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Institute
- Theodor-Boveri-Institut für Biowissenschaften (168)
- Graduate School of Life Sciences (97)
- Institut für Anorganische Chemie (60)
- Institut für Psychologie (54)
- Medizinische Klinik und Poliklinik I (54)
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- Institut für Informatik (43)
- Klinik und Poliklinik für Allgemein-, Viszeral-, Gefäß- und Kinderchirurgie (Chirurgische Klinik I) (39)
Sonstige beteiligte Institutionen
- Ökologische Station Fabrikschleichach (2)
- Airbus Defence and Space GmbH (1)
- Apotheke, Universitätsklinikum Würzburg (1)
- Biomedical Center Munich, Department of Physiological Chemistry, Ludwig-Maximilians-Universität München (1)
- Bundeswehr Institute of Radiobiology affiliated to the University of Ulm, Munich, Germany (1)
- Clinical Trial Center (CTC) / Zentrale für Klinische Studien Würzburg (ZKSW) (1)
- Cologne Game Lab (1)
- DFG Forschungsgruppe 2757 / Lokale Selbstregelungen im Kontext schwacher Staatlichkeit in Antike und Moderne (LoSAM) (1)
- Department of Cellular Biochemistry, University Medical Center Göttingen (1)
- Department of English, Jamia Millia Islamia (A Central University), New Delhi (1)
These days, we are living in a digitalized world. Both our professional and private lives are pervaded by various IT services, which are typically operated using distributed computing systems (e.g., cloud environments). Due to the high level of digitalization, the operators of such systems are confronted with fast-paced and changing requirements. In particular, cloud environments have to cope with load fluctuations and respective rapid and unexpected changes in the computing resource demands. To face this challenge, so-called auto-scalers, such as the threshold-based mechanism in Amazon Web Services EC2, can be employed to enable elastic scaling of the computing resources. However, despite this opportunity, business-critical applications are still run with highly overprovisioned resources to guarantee a stable and reliable service operation. This strategy is pursued due to the lack of trust in auto-scalers and the concern that inaccurate or delayed adaptations may result in financial losses.
To adapt the resource capacity in time, the future resource demands must be "foreseen", as reacting to changes once they are observed introduces an inherent delay. In other words, accurate forecasting methods are required to adapt systems proactively. A powerful approach in this context is time series forecasting, which is also applied in many other domains. The core idea is to examine past values and predict how these values will evolve as time progresses. According to the "No-Free-Lunch Theorem", there is no algorithm that performs best for all scenarios. Therefore, selecting a suitable forecasting method for a given use case is a crucial task. Simply put, each method has its benefits and drawbacks, depending on the specific use case. The choice of the forecasting method is usually based on expert knowledge, which cannot be fully automated, or on trial-and-error. In both cases, this is expensive and prone to error.
Although auto-scaling and time series forecasting are established research fields, existing approaches cannot fully address the mentioned challenges: (i) In our survey on time series forecasting, we found that publications on time series forecasting typically consider only a small set of (mostly related) methods and evaluate their performance on a small number of time series with only a few error measures while providing no information on the execution time of the studied methods. Therefore, such articles cannot be used to guide the choice of an appropriate method for a particular use case; (ii) Existing open-source hybrid forecasting methods that take advantage of at least two methods to tackle the "No-Free-Lunch Theorem" are computationally intensive, poorly automated, designed for a particular data set, or they lack a predictable time-to-result. Methods exhibiting a high variance in the time-to-result cannot be applied for time-critical scenarios (e.g., auto-scaling), while methods tailored to a specific data set introduce restrictions on the possible use cases (e.g., forecasting only annual time series); (iii) Auto-scalers typically scale an application either proactively or reactively. Even though some hybrid auto-scalers exist, they lack sophisticated solutions to combine reactive and proactive scaling. For instance, resources are only released proactively while resource allocation is entirely done in a reactive manner (inherently delayed); (iv) The majority of existing mechanisms do not take the provider's pricing scheme into account while scaling an application in a public cloud environment, which often results in excessive charged costs. Even though some cost-aware auto-scalers have been proposed, they only consider the current resource demands, neglecting their development over time. For example, resources are often shut down prematurely, even though they might be required again soon.
