Giant vesicles may contain several spatial compartments formed by phase separation within their enclosed aqueous solution. This phenomenon might be related to molecular crowding, fractionation and protein sorting in cells. To elucidate this process we used two chemically dissimilar polymers, polyethylene glycol (PEG) and dextran, encapsulated in giant vesicles. The dynamics of the phase separation of this polymer solution enclosed in vesicles is studied by concentration quench, i.e. exposing the vesicles to hypertonic solutions. The excess membrane area, produced by dehydration, can either form tubular structures (also known as tethers) or be utilized to perform morphological changes of the vesicle, depending on the interfacial tension between the coexisting phases and those between the membrane and the two phases. Membrane tube formation is coupled to the phase separation process. Apparently, the energy released from the phase separation is utilized to overcome the energy barrier for tube formation. The tubes may be absorbed at the interface to form a 2-demensional structure. The membrane stored in the form of tubes can be retracted under small tension perturbation. Furthermore, a wetting transition, which has been reported only in a few experimental systems, was discovered in this system. By increasing the polymer concentration, the PEG-rich phase changed from complete wetting to partial wetting of the membrane. If sufficient excess membrane area is available in the vesicle where both phases wet the membrane, one of the phases will bud off from the vesicle body, which leads to the separation of the two phases. This wetting-induced budding is governed by the surface energy and modulated by the membrane tension. This was demonstrated by micropipette aspiration experiments on vesicles encapsulating two phases. The budding of one phase can significantly decrease the surface energy by decreasing the contact area between the coexisting phases. The elasticity of the membrane allows it to adjust its tension automatically to balance the pulling force exerted by the interfacial tension of the two liquid phases at the three-phase contact line. The budding of the phase enriched with one polymer may be relevant to the selective protein transportation among lumens by means of vesicle in cells. / In der wässrigen Lösung im Inneren von Riesenvesikeln können sich mehrere, räumlich getrennte Phasen ausbilden. Dieses Phänomen könnte im Zusammenhang stehen mit wichtigen Prozessen innerhalb von Zellen, wie etwa Fraktionierung und Sortieren von Proteinen, oder etwa das sog. “Molecular Crowding”. Wir studieren diesen Prozess am Beispiel von zwei unterschiedlichen Polymeren, Polyethylen Glycol (PEG) und Dextran, innerhalb von Riesenvesikeln. Die Dynamik der Phasentrennung dieser eingeschlossenen Polymerlösung lässt sich untersuchen, indem man die Vesikel einer hypertonischen Lösung aussetzt. Durch die Dehydrierung entsteht dabei überschüssige Membranfläche. Je nach Grenzflächenspannung zwischen den koexistierenden Phasen, sowie zwischen der Membran und den beiden Phasen, wird diese überschüssige Fläche entweder zur Ausbildung röhrchenartiger Strukturen verwendet, oder aber es stellen sich morphologische Veränderungen am Vesikel ein. Die Ausbildung der Membranröhrchen ist offenbar gekoppelt an den Phasentrennungsprozess: Die Energie, die bei Phasentrennung frei wird, dient offenbar dazu, die Energiebarriere der Röhrchenbildung zu überwinden. Die Röhrchen können an der Grenzfläche absorbiert werden und dort eine zweidimensionale Struktur ausbilden. Durch kleine Störungen in der Spannung kann die in Form von Röhrchen gespeicherte Membran wieder in deren Oberfläche zurückgezogen werden. Desweiteren wurde in diesem System ein Benetzungsübergang entdeckt, der bisher nur in wenigen experimentellen Systemen beobachtet werden konnte: Erhöht man die Polymerkonzentration, so geht die PEG-reiche Phase von vollständiger zu unvollständiger Benetzung der Membran über. Steht in einem Vesikel, in dem beide Phasen die Membran benetzen, ausreichend überschüssige Membranfläche zur Verfügung, so wird sich eine Phase aus dem Vesikelkörper herauswölben, was zur Trennung der beiden Phasen führt. Dieser benetzungsinduzierte Auswölbungsprozess wird durch die Oberflächenenergie bestimmt und von der Membranspannung moduliert. Dies konnte experimentell an Vesikeln gezeigt werden, die zwei Phasen beinhalten, indem durch eine Mikropipette ein Unterdruck erzeugt wurde. Die Oberflächenenergie kann durch Auswölbung einer der Phasen signifikant verringert werden, da die Kontaktfläche zwischen den koexistierenden Phasen verkleinert wird. Die Elastizität der Membran erlaubt es, die Spannung automatisch anzupassen, sodass die ziehende Kraft ausgeglichen wird, die durch die Grenzflächenspannung der beiden flüssigen Phasen an der drei-Phasen Kontaktlinie ausgeübt wird. Die Auswölbung einer durch Polymere angereicherten Phase könnte relevant sein für den selektiven Transport von Proteinen mit Vesikeln in der Zelle.

Das Ziel der Arbeit bestand darin, ein verbessertes Verfahren zur Ausbildung einer Polyurethanschicht mit poröser Kapillarstruktur zu erarbeiten. Die Fällung (Koagulation) einer Polyurethanlösung erfolgt durch kontrollierte Freisetzung von Fällmittel aus einem zugesetzten, thermisch sensiblen und porös umhüllten Hydrogel. Bei Erwärmung auf eine stoffspezifische Temperatur setzt das Hydrogel Wasser frei, das die Fällung initiiert. Gegenüber der Fällung in einem Fällbad erzielt diese Verfahrensweise deutliche Verbesserungen. Die umhüllten Gele wurden mit Hilfe rheologischer, thermoanalytischer und weiterer Untersuchungsmethoden umfassend charakterisiert. Dadurch gelang es, eine Hüllenstruktur auszuwählen, die den spontanen Austausch von Löse- und Fällmittel verhindert und gleichzeitig die Wasserfreisetzung gewährleistet.

