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Thermoresponsive Zellkultursubstrate für zeitlich-räumlich gesteuertes Auswachsen neuronaler Zellen
(2019)
Ein wichtiges Ziel der Neurowissenschaften ist das Verständnis der komplexen und zugleich faszinierenden, hochgeordneten Vernetzung der Neurone im Gehirn, welche neuronalen Prozessen, wie zum Beispiel dem Wahrnehmen oder Lernen wie auch Neuropathologien zu Grunde liegt. Für verbesserte neuronale Zellkulturmodelle zur detaillierten Untersuchung dieser Prozesse ist daher die Rekonstruktion von geordneten neuronalen Verbindungen dringend erforderlich. Mit Oberflächenstrukturen aus zellattraktiven und zellabweisenden Beschichtungen können neuronale Zellen und ihre Neuriten in vitro strukturiert werden. Zur Kontrolle der neuronalen Verbindungsrichtung muss das Auswachsen der Axone zu benachbarten Zellen dynamisch gesteuert werden, zum Beispiel über eine veränderliche Zugänglichkeit der Oberfläche.
In dieser Arbeit wurde untersucht, ob mit thermoresponsiven Polymeren (TRP) beschichtete Zellkultursubstrate für eine dynamische Kontrolle des Auswachsens neuronaler Zellen geeignet sind. TRP können über die Temperatur von einem zellabweisenden in einen zellattraktiven Zustand geschaltet werden, womit die Zugänglichkeit der Oberfläche für Zellen dynamisch gesteuert werden kann. Die TRP-Beschichtung wurde mikrostrukturiert, um einzelne oder wenige neuronale Zellen zunächst auf der Oberfläche anzuordnen und das Auswachsen der Zellen und Neuriten über definierte TRP-Bereiche in Abhängigkeit der Temperatur zeitlich und räumlich zu kontrollieren. Das Protokoll wurde mit der neuronalen Zelllinie SH-SY5Y etabliert und auf humane induzierte Neurone übertragen. Die Anordnung der Zellen konnte bei Kultivierung im zellabweisenden Zustand des TRPs für bis zu 7 Tage aufrecht erhalten werden. Durch Schalten des TRPs in den zellattraktiven Zustand konnte das Auswachsen der Neuriten und Zellen zeitlich und räumlich induziert werden. Immunozytochemische Färbungen und Patch-Clamp-Ableitungen der Neurone demonstrierten die einfache Anwendbarkeit und Zellkompatibilität der TRP-Substrate.
Eine präzisere räumliche Kontrolle des Auswachsens der Zellen sollte durch lokales Schalten der TRP-Beschichtung erreicht werden. Dafür wurden Mikroheizchips mit Mikroelektroden zur lokalen Jouleschen Erwärmung der Substratoberfläche entwickelt. Zur Evaluierung der generierten Temperaturprofile wurde eine Temperaturmessmethode entwickelt und die erhobenen Messwerte mit numerisch simulierten Werten abgeglichen. Die Temperaturmessmethode basiert auf einfach zu applizierenden Sol-Gel-Schichten, die den temperatursensitiven Fluoreszenzfarbstoff Rhodamin B enthalten. Sie ermöglicht oberflächennahe Temperaturmessungen in trockener und wässriger Umgebung mit hoher Orts- und Temperaturauflösung. Numerische Simulationen der Temperaturprofile korrelierten gut mit den experimentellen Daten. Auf dieser Basis konnten Geometrie und Material der Mikroelektroden hinsichtlich einer lokal stark begrenzten Temperierung optimiert werden. Ferner wurden für die Kultvierung der Zellen auf den Mikroheizchips eine Zellkulturkammer und Kontaktboard für die elektrische Kontaktierung der Mikroelektroden geschaffen.
