bullmann, Torsten; Shibata, TatsuoPIP3 dynamics observed in membranes are responsible for the protruding edge formation in cancer and amoeboid cells. The mechanisms that maintain those PIP3 domains in three-dimensional space remain elusive, due to limitations in observation and analysis techniques. Recently, a strong relation between the cell geometry, the spatial confinement of the membrane, and the excitable signal transduction system has been revealed by Hörning and Shibata (2019) using a novel 3D spatiotemporal analysis methodology that enables the study of membrane signaling on the entire membrane (Hörning and Shibata, 2019). Here, using 3D spatial fluctuation and phase map analysis on actin polymerization inhibited Dictyostelium cells, we reveal a spatial asymmetry of PIP3 signaling on the membrane that is mediated by the contact perimeter of the plasma membrane - the spatial boundary around the cell-substrate adhered area on the plasma membrane. We show that the contact perimeter guides PIP3 waves and acts as a pinning site of PIP3 phase singularities, that is, the center point of spiral waves. The contact perimeter serves as a diffusion influencing boundary that is regulated by a cell size- and shape-dependent curvature. Our findings suggest an underlying mechanism that explains how local curvature can favor actin polymerization when PIP3 domains get pinned at the curved protrusive membrane edges in amoeboid cells.