To address the mentioned challenges and the shortcomings of existing work, this thesis presents three contributions: (i) The first contribution-a forecasting benchmark-addresses the problem of limited comparability between existing forecasting methods; (ii) The second contribution-Telescope-provides an automated hybrid time series forecasting method addressing the challenge posed by the "No-Free-Lunch Theorem"; (iii) The third contribution-Chamulteon-provides a novel hybrid auto-scaler for coordinated scaling of applications comprising multiple services, leveraging Telescope to forecast the workload intensity as a basis for proactive resource provisioning. In the following, the three contributions of the thesis are summarized:
Contribution I - Forecasting Benchmark
To establish a level playing field for evaluating the performance of forecasting methods in a broad setting, we propose a novel benchmark that automatically evaluates and ranks forecasting methods based on their performance in a diverse set of evaluation scenarios. The benchmark comprises four different use cases, each covering 100 heterogeneous time series taken from different domains. The data set was assembled from publicly available time series and was designed to exhibit much higher diversity than existing forecasting competitions. Besides proposing a new data set, we introduce two new measures that describe different aspects of a forecast. We applied the developed benchmark to evaluate Telescope.
Contribution II - Telescope
To provide a generic forecasting method, we introduce a novel machine learning-based forecasting approach that automatically retrieves relevant information from a given time series. More precisely, Telescope automatically extracts intrinsic time series features and then decomposes the time series into components, building a forecasting model for each of them. Each component is forecast by applying a different method and then the final forecast is assembled from the forecast components by employing a regression-based machine learning algorithm. In more than 1300 hours of experiments benchmarking 15 competing methods (including approaches from Uber and Facebook) on 400 time series, Telescope outperformed all methods, exhibiting the best forecast accuracy coupled with a low and reliable time-to-result. Compared to the competing methods that exhibited, on average, a forecast error (more precisely, the symmetric mean absolute forecast error) of 29%, Telescope exhibited an error of 20% while being 2556 times faster. In particular, the methods from Uber and Facebook exhibited an error of 48% and 36%, and were 7334 and 19 times slower than Telescope, respectively.
Contribution III - Chamulteon
To enable reliable auto-scaling, we present a hybrid auto-scaler that combines proactive and reactive techniques to scale distributed cloud applications comprising multiple services in a coordinated and cost-effective manner. More precisely, proactive adaptations are planned based on forecasts of Telescope, while reactive adaptations are triggered based on actual observations of the monitored load intensity. To solve occurring conflicts between reactive and proactive adaptations, a complex conflict resolution algorithm is implemented. Moreover, when deployed in public cloud environments, Chamulteon reviews adaptations with respect to the cloud provider's pricing scheme in order to minimize the charged costs. In more than 400 hours of experiments evaluating five competing auto-scaling mechanisms in scenarios covering five different workloads, four different applications, and three different cloud environments, Chamulteon exhibited the best auto-scaling performance and reliability while at the same time reducing the charged costs. The competing methods provided insufficient resources for (on average) 31% of the experimental time; in contrast, Chamulteon cut this time to 8% and the SLO (service level objective) violations from 18% to 6% while using up to 15% less resources and reducing the charged costs by up to 45%.
The contributions of this thesis can be seen as major milestones in the domain of time series forecasting and cloud resource management. (i) This thesis is the first to present a forecasting benchmark that covers a variety of different domains with a high diversity between the analyzed time series. Based on the provided data set and the automatic evaluation procedure, the proposed benchmark contributes to enhance the comparability of forecasting methods. The benchmarking results for different forecasting methods enable the selection of the most appropriate forecasting method for a given use case. (ii) Telescope provides the first generic and fully automated time series forecasting approach that delivers both accurate and reliable forecasts while making no assumptions about the analyzed time series. Hence, it eliminates the need for expensive, time-consuming, and error-prone procedures, such as trial-and-error searches or consulting an expert. This opens up new possibilities especially in time-critical scenarios, where Telescope can provide accurate forecasts with a short and reliable time-to-result.