Cononsolvency occurs if a mixture of two good solvents causes the collapse or demixing of polymers into a polymer-rich phase in a certain range of compositions of these two solvents. The better solvent is usually called cosolvent and another common solvent is called solvent. So far, the phase-transition mechanism behind cononsolvency is still rather controversially debated in literature. In this thesis, I experimentally investigated the cononsolvency effect of poly(N-isopropylacrylamide) (PNiPAAm) brushes with different grafting density in aqueous alcohol mixtures. I have used Vis-spectroscopic ellipsometry measurements and proved the hypothesis that the cononsolvency transition of PNiPAAm brushes consists of a volume phase-like equilibrium transition. I found a strong collapse transition in PNiPAAm brushes followed by a reentry behavior as observed by ellipsometry measurements. Using a series of alcohols with increasing alkyl-chain length I have demonstrated that the cononsolvency effect is enhanced and shifted to smaller volume fractions of the alcohol. Particularly for the alcohol with increasing hydrophobic property this is correlated with an increasing tendency of demixing between the cosolvent and water. This is apparently in contrast to the hypothesis of strongly associative solvents being the origin of the cononsolvency effect. The hypothesis of preferential adsorption, on the other hand, can account for this case by assuming an increasing hydrophobically driven adsorption of the cosolvent on the polymer chains. The recently proposed adsorption-attraction model based on the concept of preferential adsorption, can be used to predict the corresponding phase-transition behavior. In particularly the model predictions for variation of the grafting density is in agreement with the experimental findings. However, to reflect the imperfect mixing of the longer alcohols in water as well as finite miscibility of the polymers in the common solvent, extensions of the model have to be considered. I have shown that the simplest extension of the model taking into account the Flory-Huggins parameter for polymer and water can account for the qualitative changes observed for temperature changes in my experiments. Both a theoretical analysis and experimental observations show that the phase-transition mechanism of cononsolvency depends on the relative strengths of various interactions in the polymer solutions. A cononsolvency transition can be driven by a strong cosolvent-solvent attraction or by the preferential adsorption of cosolvent onto the polymer. By an extension of the adsorption-attraction model, I report on a comprehensive and quantitative theoretical study of the cononsolvency effect of neutral polymers such as PNiPAAm brushes, macro-gels and single long chains. The extended adsorption-attraction model is able to describe and predict the phase-transition behaviors of these systems in various aqueous alcohol solutions quantitatively. My analysis showed that besides the dominant role of polymer-cosolvent preferential adsorption and the monomer-cosolvent-monomer triple contacts (cosolvent-assisted temporary cross-linking effect) that define the strength of the collapse-transition in the cosolvent-poor region, other effects are shown to be of relevance: The non-ideal mixing between polymer and solvent plays a role in shifting the collapse transition to the lower-concentration region of cosolvent, and an increase of the demixing tendency between cosolvent and solvent on the polymer chains reduces the window width of the cononsolvency transition. Using data from my own experiments and literature I can show that the cononsolvency response of brushes, gels and single long polymer chain can be consistently described with the same model. The model parameters are consistent with their microscopic interpretation. In addition, weakening of the cononsolvency transition in cosolvent-poor aqueous solutions at high hydrostatic pressure can be explained by the suppression of demixing tendency between cosolvent and water, and between polymer and water in the case of PNiPAAm. An investigation of the grafting-density effect in the cononsolvency transition of grafted PNiPAAm polymer, showed that a decrease of grafting density at the collapse state as well as the temperature is fixed, the swollen polymer chains can show various morphologies not limited to collapse brush. In addition, my experimental results clearly showed that the strongest collapse state can be only realized by polymer brushes with moderate grafting densities. My results display the universal character of the cononsolvency effect with respect to series of cosolvents and show that PNiPAAm brushes display a well-defined and sharp collapse transition. This is most pronounced for 1-propanol as cosolvent which is still fully miscible in water. Potential applications are switches built from implementation of brushes in pores and similar concave geometries can be realized by harnessing the cononsolvency effect of stimuli-responsive polymers such as PNiPAAm. As an example of application of cononsolvency effect of grafted polymers, different molecular-weight PNiPAAm polymers are grafted around the rim of solid-state nanopores by using grafting-to method. I demonstrate that small amounts of ethanol admixed to an aqueous solution can trigger the translocation of fluorescence DNA through polymer-decorated nanopores. I can identify the cononsolvency effect as being responsible for this observation which causes an abrupt collapse of the brush by increasing the alcohol content of the aqueous solution followed by a reswelling at higher alcohol concentration. For the first time, I provide a quantitative method to estimate hydrodynamic thickness of a polymer layer which is grafted around the rim of nanopores. Regardless of the grafting density of a grafted PNiPAAm polymer layer around the rim of nanopores, in the alcohol-tris buffer mixtures, the polymer layer displays solvent-composition responsive behaviors in the range of metabolic pH values and room temperatures. Although in this study PNiPAAm was chosen as a model synthetic polymer, I believe in that the conclusions made for PNiPAAm can be also in general extended to other synthetic polymers as well as to biopolymers such as proteins. As a proof of concept of using synthetic polymers to mimic biological functions of cell-membrane channels, my study clearly transpired that cononsolvency effect of polymers can be used as a trigger to change the size of nanopores in analogy to the opening and closure of the gates of cell-membrane channels.:Chapter 1 Background and motivation 4 1.1 