The use of living radical polymerization methods has shown significant potential to control grafting of polymers from inert polymeric substrates. The objective of this thesis is to create advanced substrates for use in combinatorial chemistry applications through the use of g-radiation as a radical source, and the use of RAFT, ATRP and RATRP living radical techniques to control grafting polymerization. The substrates grafted were polypropylene SynPhase lanterns from Mimotopes and are intended to be used as supports for combinatorial chemistry. ATRP was used to graft polymers to SynPhase lanterns using a technique where the lantern was functionalized by exposing the lanterns to gamma-radiation from a 60Co radiation source in the presence of carbon tetra-bromide, producing short chain polystyrene tethered bromine atoms, and also with CBr4 directly functionalizing the surface. Styrene was then grafted off these lanterns using ATRP. MMA was graft to the surface of SynPhase lanterns, using g-radiation initiated RATRP at room temperature. It was found that the addition of the thermal initiator, AIBN, successfully increased the concentration of radicals to a level where we could achieve proper control of the polymerization. RAFT was used to successfully control the grafting of styrene, acrylic acid and N,N???-dimethylacrylamide to polypropylene SynPhase Lanterns via a -initiated RAFT agent mediated free radical polymerization process using cumyl phenyldithioacetate and cumyl dithiobenzoate RAFT agents. Amphiphilic brush copolymers were produced with a novel combined RAFT and ATRP system. Polystyrene-co-poly(vinylbenzyl chloride) created using gamma-radiation and controlled with the RAFT agent PEPDA was used as a backbone. The VBC moieties were then used as initiator sites for the ATRP grafting of t-BA to give a P(t-BA) brush that was then hydrolyzed to produce a PAA brush polymer. FMOC loading tests were conducted on all these lanterns to assess their effectiveness as combinatorial chemistry supports. It was found that the loading could be controlled by adjusting the graft ratio of the lanterns and had a comparable loading to those commercially produced by Mimotopes.

It was demonstrated for the first time that RAFT polymerizations of NIPAAm can be carried out directly in water at room temperature without photo initiator under UV radiation. Under these conditions, the controlled/living features could be proven for a large range of monomer/RAFT agent ratios. Moreover, even at a monomer conversion exceeding 80%, polymerization control (PDI<1.2) is maintained. It is also demonstrated that the RAFT polymerization of AA can be carried out without photo initiator in water at ambient temperature in the presence of TRITT at short wavelength. At these wavelengths, the controlled/ living characteristics is maintained even at a monomer to polymer conversions exceeding 80%. UV/Vis spectrometry was employed to monitor the functional group (-S(C=S)S-) changes of the employed trithiocarbonate RAFT agent S,S???-Bis(??,?????-dimethyl-acetic acid)-trithiocarbonate (TRITT) in aqueous solution when exposed to UV radiation. It is shown that the degradation pattern of TRITT alone as well as TRITT in the presence of NIPAAm deviate from each other. Surprisingly, it is found that TRITT completely decomposed at 254 nm while the addition of monomer prevented the decomposition of TRITT at the same wavelength. Nuclear magnetic resonance (NMR) techniques were applied to study the decomposition products of TRITT in solution without the addition of monomer. Methanol-d4 was selected as the solvent. In addition, high-resolution soft ionization mass spectrometry techniques were used to map the product species generated during UV radiation induced RAFT polymerizations of NIPAAm and AA in aqueous media, allowing for the tentative assignment of end groups. The NMR analysis suggests that the decomposition of TRITT in methanol-d4 under UV radiation has three cleavage patterns. These three cleavage patterns (described in the current thesis in detail) all occur at the ???S(C=S)S- group, which is the weakest structural unit in TRITT molecule. iii However, polymerization occurs prior to decomposition, if monomer is present. The mass spectrometric analysis suggests that the initial radicals result from the dissociation of TRITT, as well as monomer. Trithiocarbonate end group degradation leading to the formation of thiol terminated chains is also occurring. In the case of NIPAAm polymerization, a peak which may be associated with a cross termination product of the intermediate radical was observed under both 302 nm and 254 nm wavelength irradiation. Interestingly, this peak does not occur in AA polymerization at any wavelength (nor is it expected to form under conventional RAFT conditions and was not observed in previous mass spectrometry studies in thermal or ??-initiated polymerizations of NIPPAm with TRITT) and thus this assignment should be treated as very tentative only.