Although Telescope was applied for this thesis in the field of cloud computing, there is absolutely no limitation regarding the applicability of Telescope in other domains, as demonstrated in the evaluation. Moreover, Telescope, which was made available on GitHub, is already used in a number of interdisciplinary data science projects, for instance, predictive maintenance in an Industry 4.0 context, heart failure prediction in medicine, or as a component of predictive models of beehive development. (iii) In the context of cloud resource management, Chamulteon is a major milestone for increasing the trust in cloud auto-scalers. The complex resolution algorithm enables reliable and accurate scaling behavior that reduces losses caused by excessive resource allocation or SLO violations. In other words, Chamulteon provides reliable online adaptations minimizing charged costs while at the same time maximizing user experience.
Fluorescence microscopy is a form of light microscopy that has developed during the 20th century and is nowadays a standard tool in Molecular and Cell biology for studying the structure and function of biological molecules. High-resolution fluorescence microscopy techniques, such as dSTORM (direct Stochastic Optical Reconstruction Microscopy) allow the visualization of cellular structures at the nanometre scale (10−9 m). This has already made it possible to decipher the composition and function of various biopolymers, such as proteins, lipids and nucleic acids, up to the three-dimensional (3D) structure of entire organelles. In practice, however, it has been shown that these imaging methods and their further developments still face great challenges in order to achieve an effective resolution below ∼ 10 nm. This is mainly due to the nature of labelling biomolecules. For the detection of molecular structures, immunostaining is often performed as a standard method. Antibodies to which fluorescent molecules are coupled, recognize and bind specifcally and with high affnity to the molecular section of the target structure, also called epitope or antigen. The fluorescent molecules serve as reporter molecules which are imaged with the use of a fluorescence microscope. However, the size of these labels with a length of about 10-15 nm in the case of immunoglobulin G (IgG) antibodies, cause a detection of the fluorescent molecules shifted to the real position of the studied antigen. In dense regions where epitopes are located close to each other, steric hindrance between antibodies can also occur and leads to an insuffcient label density. Together with the shifted detection of fluorescent molecules, these factors can limit the achievable resolution of a microscopy technique. Expansion microscopy (ExM) is a recently developed technique that achieves a resolution improvement by physical expansion of an investigated object. Therefore, biological samples such as cultured cells, tissue sections, whole organs or isolated organelles are chemically anchored into a swellable polymer. By absorbing water, this so-called superabsorber increases its own volume and pulls the covalently bound biomolecules isotropically apart. Routinely, this method achieves a magnifcation of the sample by about four times its volume. But protocol variants have already been developed that result in higher expansion factors of up to 50-fold. Since the ExM technique includes in the frst instance only the sample treatment for anchoring and magnifcation of the sample, it can be combined with various standard methods of fluorescence microscopy. In theory, the resolution of the used imaging technique improves linearly with the expansion factor of the ExM treated sample. However, an insuffcient label density and the size of the antibodies can here again impair the effective achievable resolution. The combination of ExM with high-resolution fluorescence microscopy methods represents a promising strategy to increase the resolution of light microscopy. In this thesis, I will present several ExM variants I developed which show the combination of ExM with confocal microscopy, SIM (Structured Illumination Microscopy), STED (STimulated Emission Depletion) and dSTORM. I optimized existing ExM protocols and developed different expansion strategies, which allow the combination with the respective imaging technique. Thereby, I gained new structural insights of isolated centrioles from the green algae Chlamydomonas reinhardtii by combining ExM with STED and confocal microscopy. In another project, I combined 3D-SIM imaging with ExM and investigated the molecular structure of the so-called synaptonemal complex. This structure is formed during meiosis in eukaryotic cells and contributes to the exchange of genetic material between homologous chromosomes. Especially in combination with dSTORM, the ExM method showed its high potential to overcome the limitations of modern fluorescence microscopy techniques. In this project, I expanded microtubules in mammalian cells, a polymer of the cytoskeleton as well as isolated centrioles from C. reinhardtii. By labelling after expansion of the samples, I was able to signifcantly reduce the linkage error of the label and achieve an improved label density. In future, these advantages together with the single molecule sensitivity and high resolution obtained by the dSTORM method could pave the way for achieving molecular resolution in fluorescence microscopy