Liquid-liquid phase separation 4 1.2 Polymer phase separation in a pure solvent 5 1.3 Polymer phase separation in mixtures of two good solvents 10 1.4 Characterizing cononsolvency transition in experimental study 14 1.5 Research motivation 16 Chapter 2 Phase behaviors of PNiPAAm brushes in alcohol/water mixtures: A combined experimental and theoretical study 17 2.1 Introduction 17 2.2 Materials and Methods 17 2.2.1 Materials 17 2.2.2 Preparation of Polymer Brushes 18 2.2.3 VIS-Spectroscopic Ellipsometry Measurement 18 2.2.4 Determining a polymer brush’s overlap grafting density 19 2.2.5 Test of PNiPAAm solubility in short-chain polyols 20 2.3 The adsorption-attraction model 20 2.4 Equilibrium behavior of cononsolvency transition of PNiPAAm brushes 22 2.5 Role of volume of solvent molecules in the swelling of PNiPAAm brushes 24 2.6 Cononsolvency transition of PNiPAAm brushes in aqueous solutions of a series of alcohol 24 2.7 Isomer effect of alcohol in the cononsolvency transition of PNiPAAm brushes 27 2.8 Role of alcohol-water interaction in the cononsolvency transition of PNiPAAm polymers 28 2.9 Temperature effect in the cononsolvency transition of PNiPAAm brushes 30 2.10 Grafting-density effect in the cononsolvency transition of PNiPAAm brushes 33 2.11 Octopus-shape-micelle morphology of grafted PNiPAAm polymers 34 2.12 Chapter summary 35 2.13 Chapter appendix 37 2.13.1 Data extraction and reprocessing for the molar Gibbs free energy of mixing 37 2.13.2 Temperature effect in the cononsolvency transition of PNiPAAm gels 37 Chapter 3 The extended adsorption-attraction model 41 3.1 Introduction 41 3.2 An extension of the adsorption-attraction model 43 3.3 Numerical solution of the extended adsorption-attraction model 47 3.4 Validation of the extended adsorption-attraction model 50 3.4.1 Cononsolvency transition of polymer brushes and macro-gels in different alcohol-water mixtures 51 3.4.2 An analysis of the enthalpic interaction between cosolvent and solvent 57 3.4.3 The window width of the cononsolvency transition 60 3.4.4 Pressure effect in the cononsolvency transition of PNiPAAm polymers 61 3.4.5 Cononsolvency transition of a single long polymer 65 3.5 Chapter summary 66 3.6 Chapter appendix 67 3.6.1 Chemical potential change of mixing two components 67 3.6.2 The Enthalpic Wilson model 68 3.6.3 Estimation of effective Flory-interaction parameter 73 3.6.4 Crosslink-density effect in the cononsolvency transition of poly(N-isopropylacrylamide) micro-gel and macro-gel 74 3.6.5 Pressure effect on the dimensionless chemical potential change (μ) 75 3.6.6 Pressure effect on the cosolvent-solvent interaction (χcs) 76 3.6.7 Pressure effect on the polymer-solvent interaction (χps) 77 3.6.8 Chemical potential change of DMSO/water mixtures 78 Chapter 4 Gating the translocation of DNA through poly(N-isopropylacrylamide) decorated nanopores using the cononsolvency effect in aqueous environments 80 4.1 Introduction 80 4.2 Methods 80 4.2.1 Preparation of polymer-grafted gold membrane 80 4.2.2 Translocation experiments of fluorescence λ-DNA through nanopores 82 4.2.3 Method of identification and counting of DNA translocation events 84 4.3 Results and discussion 86 4.3.1 Grafting density effect on the swollen behaviors of PNiPAAm polymers around the rim of nanopores 86 4.3.2 Switching effect of polymer chains around the rim of nanopores in the tri-buffer/ethanol mixtures 88 4.3.3 Switching effect of polymer brushes on the flat surface in the tri-buffer/ethanol mixtures 92 4.3.4 An attempt of numerical fit of experimental data using the extended adsorption-attraction model 94 4.4 Chapter summary 95 4.5 Chapter appendix 96 4.5.1 An estimation of grafting density 96 4.5.2 The method of processing data 97 Chapter 5 Concluding remarks and outlooks 100 5.1 Concluding remarks 100 5.2 Outlooks: A preliminary discussion of the cononsolvency transition of polymer solutions 102 References and notes 108 List of figures 119 List of tables 128 Acknowledgements 130 List of publications 131 Erklärung 132

All living things are driven by chemical reactions. Reactions provide energy and transform matter. Thus, maintaining the system out of equilibrium. However, these chemical reactions have to be organized in space. One way for this spatial organization is via the process of phase separation. Motivated by the recent discovery of liquid-like droplets in cells, this thesis studies the organization of chemical reactions in phase-separated systems, with and without broken detailed balance. After introducing the underlying thermodynamic principles, we generalize mass-action kinetics to systems with homogeneous compartments formed by phase separation. Here, we discuss the constraints resulting from phase equilibrium on chemical reactions. We study the relaxation kinetics towards thermodynamic equilibrium and investigate non-equilibrium states that arise when detailed balance is broken in the rates of reactions such that phase and chemical equilibria contradict each other. We then turn to spatially continuous systems with spatial gradients within formed compartments. We derive thermodynamic consistent dynamical equations for reactions and diffusion processes in such systems. Again, we study the relaxation kinetics towards equilibrium and discuss non-equilibrium states. We investigate the dynamics of droplets in the presence of reactions with broken detailed balance. Furthermore, we introduce active droplet systems maintained away from equilibrium via coupling to reservoirs at their boundaries and organizing reactions solely within droplets. Here, detailed balance is only broken at the boundaries. Nevertheless, stationary chemically active droplets exist in open systems, and droplets can divide. To quantitatively study chemically active droplet systems in multi-component mixtures, we introduce an effective description. Therefore, we couple linearized reaction-diffusion equations via a moving interface within a sharp interface limit. At the interface, the boundary conditions are set by a local phase equilibrium and the continuity of fluxes. Equipped with these tools, we introduce and study protocell models of chemically active droplets. We explicitly model these protocells’ nutrient and waste dynamics, leading to simple models of their metabolism. Next, we study the energetics of these droplets and identify processes responsible for growth or shrinkage and maintaining the system out of equilibrium. Furthermore, we discuss the energy balance leading to the heating and cooling of droplets. Finally, we show why chemically active droplets do not spontaneously divide in two-dimensional systems with bulk-driven