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Applications of Fluorescent Displacement-based Sensors for Monitoring Time-Resolved Cnages in Analyte Concentrations
No full textThe present doctoral thesis describes the development of novel fluorescence-based sensors for real-time monitoring of analyte concentration changes. Water-soluble macrocycles, such as cyclodextrins, calixarenes, cyclophanes, or cucurbiturils, and their ability to encapsulate biologically and environmentally relevant analytes, as well as fluorescent dyes are the basis of such sensing systems. The first part of the thesis focuses on cucurbiturils, highly symmetric and rigid pumpkin-shaped macrocycles, comprised of two carbonyl-lined portals and a hydrophobic cavity. Herein, the reversible encapsulation of neutral guests, such as volatile hydrocarbons by cucurbiturils has been investigated using a fluorescent indicator displacement approach. Measurements in salt-free aqueous solution have disclosed a surprisingly strong (binding constants up to 107 M-1) and highly selective binding towards the investigated hydrocarbons (differentiation of alkanes from alkenes, isoalkanes from n-alkanes, and cis- from trans-alkenes), contrasting the common conception of cucurbiturils as cation receptors. The second part of the thesis relates to the area of enzyme assay design, which allows real-time monitoring of analyte changes during biochemical transformations. The first introduced assay exploits the use of anion-receptor macrocycles, for following ATP dephosphorylation via a fluorescence-based supramolecular tandem assay. The methodology has also been exploited to screen for activators of the model enzyme employed, potato apyrase, as well as to monitor the dephosphorylation of other nucleotides. The second assay, also performed in accordance with the supramolecular tandem strategy, enables a label-free, continuous monitoring of histone tail peptide methylation in homogeneous solution. The third project employs nuclear magnetic resonance spectroscopy for experimental testing of molecules with inhibitory potential against L-aspartate-alpha-decarboxylase, an enzyme known for its critical role in the growth of microorganisms, such as Mycobacterium tuberculosis
Probing biomass-chromatographic bead interactions by AFM force spectroscopy
No full textIn this thesis the interactions between chromatographic beads, surfaces, and cells were studied. The main goal was to establish a method to study such interactions by the direct measurements of forces with an atomic force microscope.
The study is motivated by expanded bed adsorption (EBA), where bioproducts are purified from an unclarified fermentation broth by their adsorption on chromatographic beads in a fluidized bed. The unspecific deposition of biomass onto the adsorbent matrix can severely affect the process performance, leading to a poor system hydrodynamics which then decreases the success of this unit operation.
To quantify the bead-biomass interactions different chromatographic beads were attached to AFM cantilevers, and force spectroscopy experiments were performed with these colloidal probes. For establishing the method, measurements were done on technical surfaces first, later the system was applied to yeast cells. The experiments are conducted under varying conditions to study the influence of ionic strength and hydrophobicity on various chromatographic bead – surface/ biomass combinations. The measurements were compared to bead-biomass interactions modeled by using XDLVO theory. So far only qualitative comparisons between force measurements and XDLVO calculations were done. For the probing an electrostatic repulsion between a positively charged bead and a positively charged poly-L-lysine surface a good agreement was found. The measurements between hydrophobic beads and hydrophobic surfaces showed an increase of the interactions with increasing ammonium sulfate concentration, which is in good agreement with the Hofmeister series. To analyze the force curves obtained on whole cells, possible elastic contributions, e.g. from deforming cellular membranes, have to be decoupled from the interaction forces.
