Gegenstand der vorliegenden Arbeit sind ultradünne Filme organischer Moleküle auf anorganischen Substraten. Adsorbate wurden in Bedeckungen von Submonolagen bis hin zu Multilagen mittels Raster-Tunnel-Mikroskopie und Spektroskopie untersucht. Ergänzt wurde die Strukturaufklärung durch die Beugung niederenergetischer Elektronen (LEED). Im Mittelpunkt stehen in-situ Untersuchungen von im UHV aufgedampften Schichten. Darüberhinaus werden Experimente zur Erzeugung, Abbildung und Manipulation selbstassemblierter Monolagen an der flüssig-fest Grenzfläche beschrieben. Als Modell-Substanzen wurden Coronen, Zinn- und Palladium-Phthalocyanin, Naphthalocyanin sowie Trimesinsäure ausgewählt. Auf Ag(111) konnte bei geringer Bedeckung des planaren Coronens die Wechselwirkung mit dem Oberflächenzustand anhand von stehenden Elektronenwellen nachgewiesen werden. Sowohl auf Ag(111) als auch auf Graphit(0001) konnten für Coronen Monolagen kommensurable Überstrukturen gefunden werden. Für Palladium- und Zinn-Phthalocyanin offenbarte sich auf Ag(111) die Koexistenz von geordneten und ungeordneten Phasen. Wobei es für Palladium-Pc sowohl in der kristallinen als auch in der ungeordneten Phase Indizien für eine höhere Mobilität der Moleküle gibt. Im Fall des nicht planaren Zinn-Pc konnten zwei verschiedene Adsorptionsgeometrien des Moleküls mit dem STM eindeutig unterschieden werden. Die etwas größere Molekülstruktur des Naphthalocyanins bedingt eine Herauf-Skalierung der Einheitszelle, wie sie typischerweise bei Phthalocyaninen beobachtet wird. Ferner ließ sich eine eindeutige Abhängigkeit des intramolekularen Kontrastes vom Vorzeichen der Tunnelspannung durch zweimaligen Wechsel der Polarität im selben Bild zweifelsfrei belegen. Hierbei konnten die submolekularen Strukturen mit simulierten Molekülorbitalen isolierter Moleküle interpretiert werden. Bei den durch Wasserstoffbrücken-Bindungen stabilisierten offenen Netzwerken der Trimesinsäure (TMA) auf Graphit konnte je nach Lösungsmittel an der flüssig-fest Grenzfläche entweder die "Flower"- oder "Chickenwire"-Struktur eingestellt werden. Zudem konnte die Eignung dieser Schicht als Wirt-Gast-System durch die Einlagerung von C60-Fullerenen und Coronen demonstriert werden. Außerdem gelang die Manipulation einzelner C60 im TMA-Templat mit der STM-Spitze in flüssiger Umgebung bei Raumtemperatur.