reactions. Here, droplets can elongate but do not pinch off. To have a minimal two-dimensional model with droplet division, we introduce additional reactions. When these reactions are localized at the interface and dependent on its mean curvature, droplets robustly divide in 2D. In summary, this thesis contributes to the theoretical understanding of how the existence of droplets changes the kinetics of reactions and, vice versa, how chemical reactions can alter droplet dynamics.:1 Introduction 1.1 Thermodynamics of phase separation 1.1.1 Phase equilibrium in the thermodynamic limit 1.1.2 Relaxation dynamics towards equilibrium 1.1.3 Local stability of homogeneous phases 1.2 Thermodynamics of chemical reactions in homogenous mixtures 1.2.1 Conserved densities and reaction extents 1.2.2 Equilibrium of chemical reactions 1.2.3 Mass-action kinetics towards equilibrium 1.3 Simultaneous equilibrium of chemical reactions and phase separation 1.4 Chemical reactions maintained away from equilibrium 1.5 Structure of this thesis 2 Chemical reactions in compartmentalized systems 2.1 Mass-action kinetics for compartments built by phase separation 2.1.1 Dynamical equations for densities and phase volumes 2.1.2 Relaxation kinetics in a simple example 2.2 Driven chemical reactions in compartmentalized systems 2.2.1 Non-equilibrium steady states at phase equilibrium 2.2.2 The tie line selecting manifold 2.3 Discussion 3 Dynamics of concentration fields in phase-separating systems with chemical reactions 3.1 Reaction-diffusion equations for phase-separating systems 3.2 Relaxation towards thermodynamic equilibrium in spatial systems 3.2.1 Relaxation kinetics and fast diffusion 3.2.2 Relaxation kinetics with spatial gradients 3.3 Driven chemical reactions in phase-separating systems 3.3.1 Driven chemical reaction and fast diffusion 3.3.2 Non-equilibrium steady states and spatial gradients 3.3.3 Droplets growth and ripening with driven chemical reactions 3.4 Boundary-driven chemically active droplets 3.4.1 Droplets in open systems 3.4.2 Non-equilibrium steady droplets and shape instabilities 3.5 Discussion 4 Chemically active droplets in the sharp interface limit 4.1 Droplet dynamics via reaction-diffusion equations coupled by a moving interface 4.2 Stationary interface positions in spherical symmetry 4.2.1 Interface conditions in closed systems 4.2.2 Interface conditions in open systems 4.3 Shape instabilities of spherical droplets 4.4 Discussion 5 Models of protocells and their metabolism as chemically active droplets 5.1 Breaking detailed balance in protocell models 5.1.1 Boundary-driven protocell models 5.1.2 Bulk-driven protocell models 5.2 Protocell dynamics 5.2.1 Steady states droplets 5.2.2 Shape stability of spherical symmetric droplets 5.3 Energetics of protocells 5.3.1 Mass conservation and droplet growth or shrinkage 5.3.2 Energy conservation and droplet heating or cooling 5.4 Discussion 6 The role of dimensionality on droplet division 6.1 Stability of chemically active droplets in 2D vs. 3D 6.1.1 Stationary droplets in 1D, 2D and 3D 6.1.2 Elongation instability 6.1.3 Pinch-off instability 6.2 Pinch-off in 2D via curvature-dependent chemical reactions 6.2.1 Determining the mean curvature of the droplet interface 6.2.2 Chemical reactions at the interface 6.3 Discussion 7 Conclusion and Outlook A Free energy considerations B Surface tension in multi-component mixtures C Figure details Bibliography

Recently, there has been a growing drive towards the bottom-up development of synthetic cells that mimic key cellular features. A cellular feature ubiquitous amongst cells is that of compartmentalisation. Compartmentalisation enables the spatiotemporal control of biochemical reactions and is thus vital for the development of synthetic cells. To date, most synthetic cell models have utilised classical membrane bound containers as model compartments. However, recent advances in cell biology have highlighted the importance of membraneless compartments formed via liquid-liquid phase separation (LLPS) as organisation centres. It has been suggested that these organelles play a critical role in regulating cell biochemistry, yet very little is known about their interactions with enzymatic reactions. Thus, aiming to develop novel synthetic capabilities, the work presented in this thesis designs and characterises synthetic cells which include features of membraneless compartmentalisation. These systems utilise complex coacervates, a specific type of LLPS that is driven by the electrostatic attraction of oppositely charged polymers, as model membraneless compartments. These low complexity systems subsequently provide ideal platforms for systematic investigations of the interaction of membraneless coacervate compartments with enzymatic reactions. In Chapter 3 and 4, I focus on developing a responsive synthetic cell system that recapitulates features of membrane-bound and membraneless compartmentalisation. I generate a pH-responsive system by exploiting the intrinsic pKa of cationic polylysine to trigger coacervation within a liposome. This synthetic cell is then functionalised with the enzyme formate dehydrogenase (FDH). I show that coacervate properties can be utilized to locally concentrate and activate the FDH reaction at low enzyme concentrations, thus demonstrating that membraneless compartments can activate reactions via sequestration into coacervate reaction centres. In Chapter 5, I then proceed to characterise whether the diffusive exchange of molecules across a droplet phase boundary effects enzyme dynamics. Synthetic cells constructed from emulsion droplets with coacervate sub-compartments were used as model systems with diffusive exchange, while bulk coacervate and supernatant phases were used as uncoupled model systems without exchange. I studied the FDH reaction in both models and I conclude that coupling of the phases increases reaction rates compared to an uncoupled system. When coupled, the supernatant acts as a ’sink’ removing the product NADH from the coacervate droplets. This increases the apparent reaction rate in the supernatant, while the reduction of NADH concentration in the coacervate reduces product inhibition. This demonstrated that the open phase boundary tightly couples membraneless droplets to their surroundings, which can ultimately lead to increased reaction rates both inside and outside the compartments. Finally in Chapter 6, I scrutinize enzyme kinetics of the enzymes FDH and β -galactosidase in the unique coacervate physicochemical environment using Michaelis-Menten assays in CM-Dex/PDDA bulk phase. Results show that the KM and Vmax of FDH significantly increased