High-resolution nuclear magnetic resonance (NMR) spectroscopy is used in structure elucidation and qualitative as well as quantitative examination of product components. Despite the worldwide development of numerous innovative NMR spectroscopic methods, several official methods that analyze specific substances and do not represent a holistic analysis, are still in use for the quality control of drugs, food and chemicals. Thus, counterfeit or contaminated products of inferior quality can be brought onto the market and distributed despite previous quality controls. To prevent this, three NMR spectroscopic methods have been developed within the scope of this work (1) to study the peroxide value in vegetable and animal oils, (2) for the qualitative and quantitative analysis of metal cations and (3) to determine the enantiomeric excess in chiral alcohols. In oil analysis, titration methods are used to determine the bulk quality parameters such as peroxide value, which represents the concentration of peroxides. Titrations show several drawbacks, such as the need of a large amount of sample and solvents, cross reactions and the low robustness. Thus, an alternative NMR spectroscopic method was developed to improve the peroxide analysis by using triphenylphosphine as a derivatization reagent, which reacts with peroxides in a stoichiometric ratio of 1:1 forming triphenylphosphine oxide. In the 1H-31P decoupled NMR spectrum, the signals of the unreacted triphenylphosphine and the reacted triphenylphosphine oxide are detected at 7.4 ppm and 7.8 ppm, respectively. The ratio of the two signals is used for the calculation of the peroxide concentration. 108 oil samples with a peroxide value between 1 meq/kg and 150 meq/kg were examined using the developed method. Oils with a very low peroxide value of less than 3 meq/kg showed a relative standard deviation of 4.9%, highly oxidized oils with a peroxide value of 150 meq/kg of 0.2%. The NMR method was demonstrated as a powerful technique for the analysis of vegetable and krill oils. Another 1H NMR spectroscopic method was developed for the qualitative determination of Be2+, Sr2+ and Cd2+, and for the qualitative and quantitative determination of Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ by using ethylenediamine tetraacetate (EDTA) as complexing agent. EDTA is a hexadentate ligand that forms stable chelate complexes with divalent cations. The known amount of added EDTA and the signal ratio of free and complexed EDTA are used to calculate the concentrations of the divalent cations, which makes the use of an internal standard obsolete. The use of EDTA with Be2+, Sr2+, Cd2+, Ca2+, Mg2+, Hg2+, Sn2+, Pb2+ and Zn2+ result in complexes whose signals are pH-independent, showing cation-specific chemical shifts and couplings in the 1H NMR spectrum that are used for identification and quantification. In the presented NMR method, the limit of quantification of the cations Ca2+, Mg2+, Hg2+, Sn2+, Pb2+, and Zn2+ was determined with 5-22 μg/mL. This method is applicable in the food and drug sectors. The third NMR spectroscopic method introduced an alternative determination of the enantiomer excess (ee) of the chiral alcohols menthol, borneol, 1-phenylethanol and linalool using phosgene as a derivatizing reagent. Phosgene reacts with a chiral alcohol to form carboxylic acid diesters, made of two identical (RR, SS) or two different enantiomers (RS, SR). These two different types of diastereomers can be examined by the difference of their chemical shifts. In the presented method, the integration values of the carbonyl signals in the 13C NMR spectrum are used for the determination of the enantiomer excess. The limit of quantification depends, among others, on the sample and on the non-labelled or 13C-labelled phosgene used for the analysis. In the case of menthol, a quantification limit of ee=99.1% was determined using non-labelled phosgene and ee=99.9% using 13C-labelled phosgene. The 13C NMR method was also applied for the quality control of the enantiomeric purity of borneol, 1-phenylethanol and linalool. The developed 13C NMR method represents a powerful alternative to Mosher’s reagent for investigating the enantiomeric excess in chiral alcohols. This work demonstrates the variety of possibilities of applications for the quantitative nuclear magnetic resonance spectroscopy in the chemical analysis of drugs, food and chemicals using tagging reactions such as derivatizations and complexations. The nuclear resonance spectroscopic methods developed in this research work represent powerful alternatives to the previously used quality control techniques.