Als "surface-layer" (S-Layer, SL) bezeichnet man die regelmäßig strukturierten Hüllproteinlagen auf der Oberfläche von etwa 80 % aller bisher bekannten Bakterienspezies. Sie entstehen durch Selbstassemblierung von identischen Proteinuntereinheiten, die wiederum zumeist durch nichtkovalente Wechselwirkungen mit der darunterliegenden Zellwandkomponente verknüpft sind. Trotz ihrer Diversität auf der Ebene der Primärstruktur weisen S-Layer verschiedener Bakterienarten einheitliche physikochemische Merkmale auf. Dazu zählt u.a. die Wiedereinnahme einer hochgradig strukturierten, porösen Proteinschicht nach reversibler Denaturierung. Infolge der Reassemblierung entstehen sowohl in Lösung als auch an Phasengrenzen Proteinassemblate, deren Porenanordnungen die gleiche regelmäßige Symmetrie aufweisen, wie die nativen Hüllproteine auf der Bakterienzelle. Das in seiner Domänenstruktur aufgeklärte Hüllprotein SbsC des mesophilen Bakterienstammes Geobacillus (G.) stearothermophilus ATCC 12980 zeichnet sich durch eine ausgezeichnete Synthetisierbarkeit in E. coli aus. C-terminale Fusionen, die im Falle des verstärkt grün fluoreszierenden Proteins (EGFP) bis zu 240 Aminosäuren umfassen, führten nicht zu einem Verlust der Selbstassemblierung. Darüber hinaus zeigen in vitro gebildete SbsC-Assemblate eine außergewöhnliche Stabilität gegenüber hohen Ethanolkonzentrationen. Die durch gerichtete Mutagenese erzeugten SbsC-Fusionsproteine SbsC(aa 31-1099)-HspA und SbsC(aa 31-1099)-12His besitzen in assemblierter Form im Vergleich mit dem unmodifizierten Protein eine bis zu zweimal höhere Bindungsaffinität gegenüber Platinionen. In denaturierter Form waren beide Fusionsproteine in der Lage, Nickelionen zu komplexieren. In der vorliegenden Arbeit wurde erstmals ein SL-Protein in einem eukaryontischen Mikroorganismus produziert. Das in der Hefe S. cerevisiae gebildete Fusionsprotein SbsC(aa 31-1099)-EGFP assembliert dabei im Cytosol der Wirtszellen zu röhrenförmigen Assemblaten mit regelmäßiger Symmetrie. Das bisher unbekannte SL-Protein des Stammes G. stearothermophilus DSM 13240 wurde erfolgreich heterolog in E. coli exprimiert. Die Vorläuferform besitzt im Vergleich zum maturen Protein ein 31 aa umfassendes Sekretionssignal am extremen N-Terminus. Sowohl das authentische Protein als auch das heterolog in E. coli exprimierte Vorläuferprotein zeigen eine dem SbsC-Protein vergleichbare Reassemblierungscharakteristik. Im Gegensatz dazu führte die Verkürzung der N-terminalen 30 Aminosäuren des als S13240 bezeichneten Hüllproteins im heterologen System zu einem irreversiblen Verlust der Fähigkeit zur Selbstassemblierung.

Die vorliegende Arbeit befasst sich mit synthetischen Ansätzen zur Verbesserung der Handhabung von Pd-Nanopartikeln in der heterogenen Umwelt- und Elektrokatalyse. Nanopartikuläres Pd an Magnetit sowie an Silica-Sphären mit Magnetit-Kern erreichten eine hohe Aktivität bei der Dechlorierung von Hexachlorbenzol. Im Gegensatz zu ungeträgerten Nanopartikeln gelang die Abtrennung jener mit einem Magnet aus der Reaktionslösung. Weitere Untersuchungen ergaben, dass die Shewanella oneidensis eine heterogene Keimbildung im mikrobiellen Herstellungsverfahren von Pd-Nanomaterialien vermittelte. Die Mikroorganismen waren vermutlich nicht aktiv am Elektronenübergang beteiligt. Die partiell aggregierten Produkte des mikrobiellen Verfahrens ließen sich zur Herstellung von Aerogelen durch Selbstassemblierung verwenden. Elektrodenfilme aus mikrobiell als auch chemisch synthetisiertem nanopartikulären Pd zeigten ähnliche Eigenschaften bei der elektrochemischen Oxidation von Methanol. Darüber hinaus ermöglichte die