compared to buffer, while those of β-galactosidase do not. I hypothesise that the negatively charged formate substrate of the FDH reaction interacts strongly with the positively charged PDDA, decreasing its affinity for the enzyme. Furthermore, I suggest that the coacervate environment facilitates the rate limiting hydride transfer of the reaction, thereby increasing the maximum rate. This data demonstrates that the coacervate environment itself can tune and control enzyme dynamics. In conclusion, my work establishes responsive, tunable and enzymatically active syn- thetic cellular systems with features of membraneless compartmentalisation. My results indicate that membraneless compartments can have significant impact on the dynamics of enzymatic reactions, opening up possible ways to control reaction rates in synthetic systems and suggesting plausible functions for membraneless organelles in vivo. Overall, I demonstrate that rationally designed synthetic cells provide biomimetic experimental platforms that offer insights into the influence of membraneless compartmentalisation on enzymatic reactions. Parts of the presented work have been published as two first author publications in peer-reviewed journals. / ‘Bottom-up'’ Modelle synthetischer Zellen, die Schlüsselmerkmale zellbasierten Lebens imitieren, rücken immer mehr in den Fokus. Von zentraler Bedeutung ist hier die Kompartmentbildung. Sie erst ermöglicht die räumliche und zeitliche Kontrolle biochemischer Abläufe und ist daher entscheidend bei der Entwicklung synthetischer Zellen. Bisher wurden in der Mehrzahl der synthetischen Zellmodelle klassische, membrangebundene Reaktionsräume als Modellkompartimente verwendet. Jüngste Fortschritte in der Zellbiologie belegen jedoch die Bedeutung von membranlosen Kompartimenten, die durch Flüssig-Flüssig-Phasentrennung (LLPS) gebildet werden. Es wird angenommen, dass diese membranlosen Kompartimente eine zentrale Rolle bei der Regulierung der Zellchemie spielen. Jedoch ist bisher nur sehr wenig über ihren Einfluss auf enzymatische Reaktionen bekannt und experimentell belegt. Mit dem Ziel, die Bandbreite und das Verständnis synthetischer Modelle zu erweitern, wurden in dieser Arbeit neue Methoden entwickelt und dargestellt, die membranlose Kompartmentbildung benutzen. Es wurden hierfür komplexe Koazervate eingesetzt, eine spezielle Art der LLPS, welche durch die elektrostatische Anziehung von entgegengesetzt geladenen Polymeren angetrieben wird. Diese verhältnismäßig einfachen Systeme bieten eine ideale Plattform für systematische Untersuchungen des Einflusses von membranlosen Koazervatkompartimenten auf enzymatische Reaktionen. In den Kapiteln 3 und 4 konzentrierte ich mich auf die Entwicklung eines reaktionsfähigen synthetischen Modellsystems, das die Phänomene sowohl membrangebundener als auch membranfreier Kompartmentbildung vereint. Zur Steuerung der Koazervierung innerhalb von Liposomen wurde ein pH-reaktives System verwendet, welches sich den intrinsischen pKa von kationischen Polylysin zunutze macht. Diese synthetis- che Zelle wurde im folgenden Schritt mit dem Enzym Formiat-Dehydrogenase (FDH) funktionalisiert. Ich konnte damit zeigen, dass es die Eigenschaften von Koazervaten ermöglichen, die FDH-Reaktion bei global sehr niedrigen Enzymkonzentrationen zu aktivieren. Hierbei wirken die membranlosen Koazervate in Folge einer lokal er- höhten Enzymkonzentration als Zentren gesteigerter Reaktivität. Dies geschieht durch die lokale Konzentrationserhöhung in Koazervaten, was bei LLPS auch durch den Verteilungskoeffizient beschrieben wird. Mit anderen Worten agieren diese membran- losen Kompartimente durch Sequestrierung als Reaktionszentren. Im Kapitel 5 charakterisierte ich den Einfluss von diffusivem Molekülaustausch auf die Enzymkinetik über die Koazervat-Phasengrenze hinweg. Hierbei wurden zwei Systeme miteinander verglichen. Einerseits wurde ein synthetisches Zellmodell, beste- hend aus mikrofluidisch hergestellten Wasser-in-Öl Emulsionstropfen, die Koazervate enthalten, als Modellsystem mit diffusivem Austausch zwischen den Phasen verwendet. Andererseits wurden separate, reine Koazervatphasen und reine Überstandsphasen als Modellsysteme ohne Austausch verwendet. Ich habe die FDH-Reaktion in beiden Modellsystemen untersucht und kam zu dem Schluss, dass die Kopplung der Phasen die Reaktionsgeschwindigkeiten im Vergleich zu den ungekoppelten Systemen erhöht. Bei der Kopplung wirkt die Überstandsphase als Senke, die das Produkt NADH aus den Koazervaten aufnimmt. Dies erhöht die scheinbare Reaktionsgeschwindigkeit im Überstand, während die Verringerung der NADH-Konzentration im Koazervat die Produkthemmung verringert. Dies zeigt, dass die offene Phasengrenze membranloser Kompartimente eng mit ihrer Umgebung gekoppelt ist, was als erhöhte Reaktionsraten sowohl innerhalb als auch außerhalb der Kompartimente gemessen werden kann. Schließlich untersuchte ich in Kapitel 6 die Enzymkinetik der Enzyme FDH und β- Galaktosidase in der physikalisch-chemischen Umgebung des Koazervats. Mit Hilfe von Michaelis-Menten-Experimenten in der CM-Dextran/PDDA-Bulkphase konnte gezeigt werden, dass KM und Vmax von FDH im Vergleich zum Überstand signifikant erhöht sind, wohingegen jene von β-Galaktosidase ein solches Verhalten nicht zeigen. Das führte mich zu der Hypothese, dass das negativ geladene Formiatsubstrat der FDH- Reaktion stark mit dem positiv geladenen PDDA interagiert, wodurch seine Affinität für das Enzym abnimmt. Darüber hinaus wird der ratenbegrenzende Hydridtransfer in der Umgebung des Koazervats erleichtert und es kann eine Erhöhung der Reaktionsrate beobachtet werden. Die Daten zeigen, dass abhängig vom Koazervat-Milieu die Enzymdynamik in verschiedene Richtungen gesteuert werden kann. Zusammenfassend lässt sich sagen, dass meine Arbeit reaktionsfähige, steuerbare und enzymatisch aktive synthetische Zellsysteme mit Eigenschaften membranloser Kompartmentbildung etabliert. Meine Ergebnisse deuten darauf hin, dass membranlose Kompartimente einen signifikanten Einfluss auf die Dynamik enzymatischer Reaktio- nen haben. Meine Untersuchungen eröffnen damit neuartige Wege zur Kontrolle der Reaktionsgeschwindigkeit in synthetischen Systemen und erweitern das Verständnis möglicher Funktionen membranloser Organellen in vivo. Insgesamt zeige ich, dass über- legt entworfene synthetische Zellen eine hervorragende biomimetische Plattform bieten, um Einblicke in den Einfluss von membranloser Kompartimentierung auf enzymatische Reaktionen zu gewinnen. Teile der vorgestellten Arbeit wurden als wissenschaftliche Beiträge in zwei begutachteten Journalen als Erstautor veröffentlicht.