Die vorgestellten Ergebnisse demonstrieren erstmalig das enorme Potential thermoresponsiver Zellkultursubstrate für die zeitlich und räumlich gesteuerte Formation geordneter neuronaler Verbindungen in vitro. Zukünftig könnte dies detaillierte Studien zur neuronalen Informationsverarbeitung oder zu Neuropathologien an relevanten, humanen Zellmodellen ermöglichen.
Light-switchable proteins are being used increasingly to understand and manipulate complex molecular systems. The success of this approach has fueled the development of tailored photo-switchable proteins, to enable targeted molecular events to be studied using light. The development of novel photo-switchable tools has to date largely relied on rational design. Complementing this approach with directed evolution would be expected to facilitate these efforts. Directed evolution, however, has been relatively infrequently used to develop photo-switchable proteins due to the challenge presented by high-throughput evaluation of switchable protein activity. This thesis describes the development of two genetic circuits that can be used to evaluate libraries of switchable proteins, enabling optimization of both the on- and off-states. A screening system is described, which permits detection of DNA-binding activity based on conditional expression of a fluorescent protein. In addition, a tunable selection system is presented, which allows for the targeted selection of protein-protein interactions of a desired affinity range. This thesis additionally describes the development and characterization of a synthetic protein that was designed to investigate chromophore reconstitution in photoactive yellow protein (PYP), a promising scaffold for engineering photo-controlled protein tools.
The African weakly electric fish genus Campylomormyrus is a well-investigated fish group of the species-rich family Mormyridae. They are able to generate species-specific electric organ discharges (EODs) which vary in their waveform characteristics including polarity, phase umber and duration. In mormyrid species EODs are used for communication, species discrimination and mate recognition, and it is thought hat they serve as pre-zygotic isolation mechanism driving sympatric speciation by promoting assortative mating. The EOD diversification, its volutionary effects and the link to species divergence have been examined histologically, behaviorally, and genetically. Molecular analyses are a major tool to identify species and their phenotypic traits by studying the underlying genes. The genetic variability between species further provides information from which evolutionary processes, such as speciation, can be deduced. Hence, the ultimate aim of this study is the investigation of genetic variability within the African weakly electric fish genus Campylomormyrus to better understand their sympatric speciation and comprehend their evolutionary drivers. In order to extend the current knowledge and gain more insights into its species history, karyological and genomic approaches are being pursued considering species differences. Previous studies have shown that species with different EOD duration have specific gene expression patterns and single nucleotide polymorphisms (SNPs). As EODs play a crucial role during the evolution of Campylomormyrus species, the identification of its underlying genes may suggest how the EOD diversity evolved and whether this trait is based on a complex network of genetic processes or is regulated by only a few genes. The results obtained in this study suggest that genes with non-synonymous SNPs, which are exclusive to C. tshokwe with an elongated EOD, have frequent functions ssociated with tissue morphogenesis and transcriptional regulation. Therefore, it is proposed that these processes likely co-determine EOD characteristics of Campylomormyrus species. Furthermore, genome-wide analyses confirm the genetic difference among most Campylomormyrus species. In contrast, the same analyses reveal genetic similarity among individuals of the alces-complex showing different EOD waveforms. It is therefore hypothesized that the low genetic variability and high EOD diversity represents incipient sympatric speciation. The karyological description of a Campylomormyrus species provides crucial information about chromosome number and shapes. Its diploid chromosome number of 2n=48 supports the conservation of this trait within Mormyridae. Differences have been detected in the number of bi-armed chromosomes which is unusually high compared to other mormyrid species. This high amount can be due to chromosome rearrangements which could cause genetic incompatibility and reproductive isolation. Hence an alternative hypothesis regarding processes which cause sympatric speciation is that chromosome differences are involved in the speciation process of Campylomormyrus by acting as postzygotic isolation mechanism. In summary, the karyological and genomic investigations conducted in this study contributed to the increase of knowledge about Campylomormyrus species, to the solution of some existing ambiguities like phylogenetic relationships and to the raising of new hypothesis explaining the sympatric speciation of those African weakly electric fish. This study provides a basis for future genomic research to obtain a complete picture for causes and results of evolutionary processes in Campylomormyrus.