Anwendung der fraktalen Dimension strukturelle Veränderungen abhängig von Verfahrensparametern bei der Selbstassemblierung festzustellen.:Inhaltsverzeichnis i Abbildungsverzeichnis iii Tabellenverzeichnis v Einleitung 1 1. Grundlagen 5 1.1. Eigenbewegung von Nanopartikeln in Suspension 6 1.2. Die DLVO-Theorie der Stabilität von lyophoben Kolloiden 7 1.3. Aggregation und die fraktale Dimension 11 1.4. Lichtstreuung an Kolloiden 15 1.5. Transmissionselektronenmikroskopie 19 1.6. Röntgenpulverdiffraktometrie 24 2. Edelmetall-Nanopartikel 27 2.1. Synthese von Palladium-Nanopartikeln 30 2.1.1. Reduktion mit Natriumborhydrid 30 2.1.2. Reduktion mit Citrat und Dicarboxyaceton 31 2.1.3. Keimvermitteltes Wachstum 33 2.2. pH- und Temperatur-Stabilität der Suspensionen 35 2.3. Integration in Polymerbeschichtungen 37 2.4. Resümee 41 3. Mikrobiell hergestellte Pd-Nanostrukturen 43 3.1. Dissimilatorische Metall-Reduktion 44 3.2. Eigenschaften von mikrobiellem Pd 49 3.2.1. Herstellung und Präparation 49 3.2.2. Strukturelle Eigenschaften 51 3.2.3. Umsatz und chemische Zusammensetzung 56 3.2.4. Untersuchung der organischen Bestandteile 60 3.2.5. Eigenschaften der Suspensionen 64 3.3. Kontrollversuche zur mikrobiellen Herstellung 66 3.4. Dechlorierung von Hexachlorbenzol 69 3.5. Resümee 71 4. Palladium-Magnetit-Nanokatalysatoren 75 4.1. Synthese von Magnetit-Nanopartikeln 78 4.2. Kombination von Magnetit- und Pd-Nanopartikeln 81 4.3. Abscheidung von Pd an Magnetit 84 4.4. Zwischenfazit 85 4.5. Oberflächen-modifizierte Pd-Magnetit-Komposite 86 4.6. Dechlorierung von Hexachlorbenzol 89 4.7. Resümee 91 5. Selbstassemblierung von Edelmetallnanopartikeln 93 5.1. Verfahren zur Herstellung von Pd-Hydrogelen 96 5.1.1. Variation der Verfahrensparameter 97 5.1.2. Einfluss von Temperatur und Anreicherungsfaktor 99 5.2. Aerogel-Monolithe 103 5.3. Netzwerkstrukuren aus mikrobiellem Pd 105 5.4. Elektrochemische Oxidation von Methanol 106 5.5. Resümee 111 Zusammenfassung und Ausblick 113 A. Terminologie zu Kolloiden, Aggregaten, Gelen & Co. 115 B. Experimentelle Methoden 117 B.1. Synthesevorschriften 118 B.2. Charakterisierungsmethoden 128 B.3. Elektrochemische Untersuchungen an Aerogel-Elektroden 132 Literaturverzeichnis 135 / The present work deals with synthetic approaches for the implementation of Pd-based materials in environmental and electrocatalysis. Nanoparticles of Pd either coupled to magnetite or to silica-spheres with a magnetic core showed a high activity in the dechlorination of hexachlorbenzene similar to unsupported nanoparticles. However, in contrast to unsupported nanoparticles they could be separated from the reaction solution by a magnet. Structural and chemical properties of Pd nanomaterials from a microbial synthesis were comparatively investigated. The results lead to the conclusion that the Shewanella oneidensis were not actively involved into the electron transfer and the microorganisms acted more as a substrate for heterogeneous seeding. Partially nanostructured Pd-aggregates from the microbial synthesis were further subjected to self-assembly to form noble metal aerogels. Electrode films made of both microbially and synthetically produced Pd aerogels showed similar structural and electrochemical properties in the electrooxidation of methanol. Finally, the fractal dimension was implemented as a parameter allowing to monitor the evolution of both aerogel structure and its density during the process of selfassembly.:Inhaltsverzeichnis i Abbildungsverzeichnis iii Tabellenverzeichnis v Einleitung 1 1. Grundlagen 5 1.1. Eigenbewegung von Nanopartikeln in Suspension 6 1.2. Die DLVO-Theorie der