Das Ziel der Arbeit bestand darin, ein verbessertes Verfahren zur Ausbildung einer Polyurethanschicht mit poröser Kapillarstruktur zu erarbeiten. Die Fällung (Koagulation) einer Polyurethanlösung erfolgt durch kontrollierte Freisetzung von Fällmittel aus einem zugesetzten, thermisch sensiblen und porös umhüllten Hydrogel. Bei Erwärmung auf eine stoffspezifische Temperatur setzt das Hydrogel Wasser frei, das die Fällung initiiert. Gegenüber der Fällung in einem Fällbad erzielt diese Verfahrensweise deutliche Verbesserungen. Die umhüllten Gele wurden mit Hilfe rheologischer, thermoanalytischer und weiterer Untersuchungsmethoden umfassend charakterisiert. Dadurch gelang es, eine Hüllenstruktur auszuwählen, die den spontanen Austausch von Löse- und Fällmittel verhindert und gleichzeitig die Wasserfreisetzung gewährleistet.

Die chloroplastidäre und mitochondriale Genexpression ist abhängig von einer großen Anzahl an RNA-Bindeproteinen (RBPs). Eine besonders abundante Familie sind die chloroplastidären Ribonukleoproteine (cpRNPs). Während Ziel-RNAs mehrerer cpRNPs und die Phänotypen entsprechender Mutanten beschrieben wurden, bleibt ihre molekulare Funktion weitgehend ungeklärt. In dieser Arbeit wurden Studien der cp29a Mutante durch genomweite Analysen erweitert. Diese legen nahe, dass die eigentliche Rolle von CP29A in phänotypisch erkennbarem Mutanten-Gewebe durch sekundäre Defekte maskiert wird. Um primäre Defekte zu identifizieren, wurden in vivo Bindestellen von CP29A mit einer neuen Chloroplasten-adaptierten Methode, die UV-Licht zur Quervernetzungen nutzt, bestimmt. Transkripte, die für Untereinheiten des Photosystem II und des Cytochrom-b6f-Komplexes kodieren, waren unter den Zielen von CP29A überrepräsentiert. Weiterhin wurden mehrere Bindestellen in Nachbarschaft zu Bindestellen von PPR-Proteinen identifiziert. Mit einer alternativen Methode, die chemische Quervernetzung nutzt, wurden Ziel-RNAs eines weiteren cpRNP, CP31A, identifiziert. Transkripte, die für Untereinheiten des NADH-Dehydrogenase Komplexes kodieren, waren überrepräsentiert. Diese Daten führten zu einer neuen Hypothese, die die Funktion von cpRNPs im Zusammenspiel mit PPR-Proteinen in der Prozessierung funktionell verwandter RNAs postuliert. Ein weiterer für die Genexpression relevanter Mechanismus ist die Bildung membranloser Kompartimente durch flüssig-flüssig Phasentrennung. Es wurde eine in silico Analyse durchgeführt, um organelläre Proteine mit Domänen, die auf flüssig-flüssig Phasentrennung hindeuten, zu identifizieren. Funktionen mit Bezug zu Genexpression, insbesondere RNA-Edierung, waren bei diesen Proteinen mit Prionen-ähnlichen Domänen (PLDs) überrepräsentiert. Zwei Kandidaten wurden auf ihre Neigung zur flüssig-flüssig Phasentrennung durch in vitro Experimente und in vivo Mikroskopie untersucht. / Gene expression in chloroplasts and mitochondria relies on a large number of RNA-binding proteins (RBPs), which are involved in the processing of polycistronic precursor transcripts. A particular abundant family are the chloroplast ribonucleoproteins (cpRNPs). While target RNAs and mutant phenotypes of several cpRNPs were described, insights on their molecular function remained sparse. In this thesis, analyses of cp29a mutants were extended by genome-wide transcriptome data, which suggest that in phenotypically noticeable mutant tissue the actual role of CP29A might be masked by secondary effects. To identify primary defects, in vivo binding sites of CP29A on its target transcripts were determined using a novel chloroplast-adapted approach using crosslinking by UV-light. Identified targets of CP29A are functionally enriched in mRNAs encoding subunits of the photosystem II and the cytochrome b6f complex. Moreover, several binding sites were identified in close proximity to characterized binding sites of PPR proteins. Using an alternative approach, employing chemical crosslinking, targets of another cpRNP, CP31A, were identified. Targets are enriched in genes encoding subunits of the NADH-like dehydrogenase complex. In combination, these data led to a novel hypothesis on the molecular function of cpRNPs working together with PPR proteins in the processing of functionally related RNAs. Another increasingly recognized mechanism in gene expression is the formation of membraneless organelles by liquid-liquid phase separation. An in silico screen for organellar proteins containing domains indicative of phase separation was performed. The identified set of proteins with prion-like domains (PLDs) is enriched in functions related to gene expression, particularly RNA-editing. Two selected candidate proteins were characterized for their propensity to undergo phase separation by in vitro phase separation assays and in vivo microscopy.

For life to thrive, its fundamental units, i.e., the cells, need to reliably and robustly fulfill their function. However, cellular operability is challenged by the appearance of biological noise in the concentration of proteins and other cell components. This noise arises due to spontaneous fluctuations that are inherent to all chemical reactions. For small (mesoscopic) systems, like cells, these fluctuations can be significant and disturb cellular functions. Cells evolved mechanisms to control and reduce their internal noise. One way to reduce noise in eukaryotic cells is to exploit their internal structure and restrict noise to a particular organelle, thus reducing the noise in the rest of the cell. In recent years it was shown that many cell organelles could be formed by phase separation without the need for a membrane. Thus, it was suggested that phase separation could reduce concentration noise in cells. However, until now, any systematic investigation linking essential aspects of phase separation and concentration noise in cells has been lacking. This motivates the study of fluctuations in mesoscopic phase-separating systems. This thesis develops a generic theoretical model based on a thermodynamic description of phase separation. We consider a binary mixture that can phase separate into two phases - a liquid droplet surrounded by a phase, which we refer to as continuous phase. We merge this description with methods of stochastic chemical reactions in order to account for the active turnover of phase-separating material and, thus, for the non-equilibrium nature of living cells. The resulting framework allows us to study fluctuations due to chemical turnover and phase separation in and out of equilibrium of phase separation. We use this framework to investigate how a phase-separating system can reduce concentration noise for different reaction networks. We find that phase separation can reduce concentration noise in active mesoscopic systems like cells in both phases. When turnover dynamics are slow, concentration noise in the dilute phase can be lowered to the level of Poissonian fluctuations. For the dense phase, we find that noise can fall below the Poissonian threshold. When turnover rates become faster such that the system deviates from the equilibrium configuration, the noise reduction by phase separation becomes less efficient. We test our model on experimental data of an engineered protein expressed in living cells. We find a good agreement between the data and theory and demonstrate that phase separation is a viable mechanism for noise reduction in living cells. Thus, phase separation might play an essential part in ensuring the reliable control of cellular functions.