Glucocorticoids (Gcs) are a commonly used drug to target the glucocorticoid receptor (GR). The GR has a myriad of cellular and physiological effects, however, Gcs are clinically used for the treatment of inflammatory conditions due to the potent anti-inflammatory actions of GR. The anti-inflammatory effects come with serious side effects e.g. metabolic disease. I examine the role of lipid rafts in modulating the anti-inflammatory actions of Gcs, and the role of circadian rhythms in the control of Gc side effects. I tested the role of caveolin-1 (Cav1), a constituent of membrane lipid rafts, and its role in Gc suppression of inflammation. Gene expression analysis of mouse lung tissue showed that genetic depletion of Cav1 (CAV1KO) results in increased transactivation of Gc target genes. The increased Gc action, however, does not result in an increased effect on suppression of inflammation in a model of innate immunity: aerosolised lipopolysaccharide (LPS) induced lung inflammation or in a model of adaptive immunity: Ovalbumin. CAV1KO mice were protected from LPS induced inflammation, despite increased cytokine production. This suggests a differential response to LPS in lung parenchyma and alveolar macrophages dependent on Cav1. CAV1KO results in a pro-inflammatory phenotype in macrophages, and the opposite in parenchymal tissue. These data suggest that while Cav1 is an upstream regulator of Gc response, it does not have a strong enough effect to alter the ability of GR to repress inflammation in vivo. Gc treatment results in a strong metabolic phenotype, with aberrant energy metabolism, insulin resistance and hepatosteaotosis, I investigated how this side effect interacts with circadian rhythms, another key determinant of energy metabolism. Using transcriptomics of whole lung and liver taken during the day or the night, I demonstrate that the metabolic actions of Gc in the liver can be temporally separated, whilst maintaining consistent anti-inflammatory actions in both liver and lung. This temporal gene regulation by Gc is controlled by REV-ERB, a rhythmically expressed, orphan nuclear receptor, part of the core clock machinery, via a direct interaction with GR at key regulatory DNA loci. Genetic deletion of REV-ERB protects mice from the hepatosteotosis associated with Gc treatment. Taken together, these data suggest that Gcs are regulated upstream of the receptor by the core consitutent of membrane lipid rafts; Cav1, which modulates the Gc response in vivo. Also, that the GR action can be controlled by dosing at different times of day, separating the detrimental metabolic effects of Gcs from the beneficial anti-inflammatory effects. This is enabled through a direct interaction between GR and REV-ERB at key gene regulatory sites.

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The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches. / Die Entwicklung von zellulären Lebensformen auf der Erde basiert auf der Entstehung biologischer Lipid-Membranen. Obwohl viele Techniken zur Verfügung stehen, welche es erlauben deren biophysikalische Eigenschaften zu untersuchen, sind die Möglichkeiten, verglichen mit der Analyse von Proteinen, eher eingeschränkt. Ein Grund hierfür, ist die geringe Größe von Lipiden und deren komplexe Zusammenlagerung in eine asymmetrische dicht gepackte Lipiddoppelschicht, welche sich wie eine heterogene zweidimensionale Flüssigkeit verhält. Durch die lokale Anreicherung von Sphingolipiden und Cholesterol sind Membranen in der Lage dynamische, nanoskopische Domänen auszubilden, welche lediglich mit Techniken, welche die optische Auflösungsgrenze umgehen, detailliert untersucht werden können. Ein wesentliches Ziel meiner Arbeit war es, drei Färbeverfahren für Sphingolipide zu vergleichen, erweitern und optimieren, um eine anschliessende Untersuchung mit Hilfe der einzelmolekülsensitiven Technik dSTORM (direct stochastic optical reconstruction microscopy) zu ermöglichen. Zunächst verwendete ich das klassische Färbeverfahren der Immunfluoreszenz, um Sphingolipid-Nanodomänen auf eukaryotischen Zellen mit Hilfe von Farbstoff-gekoppelten Antikörpern zu detektieren