Stabilität von lyophoben Kolloiden 7 1.3. Aggregation und die fraktale Dimension 11 1.4. Lichtstreuung an Kolloiden 15 1.5. Transmissionselektronenmikroskopie 19 1.6. Röntgenpulverdiffraktometrie 24 2. Edelmetall-Nanopartikel 27 2.1. Synthese von Palladium-Nanopartikeln 30 2.1.1. Reduktion mit Natriumborhydrid 30 2.1.2. Reduktion mit Citrat und Dicarboxyaceton 31 2.1.3. Keimvermitteltes Wachstum 33 2.2. pH- und Temperatur-Stabilität der Suspensionen 35 2.3. Integration in Polymerbeschichtungen 37 2.4. Resümee 41 3. Mikrobiell hergestellte Pd-Nanostrukturen 43 3.1. Dissimilatorische Metall-Reduktion 44 3.2. Eigenschaften von mikrobiellem Pd 49 3.2.1. Herstellung und Präparation 49 3.2.2. Strukturelle Eigenschaften 51 3.2.3. Umsatz und chemische Zusammensetzung 56 3.2.4. Untersuchung der organischen Bestandteile 60 3.2.5. Eigenschaften der Suspensionen 64 3.3. Kontrollversuche zur mikrobiellen Herstellung 66 3.4. Dechlorierung von Hexachlorbenzol 69 3.5. Resümee 71 4. Palladium-Magnetit-Nanokatalysatoren 75 4.1. Synthese von Magnetit-Nanopartikeln 78 4.2. Kombination von Magnetit- und Pd-Nanopartikeln 81 4.3. Abscheidung von Pd an Magnetit 84 4.4. Zwischenfazit 85 4.5. Oberflächen-modifizierte Pd-Magnetit-Komposite 86 4.6. Dechlorierung von Hexachlorbenzol 89 4.7. Resümee 91 5. Selbstassemblierung von Edelmetallnanopartikeln 93 5.1. Verfahren zur Herstellung von Pd-Hydrogelen 96 5.1.1. Variation der Verfahrensparameter 97 5.1.2. Einfluss von Temperatur und Anreicherungsfaktor 99 5.2. Aerogel-Monolithe 103 5.3. Netzwerkstrukuren aus mikrobiellem Pd 105 5.4. Elektrochemische Oxidation von Methanol 106 5.5. Resümee 111 Zusammenfassung und Ausblick 113 A. Terminologie zu Kolloiden, Aggregaten, Gelen & Co. 115 B. Experimentelle Methoden 117 B.1. Synthesevorschriften 118 B.2. Charakterisierungsmethoden 128 B.3. Elektrochemische Untersuchungen an Aerogel-Elektroden 132 Literaturverzeichnis 135

Während die Menschheit bereits seit Jahrtausenden von der Brillanz von Gold und Silber im ausgedehnten Zustand fasziniert ist, bestechen ihre nanoskaligen Gegenstücke mit ihren wundervollen Farben und ihrer breiten Farbpalette. Motiviert durch diese Farben versuchten Wissenschaftler das zugrundeliegende Phänomen dieser Farben, die lokalisierte Oberflächenplasmonenresonanz, zu verstehen, was den Grundstein der Forschung im Bereich Plasmonik legte. Für die Anwendung muss diese lokalisierte Oberflächenplasmonenresonanz umfassend durch Änderung von Material, Größe, Form, Anordnung und Umgebung der Nanopartikel angepasst werden. Es scheint unausweichlich, dass dieser komplexe Parameterraum nur durch die Anwendung von künstlicher Intelligenz verstanden werden kann und die Eigenschaften von solchen komplexen Strukturen — in isolierten oder gekoppelten Strukturen — angepasst werden können. Diese Dissertation untersucht die Anpassung der Plasmonenresonanz in isolierten und gekoppelten Nanostrukturen durch Kombination von Kolloidsynthese, Anordnung und künstlicher Intelligenz. Der erste Teil behandelt die Synthese von Goldnanopartikeln mit Unterstützung des maschinellen Lernens. Durch die Nutzung von baumbasierten Lernalgorithmen wird die Wichtigkeit von bestimmten Syntheseparametern und dessen Auswirkungen auf die finalen Eigenschaften der synthetisierten Nanopartikel beleuchtet. Dabei wird gezeigt, dass der Algorithmus die zugrundeliegenden Chemiekonzepte der Synthese lernen kann, ohne sie explizit zu lehren, sondern ausschließlich durch das Lernen der Synthese- und Charakterisierungsdaten. Der zweite Teil fokussiert sich auf die Anordnung und die optische Charakterisierung von heterogenen Ketten aus Gold- und