The project aims for a better understanding of the interaction forces in chromatography and might help to improve the process quality of EBA
The European Natural Gas Sector Between Regulation and Competition
No full textOne of the major goals of European energy policy is the establishment of a single and competitive internal gas market by 2014. This PhD thesis explores in four related articles whether the efforts of restructuring the European natural gas sector have been successful in creating a regulatory framework that is well-suited to provide for a level-playing field for market players leading to competitive market outcomes. Access conditions to infrastructure facilities (pipelines and storage) are of special interest. The first paper evaluates a specific regulatory measure, i.e. ownership unbundling of transmission system operators as the strongest form of vertical separation. Applying dynamic estimators on an unbalanced panel of 18 EU countries, it turns out that the more modest legal unbundling reduces natural gas end-user prices, whereas ownership unbundling shows no impact. Looking at natural gas wholesale prices it is assessed if markets are able to generate efficient price signals. The second article tests for the spatial no arbitrage condition. Based on price developments at two German hubs and the nearby Dutch market, cointegration analysis and a state space model with time-varying coefficients are used. Though market efficiency in terms of information processing has increased, price differentials are only partly explained by transportation costs pointing at capacity constraints. In the third paper, the relationship between natural gas storage utilisation and price patterns at three major European trading points is investigated (test of the intertemporal no arbitrage condition). The results reveal that market performance differs substantially from the competitive benchmark. The last article puts the empirical results of the previous papers into a broader policy context and deduces policy recommendations
Roles of cysteine cathepsins in intestinal homeostasis
No full textCysteine cathepsins are endo-lysosomal proteases expressed in a variety of cells and tissues, including the gastrointestinal tract. Trafficking and localization studies have changed the classical view of cysteine cathepsins as enzymes acting only intracellularly and now it is well established that these proteases can also be secreted and exert extracellular functions. For example, extracellular cathepsin K has been recently indentified as an intestinal antibacterial factor with anti-inflammatory potential. Our studies on cathepsin K-deficient mice demonstrated that absence of cathepsin K is accompanied by elevated levels of other cysteine cathepsins, namely cathepsins B, L, and X. In addition, cathepsin K-deficient mice exhibited higher protein levels of collagen IV, an observation also shown for cathepsin B- and cathepsin L-deficient mice. These findings made us propose a role of cysteine cathepsins in ECM remodeling through both direct and indirect effects. Moreover, this study revealed that cathepsin K is important for the intestinal barrier function, since absence of this protease resulted in impaired distribution and expression of intercellular junction proteins. An important and very interesting observation was that cathepsin-deficiency affects each part of the gastrointestinal tract differently. For the first time each part of the small and large intestine was analyzed separately and our observations revealed that the proteolytic profile is unique and characteristic for each intestinal part. Interestingly, elevated levels of cathepsin X were found only in the duodenum and colon of cathepsin B-deficient mice when compared to wild type controls, while no alterations were observed in the case of jejunum and ileum. Hence, the conditions required for compensatory tasks of cathepsin X might be present only in specific parts of the gastrointestinal tract. Overall, this study demonstrates the key role of cysteine cathepsins in intestinal homeostasis and highlights the necessity to investigate each part of the gastrointestinal tract independently and not as an entity
Molecular Modeling and Investigation of Ultrafast Dynamics in Nano-systems
No full textIn this PhD thesis, the results of molecular dynamics simulation studies of structural properties of nano-aggregates and experimental time-resolved spectroscopy studies of exciton dynamics in nano-structures of chromophores are presented. The OPLS force field parameters of chlorophyll a, astaxanthin (a carotenoid) and phenyltrimethoxysilane molecules are developed to study their structural, physical and thermodynamic properties in solution using classical molecular dynamics simulations. Simulations of chlorophyll a in different solvents show formation of monomeric, dimeric and multimeric structures in methanol, benzene and water, respectively. The structures of the aggregates show that different functional groups present in the ring of the molecule, hydrophobicity of the phytol tail and the water molecules coordinated to the Mg of the chlorin ring play important role in aggregation. Simulations of astaxanthin in water and ethanol mixtures show formation of aggregates in the mixtures in which the water content is more than 50%. The results show that hydrophobicity of the conjugated chain in astaxanthin plays a major role in aggregation. Apart from the natural systems like light-harvesting complexes, chlorophylls and carotenoids also aggregate on surfaces. In light-harvesting complexes, the aggregation is controlled by proteins in such a way that the aggregates efficiently collect sunlight, which the plants use for photosynthesis. Such a controlled aggregation is also