Predation drives coexistence, evolution and population dynamics of species in food webs, and has strong impacts on related ecosystem functions (e.g. primary production). The effect of predation on these processes largely depends on the trade-offs between functional traits in the predator and prey community. Trade-offs between defence against predation and competitive ability, for example, allow for prey speciation and predator-mediated coexistence of prey species with different strategies (defended or competitive), which may stabilize the overall food web dynamics. While the importance of such trade-offs for coexistence is widely known, we lack an understanding and the empirical evidence of how the variety of differently shaped trade-offs at multiple trophic levels affect biodiversity, trait adaptation and biomass dynamics in food webs. Such mechanistic understanding is crucial for predictions and management decisions that aim to maintain biodiversity and the capability of communities to adapt to environmental change ensuring their persistence.
In this dissertation, after a general introduction to predator-prey interactions and tradeoffs, I first focus on trade-offs in the prey between qualitatively different types of defence (e.g. camouflage or escape behaviour) and their costs. I show that these different types lead to different patterns of predator-mediated coexistence and population dynamics, by using a simple predator-prey model. In a second step, I elaborate quantitative aspects of trade-offs and demonstrates that the shape of the trade-off curve in combination with trait-fitness relationships strongly affects competition among different prey types: Either specialized species with extreme trait combinations (undefended or completely defended) coexist, or a species with an intermediate defence level dominates. The developed theory on trade-off shapes and coexistence is kept general, allowing for applications apart from defence-competitiveness trade-offs. Thirdly, I tested the theory on trade-off shapes on a long-term field data set of phytoplankton from Lake Constance. The measured concave trade-off between defence and growth governs seasonal trait changes of phytoplankton in response to an altering grazing pressure by zooplankton, and affects the maintenance of trait variation in the community. In a fourth step, I analyse the interplay of different tradeoffs at multiple trophic levels with plankton data of Lake Constance and a corresponding tritrophic food web model. The results show that the trait and biomass dynamics of the different three trophic levels are interrelated in a trophic biomass-trait cascade, leading to unintuitive patterns of trait changes that are reversed in comparison to predictions from bitrophic systems. Finally, in the general discussion, I extract main ideas on trade-offs in multitrophic systems, develop a graphical theory on trade-off-based coexistence, discuss the interplay of intra- and interspecific trade-offs, and end with a management-oriented view on the results of the dissertation, describing how food webs may respond to future global changes, given their trade-offs.
Multifunctional reprogrammable actuators based on polymer networks with crystallizable segments
(2019)
Soft polymeric materials, which can change their shape reversibly in response to external stimuli, can serve as actuating components in robotic systems. Besides electroactive polymers (EAP), hydrogels and liquid crystalline elastomers (LCE), crosslinked crystallizable shape-memory polymers networks have been introduced recently as reprogrammable thermo-reversible actuators. The integration of additional functions in such materials will lead to multifunctional polymeric actuators, which meet the complex requirements of modern robotic applications.
The primary aim of this thesis was to achieve multifunctional reprogrammable thermo-reversible actuators based on thermoplastic polymers. Here, three different actuators providing additional functionalities such as surface modification capability (i), self-healing capability (ii) or a tailorable non-response function enabling noncontinuous multi-step motions (iii) were realized. At first, it was hypothesized that surface modifiable polymeric actuators (i) can be achieved by crosslinking of crystallizable thermoplastic terpolymers having reactive moieties, where subsequent thermomechanical programming enables reversible actuations while the sustained reactive groups allow post surface modification. For the second actuator type (ii) it was hypothesized that self-healing during reprogramming of polymeric actuators prepared by crosslinking of crystallizable linear homopolymers, can be achieved by adjusting the amount of freely interpenetrating extractable polymer moieties. Finally, it was hypothesized that thermo-reversible actuators providing a non-response function (iii) and thus enable multistep motions upon continuous normal stimulation, can be achieved by a crosslinked blend of two thermoplastic polymers with co-continuous morphology having a well-separated melting and crystallization transitions. In addition, these actuators can be physically reprogrammed by heating above all melting transitions to provide a different actuating shape.