Silbernanopartikeln. Dabei wird gezeigt, dass nahezu jede Konfiguration bis zu einer Länge von 17 auf einem Quadratzentimeter durch Beschränkungsanordnung angeordnet werden können. Dies löst die synthetische Herausforderung des exponentiell wachsenden Parameterraums, der durch die Einführung eines zweiten Bausteins in der Kette eröffnet wurde. Allerdings ist die Charakterisierung zeitaufwändig und daher für die enorme Menge an Konfigurationen nicht realisierbar. Infolgedessen können elektrodynamische Simulationen hier helfen und diese Lücke schließen. Leider sind diese Simulationen durch ihre Berechnungskomplexität beschränkt, was jedoch durch den Einsatz von rekurrenten neuronalen Netzen im letzten Teil der Dissertation abgemildert wird. Letztlich zeigt diese Dissertation wie innovative Zugänge zu diesen Herausforderungen die Synthese, Charakterisierung und Verständnis von plasmonischen Nanostrukturen ermöglichen und wie die Plasmonenresonanz in Bezug zu ihren Anwendungen angepasst werden kann. / While the brilliance of gold and silver has fascinated humankind for millennia in their bulk state, their nanoscale counterparts captivate with their beautiful colors and broad color range. Motivated by these colors, researchers pursued to understand the underlying phenomenon of these colors, the localized surface plasmon resonance, which sparked the research in the field of plasmonics. In order to be useful, this localized surface plasmon resonance needs to be extensively engineered by variation of material, size, shape, arrangement, and surrounding of the nanoparticles. To explore this complex parameter space, the use of the emerging technology of artificial intelligence seems inevitable to understand and engineer the properties of such complex structures — either in isolated or coupled structures. This thesis investigates the plasmon resonance engineering in isolated and coupled nanostructures by combining colloidal synthesis, assembly, and artificial intelligence. The first part covers the machine learning assisted synthesis of gold nanoparticles, which aims to use tree-based learning algorithms to elucidate the importance of certain synthesis parameters and how they affect the final characteristics of the synthesized nanoparticles. It is shown that the algorithm can learn the underlying concepts of the chemistry of the synthesis without explicitly teaching the algorithm, but purely learning from data that was gathered during synthesis and characterization. The second part focuses on the assembly and optical characterization of heterogeneous chains composed of gold and silver nanospheres. Applying confinement assembly, virtually any configuration up to a length of 17 can be assembled on a square centimeter, which solves the synthetic challenge that is imposed by the exponentially growing configuration space due to the introduction of a second building block in the chain. However, characterization is time-consuming and therefore not feasible for vast amounts of configurations, thus only a tiny subsample is selected for electromagnetic characterization. Consequently, electrodynamicsimulations aid this task and try to fill the gap. Unfortunately, these simulations are limited by computational complexity; however, the use of recurrent neural networks enables to mitigate this problem, as shown in the final part of this thesis. In the end, this thesis showcases how innovative approaches to these challenges can enable the synthesis, characterization, and understanding of plasmonic nanostructures and how they can be used to engineer the plasmonic resonance in accordance with their desired applications.