necessary to develop nano-antennas of these chromophores for artificial photosynthesis or other photovoltaic systems. One of the ways to control their aggregation in surfaces is to change the hydrophobicity of the surface. For this reason, a molecular model of the phenyltrimethoxysilane has been parameterized to model hydrophobic phenyl-functionalized inorganic surfaces like silica surface. Functioning of nano-assemblies of chromophores for photovoltaic application relies on formation of excitons, their motion, energy dissipation, charge separation, etc. that follow the absorption of photons. The processes like formation of excitons and charge separation are desirable while energy dissipation by vibrational relaxation are undesirable. In order to control aggregation such that the desirable functions are maximized, the different processes occurring in the nano-aggregates need to investigated. These processes, which occur in femto-second to pico-second timescales, can be studied using different techniques of time-resolved spectroscopy. However, the widely used techniques in time-resolved spectroscopy do not have spatial resolution high enough to study dynamics in individual nano-structures or nano-meter or sub-nanometer thin layers of chromophores. The experimental work presented here present the development and implementation of two techniques: near-field pump-probe technique to study the ultra-fast processes in nano-structures with 100 nm spatial resolution, and transient grating technique to study ultra-fast processes in few to sub-nanometer thin films of chromophores. Results of the investigation of exciton dynamics using the two techniques on 3,4,9,10 Perylenetetracarboxylic dianhydride show ultra-fast exciton annihilation and self-trapping of excitons at high exciton densities. The results also show that the pump-probe spectroscopy using the near field technique allows one to quantify the annihilation rate and diffusion constant of the excitons in nano-crystals. These techniques can also be used to investigate ultra-fast processes in the nano-structures of chlorophylls, carotenoids and their derivatives on functionalized surfaces
Development and Application of Novel Bioinformatics and Computational Modeling Tools for Protein Engineering Advanced Computational Tools for Protein Engineering
No full textIn the last decades, enzymatic catalysis emerges as a convenient and environmentally friendly substitute for the traditional chemical processes range from the synthesis of many pharmaceutical and agrochemical building blocks to fine and bulk chemicals, and more recently, the components of biofuel. The combination of experimental and computational methods holds particular promise in the field of enzymatic catalysis to tailor enzymes for the tasks not yet exploited by natural selection. Therefore, it is important to develop computational tools that help to exploit this goal. The scope of this thesis is to propose novel bioinformatics tools and to explore computational methods aimed to support and guide protein evolution experiments. The thesis is divided into two parts. First part of the thesis (Part I, Chapter 1 and Chapter 2) is focused on extending the benchmarking system of random mutagenesis methods (MAP: Mutagenesis Assistant Program) towards the sequence/structure and structure/function analysis and to evaluate this approach on commonly used enzymes as biocatalysts. Chapter 1 offers the comprehensive information about the computational methods used to assist protein engineering experiments. Chapter 2 describes a completely renewed and improved version of MAP server, named as MAP2.03D server that correlates the generated amino acid substitution patterns to the structural information of the target protein. Therefore, the latter helps to identify in advance the random mutagenesis method that can introduce mutations having less deleterious effect and to improve protein fitness towards an expected property, e.g. charged amino acid substitutions to increase solubility of protein in water. The capability of the server was illustrated by in-silico screening of different enzymes and the predicted results were in agreement with the experimental findings. The atomic level understanding of the subtle intertwining among structure, dynamics and function of enzymes plays an important role to rationally design new or improved functions. Second part of the thesis (Part II, Chapter 3 – 6) is based on molecular modeling approach to gain insight into the structural and dynamic properties of P450BM-3 (CYP102) complex in water and in the presence of cobalt(II)sepulchrate (CoSep) as an electron transfer (ET) mediator. P450BM-3, isolated from Bacillus megaterium is an attractive target and model system for biochemical (catalyzes the wide variety of industrially attractive substrates) and biomedical (being a bacterial model for microsomal P450s system) applications. The comprehensive theoretical aspects of MD simulation are provided in Chapter 3 with the overview about the system preparation for MD simulation and the analysis of protein conformation and dynamics in the generated trajectory. In Chapter 4, the structural and dynamic properties of P450BM-3 FMN (Flavin mononucleotide) domain as holo-protein, with the cofactor in oxidized and reduced states and as apo-protein are investigated. The results illustrate the effect of FMN cofactor and its protonation state on the conformation and dynamics of the FMN domain that can be related to ET pathway from FMN to HEME cofactor. The study is further extended to garner insight into the binding modes and the structural determinant of inter-domain ET in HEME/FMN complex of P450BM-3. MD simulations were performed on both FMN and HEME domains, isolated and in their crystallographic complex and results are reported in Chapter 5. HEME/FMN complex undergoes the rearrangement process to decrease the distance between their redox centers to promote favorable ET rate under physiological condition. In Chapter 6, MD simulation of P450BM-3 domains (isolated HEME domain and HEME/FMN complex) were performed in the presence of CoSep, as ET mediator. The results illustrate the preferential binding modes of CoSep in P450BM-3 domains and the putative ET pathways from CoSep to the iron center of HEME cofactor and are in agreement with the experimental findings