und quantifizieren. Dieses Vorgehen ermöglichte es mir, Ceramid-angereicherte Plattformen mit einer Größe von ~ 75nm auf der basalen und apikalen Membran verschiedener Zell-Linien zu identifizieren und charakterisieren. Als nächstes Verfahren verwendete ich die Klick-Chemie, um Sphingolipid-Analoge in lebenden und fixierten Zellen zu untersuchen. Eine Kombination aus Fluoreszenz-Mikroskopie und Anisotropie-Messungen erlaubte es mir Rückschlüsse über deren Zugänglichkeit und Konfiguration innerhalb der Plasmamembran zu ziehen. Hierbei lokalisierten Azid-Gruppen am Ende kurzkettiger Fettsäurereste außerhalb des hydrophoben Membrankerns, wodurch sie mittels membran-undurchlässige Farbstoffe angeklickt werden konnten. Im Gegensatz dazu, waren Azide an längeren Fettsäureresten weniger zugänglich und konjugierte Farbstoffe tauchten tiefer in die Plasmamembran ein. Durch die Einführung photoreaktiver Diazirin-Gruppen oder chemisch modifzierbarer Amin-Gruppen wurden Wege geschaffen, welche eine Immobilisierung und anschließende Analyse mit Hilfe von dSTORM ermöglichen. Schließlich nutzte ich das spezifische Bindeverhalten der nicht toxischen B Untereinheiten von Shiga- (STxB) und Cholera-Toxin (CTxB) aus, um Glycosphingolipid Nanodomänen im Kontext einer Neisseria meningitidis Infektion zu untersuchen. Unter physiologischen Bedingungen waren diese homogen in der Plasmamembran verteilt, jedoch reicherten sich CTxB-detektierbare Ganglioside um eindringende Bakterien an. Darüber hinaus konnte ich einen Zusammenhang zwischen der zellzyklusabhängigen Expression von Glycosphingolipiden und dem Eindringen der Bakterien herstellen. Eine Absättigung der Zucker an der äußeren Membran durch CTxB-Vorbehandlung reduzierte die Anzahl von invasiven Bakterien signifikant und bestätigte die Schlüsselrolle von Gangliosiden bei der Aufnahme von Bakterien. Meine Ergebnisse legen Nahe, dass das Färbeverfahren für Sphingolipide an die jeweilige Fragestellung und Mikroskopietechnik angepasst werden sollte. Im Rahmen dieser Arbeit konnten neue Werkzeuge und Protokolle geschaffen werden, die die Charakterisierung von Sphingolipid-Nanodomänen mittels dSTORM für alle drei Färbeverfahren ermöglichen.

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Today applications are often designed with a modular approach, dividing functionality into micro services rather than relying on a monolithic structure. This requires solutions for decoupled message exchange throughout the distributed system and can be achieved by implementing a message broker. In some cases, it is interesting to make the message broker itself a distributed system, which has been a development path for systems such as RabbitMQ, Apache Kafka, and others. This thesis seeks to understand the alternative approaches to implementing  a distributed solution for the message broker LavinMQ. To find out which algorithm would be the most suitable for this purpose, a comparative analysis of the most common alternatives was performed based on LavinMQ's requirements and a literature review of related comparisons. The results showed that Raft would be the best choice due to its simple but effective nature. To further investigate the consensus approach in LavinMQ, a Raft prototype was developed in the programming language Crystal. The prototype was then evaluated based on correctness and performance in terms of mean replication time and mean election time. The prototype successfully passed the correctness tests, showing that the prototype successfully achieves correctness according to LavinMQ standard. The mean election time results show that the prototype recovers from a leader failure in 216 ms and that the most effective range for the heartbeat timeout is 150 to 300 ms. The replication time test results show that the mean replication time is 84.45 ms and the most efficient interval for message replication is 0.13 ms. The performance results are consistent with the results of related work, however result in overall slower performance and indicate that some additional features and optimizations need to be implemented in order to consider the prototype for practical use.