Cognitive control is what makes goal-directed actions possible. Whenever the environment or our impulses strongly suggests a response that is incompatible with our goals, conflict arises. Such conflicts are believed to cause negative affect. Aversive consequences of conflict may be registered in a conflict monitoring module, which subsequently initiates attentional changes and action tendencies to reduce negative affect. This association suggests that behavioral adaptation might be a reflection of emotion regulation. The theoretical cornerstone of current research on emotion regulation is the process model of emotion regulation, which postulates the regulation strategies situation selection, situation modification, attentional deployment, cognitive change, and response modulation. Under the assumption that conflict adaptation and affect regulation share common mechanisms, I derived several predictions regarding cognitive control from the process model of emotion regulation and tested them in 11 experiments (N = 509). Participants engaged in situation selection towards conflict, but only when they were explicitly pointed to action-outcome contingencies (Experiments 1 to 3). I found support for a mechanism resembling situation modification, but no evidence for a role of affect (Experiments 4 to 10). Changing the evaluation of conflict had no impact on the extent of conflict adaptation (Experiment 11). Overall, there was evidence for an explicit aversiveness of cognitive conflict, but less evidence for implicit aversiveness, suggesting that conflict may trigger affect regulation processes, particularly when people explicitly have affect regulation goals in mind.
The family of trypanosomatid parasites, including the human pathogens Trypanosoma brucei and Leishmania, has evolved sophisticated strategies to survive in harmful host environments. While Leishmania generate a safe niche inside the host’s macrophages, Trypanosoma brucei lives extracellularly in the mammalian bloodstream, where it is constantly exposed to the attack of the immune system. Trypanosoma brucei ensures its survival by periodically changing its protective surface coat in a process known as antigenic variation. The surface coat is composed of one species of ‘variant surface glycoprotein’ (VSG). Even though the genome possesses a large repertoire of different VSG isoforms, only one is ever expressed at a time from one out of the 15 specialized subtelomeric ‘expression sites’ (ES). Switching the coat can be accomplished either by a recombination-based exchange of the actively-expressed VSG with a silent VSG, or by a transcriptional switch to a previously silent ES.
The conserved histone methyltransferase DOT1B methylates histone H3 on lysine 76 and is involved in ES regulation in T. brucei. DOT1B ensures accurate transcriptional silencing of the inactive ES VSGs and influences the kinetics of a transcriptional switch. The molecular machinery that enables DOT1B to execute these regulatory functions at the ES is still elusive, however. To learn more about DOT1B-mediated regulatory processes, I wanted to identify DOT1B-associated proteins.
Using two complementary approaches, specifically affinity purification and proximity-dependent biotin identification (BioID), I identified several novel DOT1B-interacting candidates. To validate these data, I carried out reciprocal co-immunoprecipitations with the most promising candidates. An interaction of DOT1B with the Ribonuclease H2 protein complex, which has never been described before in any other organism, was confirmed. Trypanosomal Ribonuclease H2 maintains genome integrity by resolving RNA-DNA hybrids, structures that if not properly processed might initiate antigenic variation. I then investigated DOT1B’s contribution to this novel route to antigenic variation. Remarkably, DOT1B depletion caused an increased RNA-DNA hybrid abundance, accumulation of DNA damage, and increased VSG switching. Deregulation of VSGs from throughout the silent repertoire was observed, indicating that recombination-based switching events occurred. Encouragingly, the pattern of deregulated VSGs was similar to that seen in Ribonuclease H2-depleted cells. Together these data support the hypothesis that both proteins act together in modulating RNA-DNA hybrids to contribute to the tightly-regulated process of antigenic variation.
The transmission of trypanosomatid parasites to mammalian hosts is facilitated by insect vectors. Parasites need to adapt to the extremely different environments encountered during transmission. To ensure their survival, they differentiate into various specialized forms adapted to each tissue microenvironment. Besides antigenic variation, DOT1B additionally affects the developmental differentiation from the mammalian-infective to the insect stage of Trypanosoma brucei. However, substantially less is known about the influence of chromatin-associated proteins such as DOT1B on survival and adaptation strategies of related Leishmania parasites. To elucidate whether DOT1B’s functions are conserved in Leishmania, phenotypes after gene deletion were analyzed. As in Trypanosoma brucei, generation of a gene deletion mutant demonstrated that DOT1B is not essential for the cell viability in vitro. DOT1B deletion was accompanied with a loss of histone H3 lysine 73 trimethylation (the lysine homologous to trypanosomal H3K76), indicating that Leishmania DOT1B is also solely responsible for catalyzing this post-translational modification. As in T. brucei, dimethylation could only be observed during mitosis/cytokinesis, while trimethylation was detectable throughout the cell cycle in wild-type cells. In contrast to the trypanosome DOT1B, LmxDOT1B was not essential for differentiation in vitro. However, preliminary data indicate that the enzyme is required for effective macrophage infection.