RNA-Bindungsproteine spielen Schlüsselfunktionen bei der post-transkriptionellen Regulation der Genexpression. Durch Bindung an RNA steuern sie die RNA-Aufbereitung, den Transport, die Stabilität und die Translation. In den letzten zehn Jahren wurden bedeutende Fortschritte bei der Aufklärung bakterieller post-transkriptioneller Mechanismen erzielt. Es wird immer deutlicher, dass diese Regulierungsebene auch bei der Pathogenese und Antibiotikaresistenz eine wichtige Rolle spielt. Die Analyse von RNA-Protein-Komplexen (RNPs) auf Proteomebene wurde durch die (m)RNA-interactome-capture Technologie vorangetrieben, die den Teil des Proteoms isoliert, welcher mit polyadenylierter (m)RNA vernetzt ist. Dies hat zur Identifizierung von Hunderten von neuen RBPs in einer Vielzahl von eukaryontischen Arten, vom Menschen bis zur Hefe, geführt. Allerdings fehlt die Poly-Adenylierung in der funktionellen RNA von Bakterien und anderen Klassen von -eukaryontischen- regulatorischen RNAs. Ziel dieser Arbeit war es, diese Einschränkung durch die Entwicklung einer neuartigen und unvoreingenommenen Methode zur Aufreinigung von UV-vernetzten RNPs in lebenden Zellen zu überwinden: PTex (Phenol-Toluol-Extraktion). Das Reinigungsprinzip basiert ausschließlich auf den physikalisch-chemischen Eigenschaften von vernetzten RNPs gegenüber ungebundenen Proteinen oder RNA; es ist dabei unparteiisch gegenüber spezifischen RNAs oder Proteinen und ermöglicht somit erstmals eine systemweite Analyse von nicht-poly-(A)-RNA-interagierenden Proteinen sowohl in eukaryontischen (HEK293) als auch in prokaryontischen (Salmonella Typhimurium) Zellen. / RNA binding proteins play key functions in post-transcriptional regulation of gene expression. By binding to RNA, they control RNA editing, transport, stability and translation. In the last decade, significant advances have been made in the elucidation of bacterial post-transcriptional mechanisms. It is becoming increasingly clear that this layer of regulation also plays an important role in pathogenesis and antibiotic resistance. The analysis of RNA-protein complexes (RNPs) at the proteome level has been driven by the (m)RNA interactome capture technology which isolates the proteome cross-linked to poly-adenylated (m)RNA. This has resulted in the identification of hundreds of novel RBPs in a diversity of eukaryotic species ranging from humans to yeast. However, poly-adenylation is absent in functional RNA from bacteria and other classes of -eukaryotic- regulatory RNAs. This work was aimed to overcome that limitation by developing a novel and unbiased method for the purification of UV-cross-linked RNPs in living cells: PTex (Phenol Toluol extraction). The purification principle is solely based on physicochemical properties of cross-linked RNPs versus unbound proteins or RNA, and it is impartial towards specific RNA or proteins; enabling for the first time a system-wide analysis of non-poly(A) RNA interacting proteins in both eukaryotic (HEK293) and prokaryotic (Salmonella Typhimurium) cells.

Silizium basierende Technologie bestimmt den technologischen Fortschritt in der Welt und ist weiterhin ein Material für die weitere Entwicklung von Schlüsseltechnologien. Die Änderung der Silizium-Materialeigenschaft der optischen und elektronische Bandlücke durch die Reduktion der Materialdimension auf die Nanometerskala ist dabei von besonders großem Interesse. Die meisten Silizium-Nanomaterialien bestehen aus Punkt-, Kugel- oder Drahtformen. Ein relativ neues Materialsystem sind dreidimensionale, durchdringende, Nano-Komposit Netzwerke aus Silizium in einer Siliziumdioxid Matrix. Die vorliegende Arbeit untersucht die Entstehung von dreidimensionalen Silizium-Nanokomposit-Netzwerken durch Abscheidung eines siliziumreichen Siliziumoxids(SiOx, mit x<2) und anschlieÿender thermischen Behandlung. Hierbei wurden die reaktive Ionenstrahl-Sputterabscheidung (IBSD), sowie das reaktive Magnetronsputtern (RMS) verglichen. Auch wurden die Unterschiede zwischen klassischer Ofen und Millisekunden-Linienlaser Behandlung untersucht. Abgeschiedene und thermisch behandelte Dünnschichten wurden hinsichtlich der integralen Zusammensetzung, Homogenität, Morphologie und Struktur mittels Rutherford-Rückstreuspektroskopie, Ramanspektroskopie, Röntgenbeugung, spektroskopische Ellipsometrie, Photospektrometrie und (Energie gefilterter) Transmissionselektronenmikroskopie untersucht. Abhängig von der Abscheidemethode und des thermischen Ausheilprozesses wurden unterschiedliche Strukturgrößen und Kristallisationsgrade erzeugt. Insbesondere wurde gezeigt, dass während der 13 ms langen Laserbearbeitung (Ofen: 90 min) wesentlich größere Strukturen (laser:~50 nm; oven:~10 nm) mit einer deutlich höheren Kristallinität (laser:~92-99%; oven:~35-80%) entstehen. Darüber hinaus erhält sich die abscheidebedingte Morphologie nach der Ofenbehandlung, verschwindet jedoch nach der Laserprozessierung. Erklärt wurde dies mit einem Prozess über die flüssige Phase während der Laserbearbeitung, im Gegensatz zu einem Festphasenprozess bei der Ofenbehandlung. Abschließend wurde gezeigt, dass absichtlich eingebrachte vertikale und horizontale Schwankungen der Zusammensetzung genutzt werden können, um definierte Silizium Nanonetzwerke mit einer dreidimensionalen quadratischen Netzstruktur herzustellen.:1 Introduction 2 Fundamentals 2.1 The silicon - silicon oxide system 2.1.1 The Si-O phase diagram 2.1.2 Chemical reaction consideration 2.2 Phase separation of binary systems 2.2.1 Phase separation regimes 2.2.2 Diffusion in solids 2.3 Different types of silicon nanostructures 2.3.1 0D - Silicon nanoparticles 2.3.2 1D - Silicon nanowires 2.3.3 3D - Silicon nanonetworks 3 Experimental methods 3.1 SiOx thin film deposition 3.1.1 SiOx thin films by ion beam sputter deposition 3.1.2 SiOx thin films by reactive magnetron sputter deposition 3.1.3 Comparison of ion beam and magnetron sputter deposition 3.2 Thermal processing of as-deposited SiOx thin films 3.2.1 Oven treatment 3.2.2 Laser treatment 3.3 Thin-film characterization 3.3.1 Rutherford backscattering 3.3.2 