White dwarf coalescences
No full textA good fraction of white dwarfs are found in binary systems and, in many cases, they will exchange mass during their lifetime. The mass transfer phase will change the course of their live to stages that single stars cannot attain. During this phase a white dwarf system can either merge or survive as a binary in a semi-detached configuration, the so-called AM CVn system. White dwarf mergers are believed to result in extreme Helium stars, R Corona Borealis stars, neutron stars and, maybe most spectacularly, type Ia supernovae. While for many years type Ia supernova were thought to result from a white dwarf star acquiring mass from a normal star, the lack of hydrogen in the spectra, the recent discoveries of supra-Chandrasekhar mass supernovae, the delay time distribution measurements, all favor the white dwarf merger model. This underlines the necessity to clarify the fate of mass-transferring double white dwarfs. In this thesis we present the results of a systematic study scanning‚ the white dwarf binary parameter space. In a first step we investigate the onset and the subsequent dynamics of mass transfer. We find that the mass transfer is long-lived, continuing for several dozen orbital periods. The second step of this study examines the parameter space region where detonations may occur during the mass transfer phase, or at surface contact when the donor is tidally disrupted and plunges into the accretor. We demonstrate that a wide variety of dynamically unstable systems are viable type Ia candidates. A large fraction of Helium-accreting binary systems explode at surface contact and/or at earlier times via detonations induced by instabilities in the accretion stream. We do not find definitive evidence for an explosion in any of the studied double Carbon-Oxygen systems. The next decade thus holds enormous promise for the study of these events, in particular with the advent of wide-field synoptic surveys allowing a detailed characterization of their explosive properties
RNA based research development, application and analysis within the MIMAS project
No full textEvery prokaryotic cell contains different sorts of ribonucleic acid (RNA) molecules, which are mainly dedicated to processing and regulating gene expression. The wide palette of functions is reflected by distinct RNA types, and each of them is represented by a complex and comprehensive research field. In particular, different culture-independent applications have attracted attention recently because the majority of microbes still resists cultivation until today. For example, the standard approach for microbial diversity studies is based on the comparative analysis of the evolutionarily conserved 16S ribosomal RNA gene (16S rDNA), and messenger RNA (mRNA) based metatranscriptomics allows culture independent gene expression analysis without prior knowledge of the present microbes or transcripts. 16S rDNA biodiversity studies, metatranscriptomics and other ‘omic’ applications play a central role within the MIMAS project, which aims at characterizing a bacterioplankton community at the long-term ecological research site Helgoland Roads. However, culture-independent applications have their limitations, and a careful design of experimental procedures is crucial to assure that these limitations do not overtly bias the results. Therefore, this thesis outlines the development and application of an improved pipeline for the analysis of metatranscriptomic data and the evaluation of PCR primers used to amplify 16S rRNA. In particular, the outcome of the latter serves as a guideline for enhanced research to find the most suitable primer pair for 16S rDNA biodiversity analysis in any habitat using any currently available sequencing technology. The methods developed were used in a multi ‘omic’ study to characterize the phylogenetic and functional potential of the microbial community. The results identified the key players of an observed bacterioplankton bloom at Helgoland Roads and provided the first insights into taxonomically distinct nutrient strategies. They indicated that Flavobacteria, Gammaproteobacteria and Alphaproteobacteria are specialized for successive degradation of different algal primary products. This provided a series of ecological niches, allowing certain community members to grow. The results helped to uncover the secret of how members of the bacterioplankton can evade extinction despite the limited resources in the habitat. The work accomplished allows future follow-up studies and furnishes scientific society with guidelines to perform accurate diversity studies. Moreover the outcome serves as basis for future ecosystem monitoring
Potentially pluripotent cells in the common marmost monkey (Callithrix Jacchus) testis