Janus particles, named after the two faced Roman god Janus, possess unique asymmetry and combine two distinct functions at their opposite sides, allowing them to target complex self-assembled architectures and materials inaccessible for homogeneous building blocks. In this study, three areas regarding the topic of Janus particles were explored: the synthesis of Janus particles, their (self-) assembly, and applications. In the first part of the work, we have drawn our attention to the optimization of the synthetic procedures concerning the preparation of Janus particles and to the extending of the current Janus particle library by adding new geometries to the list. In the case of spherical Janus particles, we have developed an easy approach to tailor the Janus ratio of the resulting particles, thus, extending the possibilities of the Pickering emulsion approach for the creation of a variety of Janus particle architectures. Additionally, a new methodology was employed to measure directly and in situ the position/contact angle of the prepared Janus particles with different Janus ratios at a water-oil interface. It was further concluded that having simply two different functionalities on a particle surface does not necessarily imply amphiphilic behavior: only in the case of large wettability contrasts our particles were in a true Janus regime. In the case of platelet-like Janus particles, we have developed a completely new approach for their large-scale synthesis, which involved a reduced number of steps compared to the spherical Janus particles. In the second part of the work, the assembly behavior of various kinds of functional spherical Janus particles was investigated depending on the nature of the Janus particles and the surrounding media conditions. Oppositely charged, uncharged amphiphilic, and charged amphiphilic Janus particles were fabricated comprising different responsive polymers on their surface, and their assembly was investigated depending on the pH value of the dispersion, the ionic strength, or the solvent. It was found that, under specific conditions, the Janus particles formed hierarchical chain-like structures in solutions, which were not observed in the case of the homogeneous particle mixtures. The obtained results indicate that the fundamental understanding of the Janus particle assembly mechanisms is crucial for the programmed formation of desired structures. In the third part of the work, we have focused on the applications of our developed hybrid hairy Janus particles and proposed two main directions that would benefit from the unique properties or architecture of the Janus particles. The first direction is based on the exploitation of the superior interfacial activity of the Janus particles and their use for interfacial catalysis. The second proposed direction for the application of Janus particles is based on their use as building blocks for functional structured surfaces. The prepared surfaces were thoroughly characterized and tested for their performance toward anti-icing as well as anti-fouling applications. Ultimately, the developed functional surfaces based on Janus particles as building blocks are very promising for their future application in the coating technology.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

This thesis is focused on the design and development of an approach, allowing the fabrication of biocompatible/biodegradable self-rolled polymer tubes, which are sensitive to stimuli at physiological conditions, can be homogenously filled with cells and are able to self-assemble into a complex 3D construct with uniaxially aligned pores. These constructs are aimed to recreate the microstructure of tissues with structural anisotropy, such as of muscles and bones. The approach consists of two steps of self-assembly. As a first step, cells are adsorbed on the top of an unfolded bilayer; triggered rolling results in a parallel encapsulation of cells inside the tubes. As a second step, the formed self-rolled tubes with encapsulated cells can be assembled in a uniaxial tubular scaffold. Three polymer systems were designed and investigated in the present work in order to allow triggered folding of the bilayer. These systems allow either reversible or irreversible tube formation. The possibility to encapsulate microobjects inside self-rolled polymer tubes was demonstrated on the example of silica particles, yeast cells and mammalian cells. At conditions when bilayer film is unfolded, particles or cells were deposited from their aqueous dispersion on the top of bilayer. An appropriate change of conditions triggers folding of the bilayer and results in encapsulation of particles or cells inside the tubes. One way swelling of an active polymer allows irreversible encapsulation of cells in a way that tubes do not unroll and cells cannot escape. It was demonstrated that encapsulated cells can proliferate and divide inside the tubes for a long period of time. Since used polymers are optically transparent, encapsulated