In this study, surface functionalizable actuators were realized from crosslinked poly[(ethylene)-co-(ethyl acrylate)-co-(maleic anhydride)] (cPEEAMA) based networks. Here crystallizable polyethylene (PE) segments should serve as actuation segments, ethyl acrylate (EA) provides elasticity to the system required for deformation, while reactive maleic anhydride (MA) will be used as chemically modifiable entities for post surface modification. Networks with varied crosslink density were prepared and its effect on thermomechanical properties as well as actuation performance was analyzed. Cyclic thermomechanical experiments were employed to investigate the actuation capability, which revealed a reversible actuation (ε׳rev) between 5 and 15%. Fourier-transform infrared spectroscopy (FTIR) measurements confirmed that MA groups were sustained at the sample surface after processing and programming, which could be modified by reaction with ethylene diamine. Such amine functionalization allows the attachment of bioactive molecules to the actuator surface, which might provide a route to actuating substrates for biotechnology.
Self-healable actuating materials were realized by poly(ε-caprolactone) (PCL) polymer networks with extractable linear PCL fractions of 5 to 60 wt%. A detailed evaluation of the actuation capabilities by cyclic experiments revealed the highest reversible change in strain of Δε = 24% for the cPCL network with 30 wt% of linear polymer. The thermal treatment of damaged samples resulted in the healing of the network when heated to 80 °C. Here a linear polymer fraction ≥ 30 wt% was necessary to achieve a self-healing efficiency of ≥ 50%. The application of such high temperatures erases the programmed actuator shape and at the same time allows to reprogram a new actuating shape. Such sustainable actuators with self-healing function are of great interest for future robotic devices.
Afore mentioned actuators operate continuously between two shapes and their movements can only be interrupted when the temperature is stopped. To overcome this limitation, noncontinuously responding actuators enabling multi-step actuation were realized from crosslinked blend networks prepared from PCL and poly[(ethylene)-co-(vinyl acetate)] (PEVA). These polymers (PCL and PEVA) were selected due to their immiscible character, where crystallizable PE and PCL segments provide two different actuation units, while vinyl acetate (VA) segment enabled sufficient elasticity of the system. A gap of 20 K in the melting and crystallization temperature of PE and PCL was achieved by selecting PEVA with 5 wt% VA content (cPCL-PEVA5) providing a co-continuous phase morphology. Cyclic thermomechanical investigations were employed to investigate noncontinuous actuation, which revealed a high Δε = 25% with a similar contribution from PCL and PE actuation units with a non-response region in the temperature range from 50 to 71 °C in heating step and 30 to 60 °C in cooling step. The actuation related to PCL part changed from 13 to 2% by altering the heating and cooling rates from 3 to 10 K·min-1. Free-standing reversible noncontinuous actuation was realized by rotating demonstrator which exhibits reversible angle change in a custom-made setup. For this purpose, cPCL-PEVA5 stripe was programmed by twisting and reversible rotational actuation was realized from 0 to 180° while pausing in the 90° position during non-response. These blends can be physically programmed to perform reversible noncontinuous actuations, while the programmed geometry can be erased by heating it to temperature above all melting transitions. By physically reprogramming of the material various different actuation modes can be obtained. Such a noncontinuous actuator would be relevant for designing interruptive actuating soft robots at continuous trigger signals.