Spectroscopic ellipsometry and photospectrometry 3.3.3 Raman spectroscopy 3.3.4 X-ray diffraction 3.3.5 Transmission electron microscopy 4 Results 4.1 Accessible SiOx compositions as a function of deposition and annealing method 4.2 Structure and properties of ion beam sputter deposited SiOx thin films before and after thermal processing 4.2.1 Phase- and microstructure of SiO0:6 thin films deposited by ion beam sputter deposition at 450°C 4.2.2 Phase- and microstructure of SiO0.6 thin films deposited by ion beam sputter deposition at room temperature 4.3 Structure and properties of reactive magnetron sputter deposited SiOx thin films before and after thermal processing 4.4 Multilayer SiOx films for the generation of defined squared mesh structures 5 Discussion 5.1 Compositional homogeneity of SiO0:6 thin films before and after thermal treatment 5.2 Phase structure of as-deposited SiOx thin films 5.3 Influence of the thermal treatment on the structural properties of percolated Si:SiO2 nanostructures 5.3.1 Observed structural properties 5.3.2 Origin of different structure sizes - liquid vs. solid state crystallization 5.4 Influence of the deposition temperature during ion beam sputtering on the structural properties of percolated Si:SiO2 nanostructures before and after thermal processing 5.5 Influence of the deposition method on the structural properties of percolated Si:SiO2 nanostructures 5.6 Formation of interface layers and electrical characterization 6 Summary and outlook 6.1 Summary 6.2 Outlook A EFTEM imaging / Silicon-based technology determines the technological progress in the world significantly and is still a material of choice for further development of key technologies. In particular the reduction of silicon structure sizes to a nanometer scale are of great interest. Most silicon nano structures are based on spherical, dot-like or cylindrical, wire-like geometries. A relatively new material system are three dimensional percolated nanocomposite networks of silicon within a silica matrix. To form any of these nano structures fast, room temperature processes are desired which also offer the possibility of structure modification by different process management. The present work studies the formation of three-dimensional silicon nanocomposite networks by the deposition of a silicon rich silicon oxide (SiO x , with x < 2) and subsequent thermal treatment. Thereby, reactive ion beam sputter deposition (IBSD) as well as reactive magnetron sputtering (RMS) was compared. As well, the differences between a conventional oven and a millisecond line-focused diode laser were studied. As-deposited and thermally treated thin films were characterized with regard to the overall mean composition, homogeneity, morphology and structure by Rutherford backscattering, Raman spectroscopy, X-ray diffraction, spectroscopic ellipsometry, photospectrometry as well as cross-sectional and energy-filtered transmission electron microscopy. Depending on the deposition method as well as the thermal treatment process different structure sizes and degrees of crystallization were achieved. Most notably it was found, that during 13 ms laser processing (oven: min. 90 min), much bigger structures (laser: ≈ 50 nm; oven: ≈ 10 nm) with a notably higher degree of crystallization (laser: ≈ 92-99%; oven: ≈ 35-80%) evolve. Moreover, the structure morphology after deposition is preserved during oven treatment but diminishes following laser processing. This was explained by a process via the liquid phase for laser processing in contrast to a solid state process during oven treatment. Finally it was shown, that intentional introduced vertical and horizontal composition fluctuations can be used to form well-defined silicon nano-networks with a three dimensional square mesh structure.:1 Introduction 2 Fundamentals 2.1 The silicon - silicon oxide system 2.1.1 The Si-O phase diagram 2.1.2 Chemical reaction consideration 2.2 Phase separation of binary systems 2.2.1 Phase separation regimes 2.2.2 Diffusion in solids 2.3 Different types of silicon nanostructures 2.3.1 0D - Silicon nanoparticles 2.3.2 1D - Silicon nanowires 2.3.3 3D - Silicon nanonetworks 3 Experimental methods 3.1 SiOx thin film deposition 3.1.1 SiOx thin films by ion beam sputter deposition 3.1.2 SiOx thin films by reactive magnetron sputter deposition 3.1.3 Comparison of ion beam and magnetron sputter deposition 3.2 Thermal processing of as-deposited SiOx thin films 3.2.1 Oven treatment 3.2.2 Laser treatment 3.3 Thin-film characterization 3.3.1 Rutherford backscattering 3.3.2 Spectroscopic ellipsometry and photospectrometry 3.3.3 Raman spectroscopy 3.3.4 X-ray diffraction 3.3.5 Transmission electron microscopy 4 Results 4.1 Accessible SiOx compositions as a function of deposition and annealing method 4.2 Structure and properties of ion beam sputter deposited SiOx thin films before and after thermal processing 4.2.1 Phase- and microstructure of SiO0:6 thin films deposited by ion beam sputter deposition at 450°C 4.2.2 Phase- and microstructure of SiO0.6 thin films deposited by ion beam sputter deposition at room temperature 4.3 Structure and properties of reactive magnetron sputter deposited SiOx thin films before and after thermal processing 4.4 Multilayer SiOx films for the generation of defined squared mesh structures 5 Discussion 5.1 Compositional homogeneity of SiO0:6 thin films before and after thermal treatment 5.2 Phase structure of as-deposited SiOx thin films 5.3 Influence of the thermal treatment on the structural properties of percolated Si:SiO2 nanostructures 5.3.1 Observed structural properties 5.3.2 Origin of different structure sizes - liquid vs. solid state crystallization 5.4 Influence of the deposition temperature during ion beam sputtering on the structural properties of percolated Si:SiO2 nanostructures before and after thermal processing 5.5 Influence of the deposition method on the structural properties of percolated Si:SiO2 nanostructures 5.6 Formation of interface layers and electrical characterization 6 Summary and outlook 6.1 Summary 6.2 Outlook A EFTEM imaging