No full textPluripotent stem cells are of outstanding interest for regenerative medicine due to their remarkable ability to generate all cell types of the body. Although pluripotent stem cells are classically derived from the inner cell mass of blastocysts, recent findings indicate that also spermatogonia can be a source for such cells. The initial aim of this project was to derive pluripotent stem cells from common marmoset monkey (Callithrix jacchus) spermatogonia. At the outset, the expression of the pluripotency factor SALL4 was investigated in marmoset embryonic stem cells and preimplantation embryos in mammalian germ line stem cells at different developmental stages and in adult mammalian spermatogonia. These experiments established SALL4 as an appropriate marker for pluripotent stem cells and undifferentiated premeiotic germ cells, including a subset of adult spermatogonia. Furthermore, these findings supported the hypothesis that SALL4-positive germ cells are a potential source of pluripotent stem cells. The experimental approach comprises the isolation and culture of spermatogonia including their eventual reprogramming and subsequent culture of spermatogonia-derived pluripotent stem cells. A prerequisite for this protocol is clear identification of the desired cells and their distinction from other cell types. Therefore, a detailed in situ analysis of marker expression in the adult primate testis was performed and an appropriate marker panel was established. All attempts to propagate spermatogonia or to derive pluripotent stem cells from testicular cells resulted in the enrichment of a fibroblast-like cell type. Initially these cells were thought to be pluripotent as two antibodies that detect the key pluripotency factor OCT-4 gave positive signals. Further analysis revealed that these signals were false-positive and a third antibody was required to display un-falsified staining. Using a standardized marker-panel and established differentiation assays, these cells were identified as testicular multipotent stromal cells (TMSCs). Importantly, TMSCs displayed the expression of many markers that are considered spermatogonia-specific within the testis, such as SSEA4, TRA-1-81, GFR-α, GPR125, THY-1 (CD90), and ITGA6. However, the germ cell-specific marker VASA and the spermatogonia-expressed factors MAGEA4, PLZF and SALL4 allowed clear distinction between marmoset spermatogonia and TMSCs. Furthermore, a culture-system for marmoset spermatogonia on irradiated TMSCs as a feeder layer was established. Although the propagation and reprogramming of marmoset spermatogonia to a pluripotent state was not achieved, this study provides for the first time a culture system for marmoset spermatogonia. Furthermore, a severe overlap in marker expression between spermatogonia and TMSCs was found. However, a panel of unequivocal markers for the identification of germ cells in culture was established, which provide a meaningful and reliable basis for future studies on cultured premeiotic (human and non-human primate) germ cells. Altogether, this work provides data which suggest that previous studies in the derivation of pluripotent stem cells from the human testes should be re-evaluated with regard to the suitability of the markers used for isolation and characterization of the cells
Mobility of fertiliser-derived uranium in arable soils and its contribution to uranium concentrations in groundwater and tap water
No full textPhosphorus (P) mineral fertilisers are found to contain high concentrations of uranium (U) (up to 206 mg U kg-1) and other trace elements (TE), such as Cd, Pb, Ni, Cu, Zn, Th, Nb, Sr, V, and rare earth elements. The content of U and other trace elements is depended on the sedimentary of igneous origin of the rock phosphate. In this study, the production of P fertilisers has been shown to contaminate top soil horizons with U and other trace elements in the close vicinity of a factory located in Southern Brazil. In contrast to this point source, agricultural P fertilisation leads to a diffuse contamination of the agro-ecosystem with U and other fertiliser-derived trace elements on a large scale. Top soil horizons of arable land accumulate fertiliser-derived U. According to the geochemical behaviour of U(VI) species under oxidising conditions, the mobilisation capacity for U in top soil horizons is considered to be high, contrary to other fertiliser-derived heavy metals (e.g. Cd). Hence, it is assumed that U can be leached to shallow groundwater and can reach fresh water resources potentially used for drinking water supply. The aims of this study were to investigate the concentration of U and other contaminants in P fertilisers, to identify geochemical processes of fertiliser-derived U mobility and mobilisation from arable top soil horizons to the groundwater, and to evaluate the origin of U in German groundwater and tap water. This study presents the broadest recent data set on regional distribution of U concentrations in German tap water to which 76 % of the German population has access. The mean U concentration was 0.68 æg L-1, the median 0.50 æg L-1. 1.3 % or 1 million of the 80.6 million inhabitants in Germany are exposed to U concentrations in tap water which are higher than the German drinking water threshold limit of 10 æg L-1. The regional distribution of U concentrations largely agrees with the geological setting reported for mineral waters, however, in addition evidence for fertiliser-derived U in tap water was found in certain areas in Northern and Southern Germany. To test the hypothesized fertiliser-derived U leaching to groundwater, samples from different depths were taken in an area intensively used for cropping production in Lower Saxony. The correlation between fertiliser-derived nitrate (NO3-) and U especially in shallow (< 15 m) groundwater samples indicates the anthropogenic origin of U. However, there is not yet a clear picture of quantities and processes of U leaching from fertilised soil into groundwater. The future aim is to investigate these processes of U migration via isotope fingerprinting and geochemical modelling to provide a solid foundation for future risk assessment