In conclusion, this study demonstrated that the identification of protein networks and the characterization of protein functions of orthologous proteins from related parasites are effective tools to improve our understanding of the parasite survival strategies. Such insights are a necessary step on the road to developing better treatments for the devastating diseases they cause.
Salt formation is a routinely used strategy for poorly water-soluble drugs and traditionally performed with small inorganic counterions. High energy crystal lattices as well as effects on the local pH within the aqueous boundary layer during dissolution drive the increased dissolution rate and apparent solubility. Ionic liquids however, by definition low melting ionic salts with often large organic counterions, combine an increased dissolution rate with solubilization of the drug by the counterion itself. Long lasting supersaturation profiles of increased kinetic solubility were reported for several drugs formulated as ionic liquids increasing their overall bioavailability. Furthermore, aggregation and micellization between highly lipophilic compounds and amphiphilic bile acids was described before, demonstrating the capabilities of the human body itself to utilize solubilization of poorly water-soluble compounds. Development of novel counterions not only tailoring the desired physicochemical properties e.g. dissolution rate of the parent drug but adding – in a best-case scenario synergistic – pharmacological activity has been driven forward in the last years. However, salt formation can only be applied for ionizable i.e. acidic or basic compounds. While co-crystals can be used as a nonionized alternative, their formation is not always successful leading to an urgent need for other formulation strategies. In these lines, development of 2D and 3D printing techniques has been ongoing for the last decades and their pharmaceutical application has been demonstrated. The versatile nature and commercial availability allow a decentralized production further elaborating this technique for a highly flexible and patient-oriented supply with medication.
This thesis focuses on the theoretical background and potential application of salt formation in the pharmaceutical development of a drug candidate. The first section presents the current knowledge and state of the art in preparation of low melting ionic liquids i.e. salts and is translated to the in vitro investigation of molecular interaction between the poorly water-soluble drug imatinib and components of the human intestinal fluid in the second section. Development of novel antibiotic counterions and assessment of their potential use in pharmaceutical formulations with fluoroquinolones is described in the last two sections.
Chapter I describes the application of low melting ionic liquids in pharmaceutical formulation and details their development in the last two decades from versatile organic solvents in chemical synthesis towards amorphous strategies for drug delivery. The chapter gives a general overview on molecular structure and physicochemical properties of several drug containing ionic liquids and details the mechanisms which attribute to a typically fast dissolution, increased aqueous solubility as well as enhanced permeation which was reported in several publications.
Chapter II translates the increased aqueous solubility of drugs by an organic counterion to the human gastrointestinal tract with taurocholate and lecithin as main drivers for the solubilization of highly lipophilic and poorly water-soluble drugs. Investigation of the interaction of imatinib – a poorly water-soluble weak base – with fasted- and fed state simulated intestinal fluids revealed a complex interplay between the components of the intestinal fluid and the drug. Mixed vesicles and micelles were observed in concentration dependent aggregation assays and revealed differences in their size, molecular arrangement as well as composition, depending on the tested drug concentration. Overall, the study outlines the effective interaction of weakly basic drugs with taurocholate and lecithin to minimize recrystallization during intestine passage finally leading to favorable supersaturation profiles.
Chapter III focuses on the development of novel antibiotic counterions which potentially move the evolution of ionic liquids from a pharmaceutical salt with tailored physicochemical properties to a synergistic combination of two active pharmaceutical ingredients. The natural occurring anacardic acid derived from the cashew nut shell inspired a series of antibacterial active acidic compounds with increasing alkyl chain length. Their physicochemical properties, antibacterial activity, bacterial biofilm inhibition and cytotoxicity were detailed and in vivo activity in a Galleria mellonella model was assessed. This group of anacardic acid derivatives is synthetically accessible, easily modifiable and yielded two compounds with favorable activity and physicochemical profile for further drug development.