cells can be easily observed using optical and fluorescent microscopy. Reversible swelling of an active polymer provides the possibility to release encapsulated objects. It was demonstrated that in aqueous media microtubes possessing small amount of negatively charged groups on external walls self-assemble in the presence of oppositely charged microparticles that results in a formation of 3D constructs. In obtained aggregates tubes and therefore pores were well-aligned and the orientation degree was extremely high. Moreover, the approach allows the design of porous materials with complex architectures formed by tubes of different sorts. The assembly of cell-laden microtubes results in a formation of uniaxial tubular scaffold homogeneously filled with cells. The results presented in this work demonstrate that the proposed approach is of practical interest for biotechnological applications. Self-rolled tubes can be filled with cells during their folding providing the desired homogeneity of filling. Individual tubes of different diameters could be used to investigate cell behaviour in confinement in conditions of structural anisotropy as well as to mimic blood vessels. Due to their directionality tubes could be used to guide the growth of cells that is of interest for regeneration of neuronal tissue. Reversibly foldable films allow triggered capture and release of the cells that could be implemented for controlled cell delivery. In perspective, self-assembled 3D constructs with aligned pores could be used for bottom-up engineering of the scaffolds, mimicking such tissues as cortical bone and skeletal muscle, which are characterized by repeating longitudinal units. Such constructs can be also considered as a good alternative of traditional 2D flat cell culture.

Janus particles, named after the two faced Roman god Janus, possess unique asymmetry and combine two distinct functions at their opposite sides, allowing them to target complex self-assembled architectures and materials inaccessible for homogeneous building blocks. In this study, three areas regarding the topic of Janus particles were explored: the synthesis of Janus particles, their (self-) assembly, and applications. In the first part of the work, we have drawn our attention to the optimization of the synthetic procedures concerning the preparation of Janus particles and to the extending of the current Janus particle library by adding new geometries to the list. In the case of spherical Janus particles, we have developed an easy approach to tailor the Janus ratio of the resulting particles, thus, extending the possibilities of the Pickering emulsion approach for the creation of a variety of Janus particle architectures. Additionally, a new methodology was employed to measure directly and in situ the position/contact angle of the prepared Janus particles with different Janus ratios at a water-oil interface. It was further concluded that having simply two different functionalities on a particle surface does not necessarily imply amphiphilic behavior: only in the case of large wettability contrasts our particles were in a true Janus regime. In the case of platelet-like Janus particles, we have developed a completely new approach for their large-scale synthesis, which involved a reduced number of steps compared to the spherical Janus particles. In the second part of the work, the assembly behavior of various kinds of functional spherical Janus particles was investigated depending on the nature of the Janus particles and the surrounding media conditions. Oppositely charged, uncharged amphiphilic, and charged amphiphilic Janus particles were fabricated comprising different responsive polymers on their surface, and their assembly was investigated depending on the pH value of the dispersion, the ionic strength, or the solvent. It was found that, under specific conditions, the Janus particles formed hierarchical chain-like structures in solutions, which were not observed in the case of the homogeneous particle mixtures. The obtained results indicate that the fundamental understanding of the Janus particle assembly mechanisms is crucial for the programmed formation of desired structures. In the third part of the work, we have focused on the applications of our developed hybrid hairy Janus particles and proposed two main directions that would benefit from the unique properties or architecture of the Janus particles. The first direction is based on the exploitation of the superior interfacial activity of the Janus particles and their use for interfacial catalysis. The second proposed direction for the application of Janus particles is based on their use as building blocks for functional structured surfaces. The prepared surfaces were thoroughly characterized and tested for their performance toward anti-icing as well as anti-fouling applications. Ultimately, the developed functional surfaces based on Janus particles as building blocks are very promising for their future application in the coating technology.