Force plays a fundamental role in the regulation of biological processes. Cells can sense the mechanical properties of the extracellular matrix (ECM) by applying forces and transmitting mechanical signals. They further use mechanical information for regulating a wide range of cellular functions, including adhesion, migration, proliferation, as well as differentiation and apoptosis. Even though it is well understood that mechanical signals play a crucial role in directing cell fate, surprisingly little is known about the range of forces that define cell-ECM interactions at the molecular level.
Recently, synthetic molecular force sensor (MFS) designs have been established for measuring the molecular forces acting at the cell-ECM interface. MFSs detect the traction forces generated by cells and convert this mechanical input into an optical readout. They are composed of calibrated mechanoresponsive building blocks and are usually equipped with a fluorescence reporter system. Up to date, many different MFS designs have been introduced and successfully used for measuring forces involved in the adhesion of mammalian cells. These MFSs utilize different molecular building blocks, such as double-stranded deoxyribonucleic acid (dsDNA) molecules, DNA hairpins and synthetic polymers like polyethylene glycol (PEG). These currently available MFS designs lack ECM mimicking properties.
In this work, I introduce a new MFS building block for cell biology applications, derived from the natural ECM. It combines mechanical tunability with the ability to mimic the native cellular microenvironment. Inspired by structural ECM proteins with load bearing function, this new MFS design utilizes coiled coil (CC)-forming peptides. CCs are involved in structural and mechanical tasks in the cellular microenvironment and many of the key protein components of the cytoskeleton and the ECM contain CC structures. The well-known folding motif of CC structures, an easy synthesis via solid phase methods and the many roles CCs play in biological processes have inspired studies to use CCs as tunable model systems for protein design and assembly. All these properties make CCs ideal candidates as building blocks for MFSs. In this work, a series of heterodimeric CCs were designed, characterized and further used as molecular building blocks for establishing a novel, next-generation MFS prototype.
A mechanistic molecular understanding of their structural response to mechanical load is essential for revealing the sequence-structure-mechanics relationships of CCs. Here, synthetic heterodimeric CCs of different length were loaded in shear geometry and their mechanical response was investigated using a combination of atomic force microscope (AFM)-based single-molecule force spectroscopy (SMFS) and steered molecular dynamics (SMD) simulations. SMFS showed that the rupture forces of short heterodimeric CCs (3-5 heptads) lie in the range of 20-50 pN, depending on CC length, pulling geometry and the applied loading rate (dF/dt). Upon shearing, an initial rise in the force, followed by a force plateau and ultimately strand separation was observed in SMD simulations. A detailed structural analysis revealed that CC response to shear load depends on the loading rate and involves helix uncoiling, uncoiling-assisted sliding in the direction of the applied force and uncoiling-assisted dissociation perpendicular to the force axis.
The application potential of these mechanically characterized CCs as building blocks for MFSs has been tested in 2D cell culture applications with the goal of determining the threshold force for cell adhesion. Fully calibrated, 4- to 5-heptad long, CC motifs (CC-A4B4 and CC-A5B5) were used for functionalizing glass surfaces with MFSs. 3T3 fibroblasts and endothelial cells carrying mutations in a signaling pathway linked to cell adhesion and mechanotransduction processes were used as model systems for time-dependent adhesion experiments. A5B5-MFS efficiently supported cell attachment to the functionalized surfaces for both cell types, while A4B4-MFS failed to maintain attachment of 3T3 fibroblasts after the first 2 hours of initial cell adhesion. This difference in cell adhesion behavior demonstrates that the magnitude of cell-ECM forces varies depending on the cell type and further supports the application potential of CCs as mechanoresponsive and tunable molecular building blocks for the development of next-generation protein-based MFSs.This novel CC-based MFS design is expected to provide a powerful new tool for observing cellular mechanosensing processes at the molecular level and to deliver new insights into the mechanisms and forces involved. This MFS design, utilizing mechanically tunable CC building blocks, will not only allow for measuring the molecular forces acting at the cell-ECM interface, but also yield a new platform for the development of mechanically controlled materials for a large number of biological and medical applications.