Chapter IV outlines the potential application of anacardic acid derivatives in pharmaceutical formulations by salt formation with fluoroquinolone antibiotics as well as novel techniques such as 2D/3D printing for preparation of drug imprinted products. Despite anacardic acid derivatives demonstrated promising physicochemical properties, salt formation with fluoroquinolone antibiotics was not feasible. However, 2D/3D printed samples with anacardic acid derivative alone or in combination with ciprofloxacin demonstrated physical compatibility between drug and matrix as well as antibacterial activity against three S. aureus strains in an agar diffusion assay. Conclusively, drug printing can be applied for the herein tested compounds, but further process development is necessary.
In summary, preparation of low melting ionic liquids, salts or co-crystals is an appropriate strategy to increase the aqueous solubility of poorly water-soluble drugs and tailor physicochemical properties. The counterion itself solubilizes the drug and furthermore potentially interferes with the complex micellar environment in the human intestine. However, salt formation as routinely used formulation strategy is not feasible in every case and development of alternative techniques is crucial to hurdle challenges related to unfavorable physicochemical properties. The outlined techniques for 2D/3D drug printing provide versatile production of drug products while extending the design space for novel drug development.
Fluids in Gravitational Fields – Well-Balanced Modifications for Astrophysical Finite-Volume Codes
(2021)
Stellar structure can -- in good approximation -- be described as a hydrostatic state, which which arises due to a balance between gravitational force and pressure gradient. Hydrostatic states are static solutions of the full compressible Euler system with gravitational source term, which can be used to model the stellar interior. In order to carry out simulations of dynamical processes occurring in stars, it is vital for the numerical method to accurately maintain the hydrostatic state over a long time period. In this thesis we present different methods to modify astrophysical finite volume codes in order to make them \emph{well-balanced}, preventing them from introducing significant discretization errors close to hydrostatic states. Our well-balanced modifications are constructed so that they can meet the requirements for methods applied in the astrophysical context: They can well-balance arbitrary hydrostatic states with any equation of state that is applied to model thermodynamical relations and they are simple to implement in existing astrophysical finite volume codes. One of our well-balanced modifications follows given solutions exactly and can be applied on any grid geometry. The other methods we introduce, which do no require any a priori knowledge, balance local high order approximations of arbitrary hydrostatic states on a Cartesian grid. All of our modifications allow for high order accuracy of the method. The improved accuracy close to hydrostatic states is verified in various numerical experiments.
In recent years <mi>PI</mi>-conjugated organoboron polymers and BN-doped polycyclic aromatic hydrocarbons have attracted a lot of interest due to their great potential in organic electronics. However, there are only few known examples of conjugated polymers with BN units in their main chain. Within this work silazane cleavage with silicon-boron (Si/B) exchange for the synthesis of a novel class of inorganic-organic hybrid polymers is demonstrated. These polymers consist of alternating NBN and para-phenylene units in the main chain. Photophysical studies and TD-DFT calculations for the polymer and molecular model systems were carried out, revealing a low extent of <mi>PI</mi>-conjugation across the NBN units. The new polymers can be used as macromolecular polyligands by a cross-linking reaction with a ZrIV compound. In the next chapter the synthesis and characterization of the first poly(p-phenylene iminoborane) is presented. This novel inorganic–organic hybrid polymer can be described as a BN analogue of the well-known poly(p-phenylene vinylene) (PPV) and is also accessible using the previously described Si/B exchange as synthetic strategy. Photophysical investigations and TD-DFT calculations on the polymer and corresponding model oligomers provide clear evidence for <mi>PI</mi>-conjugation across the B=N units and extension of the conjugation path with increasing chain length. Furthermore, a possible application of Si/B exchange for the synthesis of polysulfoximines was explored. Herein, diaryl sulfoximines and a p-phenylene bisborane serve as building blocks for new BN- and BO-doped alternating inorganic–organic hybrid copolymers. While the BN-linked polymers were accessible by a facile silicon/boron exchange protocol, the synthesis of polymers with B–O linkages in the main chain is achieved by salt elimination. In the last chapter the concept of Si/B exchange was investigated for the synthesis of BP-linked oligomers. Herein oligomers with sterically less demanding substituents (substituents: 2,4,6-trimethylphenyl or 2,4,6-tri-iso-propylphenyl) at the phosphorus are accessible using Si/B exchange, but the oligomer with Mes* (2,4,6-tri-tert-butylphenyl) as substituent needed a salt elimination pathway to give the desired product. Experimental data and theoretical investigations indicate, that the P-substituent has a high influence on the geometry of the phosphorus center and therefore on the possible conjugation over the BP units.