S-layer proteins of S. ureae ATCC 13881 form on the cell surface an S-layer lattice with p4 square type symmetry and a period of about 13.5 nm. These lattices were shown to be the excellent nanotemplates for deposition of regular metal clusters. The synthesis of the S. ureae S-layer protein is highly efficient, the protein accounts for approximately 10-15 % of the total cell protein content, judged by the SDS-PAGE results. Besides, the S-layer protein production is tightly regulated, since only negligible amounts of S-layer proteins are observed in the medium at different cell growth phases. At the same time, mechanisms of the regulation of S-layer protein synthesis are poorly understood. As several hundreds of S-layer proteins are produced per second during the cell growth, the S-layer gene promoters are among the strongest prokaryotic promoters at all. However, little is known about factors regulating the expression of S-layer genes, furthermore, no experimental identification of other upstream regulatory sequences except for -35/-10 and RBS sequences was presented to our knowledge to date. A sequence of the S-layer gene of S. ureae ATCC 13881, encoding the previously described S-layer protein, was identified in this work by combination of different approaches. The largest part of the gene, excluding its upstream regulatory and ORF 5’ regions, was isolated from a genomic library by hybridization. The sequence of the isolated fragment proved to contain additionally an 1.9 kb non-coding region and an incomplete 0.8 kb ORF region in its 3’-part. No RBS sequence and apparent promoter regions could be identified in front of the latter sequence, suggesting that it might represent a pseudogene sequence. The sequences of the 5’ and upstream regions of the S. ureae ATCC 13881 S-layer gene were identified by combination of PCR-sequencing and chromosome walking. Totally, a sequence of the 6.4 kb long region of S. ureae genomic DNA was established. The sequence of the S. ureae S-layer protein was deduced from the respective gene sequence and agreed with the peptide sequences, obtained after N-terminal sequencing of tryptic peptides of the S. ureae ATCC 13881 S-layer protein. For the protein the name SslA was proposed, which is an abbreviation for “Sporosarcina ureae S-layer protein A”. Several specific features were observed in gene organisation of sslA, which are also characteristic for other S-layer genes. The distance between the -35/-10 region and the ATG initiation codon is unusually long and a 41 bp palindromic sequence is present in the immediate vicinity of the -35/-10 region. Besides, a distant location of the rho-independent transcription terminator, which is 647 bp remote from the stop codon, will result in the mRNA transcripts with unusually long trailer region. Both the long 5’ UTR and the long 3’ trailer may have a regulatory function, either by conferring increased mRNA stability and/or by affecting translation efficiency. Potentially these sequences may define the binding sites of regulatory proteins. For example, palindromic sequences constitute the regulatory sites in several bacterial operons and may act as the binding sites of regulatory dimeric proteins. In respect to the conservation of the sslA sequence high similarity to the sequences of other functional S-layer genes, especially the slfA and slfB genes of B. sphaericus, was observed, whereas the results of phylogenetic analysis support the hypothesis that S-layer genes may have evolved via the lateral gene transfer. Based on the sslA sequence, several recombinant proteins with truncations of the terminal protein parts or C-terminal fusion of either EGFP or histidine tags were constructed. For all the truncated or EGFP-fusion SslA derivatives high level overexpression in E. coli was possible. For native SslA a moderate level of expression was observed suggesting that its high intracellular concentration may downregulate the protein synthesis. Interestingly, fluorescence microscopy indicates the same intracellular localization for heterologously produced recombinant proteins with fusions of EGFP either to the precursor or to the native SslA protein, suggesting that SslA secretion signal is not functional in E. coli. Heterologously produced SslA derivatives with truncations of N-, C- or both N- and C-terminal parts were shown to self- assemble in vitro, although the size of self-assembly structures was different from that observed upon the self-assembly of the native SslA. In the latter case extended self-assembly layers with the size up to 5x10 µm were observed, with a surface area of up to two orders of magnitude higher than that of S-layer patches, routinely isolated from S. ureae surface. Dependent on the applied recrystallization conditions preferential formation of single- or multilayer self-assembly structures was observed.