Bacterial small non-coding RNAs (sRNAs) play fundamental roles in controlling and finetuning gene expression in a wide variety of cellular processes, including stress responses, environmental signaling and virulence in pathogens. Despite the identification of hundreds of sRNA candidates in diverse bacteria by genomics approaches, the mechanisms and regulatory capabilities of these posttranscriptional regulators have most intensively been studied in Gram-negative Gammaproteobacteria such as Escherichia coli and Salmonella. So far, almost nothing is known about sRNA-mediated regulation (riboregulation) in Epsilonproteobacteria, including the major human pathogen Helicobacter pylori. H. pylori was even thought to be deficient for riboregulation as none of the sRNAs known from enterobacteria are conserved in Helicobacter and since it lacks the major RNA chaperone Hfq, which is crucial for sRNA function as well as stability in many bacteria. Nonetheless, more than 60 cis- and trans-acting sRNA candidates were recently identified in H. pylori by a global RNA sequencing approach, indicating that this pathogen, in principle, has the capability to use riboregulation for its gene expression control. However, the functions and underlying mechanisms of H. pylori sRNAs remained unclear.
This thesis focused on the first functional characterization and target gene identification of a trans-acting sRNA, RepG (Regulator of polymeric G-repeats), in H. pylori. Using in-vitro and in-vivo approaches, RepG was shown to directly base-pair with its C/Urich terminator loop to a variable homopolymeric G-repeat in the 5’ untranslated region (UTR) of the tlpB mRNA, thereby regulating expression of the chemotaxis receptor TlpB. While the RepG sRNA is highly conserved, the length of the G-repeat in the tlpB mRNA leader varies among different H. pylori isolates, resulting in a strain-specific tlpB regulation. The modification of the number of guanines within the G-stretch in H. pylori strain 26695 demonstrated that the length of the homopolymeric G-repeat determines the outcome of posttranscriptional control (repression or activation) of tlpB by RepG. This lengthdependent targeting of a simple sequence repeat by a trans-acting sRNA represents a new twist in sRNA-mediated regulation and a novel mechanism of gene expression control, since it uniquely links phase variation by simple sequence repeats to posttranscriptional regulation.
In almost all sequenced H. pylori strains, tlpB is encoded in a two gene operon upstream of HP0102, a gene of previously unknown function. This study provided evidence that HP0102 encodes a glycosyltransferase involved in LPS O-chain and Lewis x antigen production. Accordingly, this glycosyltransferase was shown to be essential for mice colonization by H. pylori. The coordinated posttranscriptional regulation of the tlpB-HP0102 operon by antisense base-pairing of RepG to the phase-variable G-repeat in the 5’ UTR of the tlpB mRNA allows for a gradual, rather than ON/OFF, control of HP0102 expression, thereby affecting LPS biosynthesis in H. pylori. This fine-tuning of O-chain and Lewis x antigen expression modulates H. pylori antibiotics sensitivity and thus, might be advantageous for Helicobacter colonization and persistence.
Whole transcriptome analysis based on microarray and RNA sequencing was used to identify additional RepG target mRNAs and uncover the physiological role of this riboregulator in H. pylori. Altogether, repG deletion affected expression of more than 40 target gene candidates involved various cellular processes, including membrane transport and adhesion, LPS modification, amino acid metabolism, oxidative and nitrosative stress, and nucleic acid modification. The presence of homopolymeric G-repeats/G-rich sequences in almost all target mRNA candidates indicated that RepG hijacks a conserved motif to
recognize and regulate multiple target mRNAs in H. pylori.
Overall, this study demonstrates that H. pylori employs riboregulation in stress response and virulence control. In addition, this thesis has successfully established Helicobacter as a new model organism for investigating general concepts of gene expression control by Hfq-independent sRNAs and sRNAs in bacterial pathogens.