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Impact of global gene regulators in E. coli on cellular proteome and metabolic function
No full textAlmost sixty yearsafter the determination of the DNA helix structure and ten years after the human genome project gene regulation remains still obscure in many aspects. Although many regulatory interactions of regulatory proteins are known as well as their metabolic impact (e.g. lac operon), holistic concepts of regulation are still mainly uncovered or attributed to single effectors. Nevertheless, certain concepts were already revealed showing for example boolean like logics in the organization of the Escherichia coli regulatory network also found in neuronal networks and computer chips supporting the view of an information processing system. According to the last example transcription is mostly regarded as the complex interaction of regulators and targets integrated in the transcriptional regulatory network (TRN). Each regulator to target connection was inferred by deletion of the regulator, complementation in trans e.g. on a plasmid and confirmed by in vitro studies in a minimal factor context. The studies were performed under certain environmental conditions and are therefore in principle only valid under these conditions. The TRN can therefore be considered as the sum of possible regulatory interactions known down to the present day. The architecture of the network is mainly considered as hierarchical. Within the hierarchy, comprehensive regulatory overlaps were found called DORs. These overlaps exhibit more or less specific functions. We investigated the regulatory overlaps of major regulators (σ-factors and NAPs) regulons creating so called couplons. Gene ontology analysis showed an enrichment of specific metabolic function compared to random subsets taken from individual regulons. Moreover, the presence of highly overlapping regulons implies the reciprocal regulation of the involved regulators. These regulators are in turn organized in a heterarchically organized network of major regulators, constructing a decision making organization coordinating downstream regulation. This is supported by noise damping feedforward connections to couplons on the next hierarchy level most permeable for consistent signal sent by the upstream heterarchical network. However the majority of genes is not yet included in the TRN but also show complex transcription patterns. We show indications, that these genes may not be integrated into the TRN in a digital way. Here digital refers to a on-off target behavior dependent on a one to one relation of regulator and target. It has been shown, that supercoilii ing of the DNA is besides the TRN a global regulator of transcription driving growth related metabolic pathways and controlling anabolism/catabolism ratios. However the analysis was based on pure microarray data lacking relevance for the proteome composition. Therefore we performed a proteome analysis based on the experimental design of previous microarray experiments and find a strong correlation of transcriptome and proteome with a temporal delay of 20 minutes. Moreover, we find the same metabolic pathways activated on the proteomics level, proving the functional relevance of supercoiling changes. Analysing different gene sets under altered supercoiling conditions we found a diametrally opposed transcription pattern of TRN regulated genes and the complementary set of isolated genes during altered supercoiling supporting previous observations of two distinct types of information (TRN and gene neighborhood) executing regulatory control. However, we find regulators reacting on supercoiling similarly to isolated genes connecting supercoiling sensitive isolated genes with TRN regulators and in fact the TRN. Moreover, major TRN regulators also constituting major structural proteins like HU, FIS or H-NS show non-random distribution of binding sites. These proteins are also involved in buffering supercoiling dynamics and therefore coordinate gene regulation and chromosome topology. Although protein binding sites may play a structural role, like matS sites for the terminus macrodomain formation, or HU for transcription foci formation, the chromosome compaction itself has major effects on regulatory control. HU mutants have shown to create the chromosomal replication origin/terminus dependent transcription differences. The genes in the origin region were more active than in the wild type and the opposite was found for the terminus. In accordance with the HU transcription pattern also the binding sites of σ-factors show clear spatial gradients. Especially the antagonists σ70 and σ38 show a diametral gradient from origin to terminus indicating a spatial coding of temporal gene expression patterns. In this study we describe a spatio-temporal transcription program based on gradients of different DNA features monitored throughout the growth cycle of Escherichia coli. We show that gene order is conserved in the γ-Proteobacteria implementing a temporal program in space starting from origin of replication in exponential phase to the terminus in stationary phase. We finally propose that supercoiling, GC-content, gene orientation and binding site distributions coordinate of the spatio-temporal gene expression program
Time-Dependent Atomistic View on the Electronic Relaxations in Light-Harvesting Systems
No full textPhotosynthesis is certainly one of the most important processes of energy conversion on earth. Plants, algae and some type of bacteria harvest the light energy and convert it into chemical energy. Subsequent to the initial absorption of light, which induces an excitation in a pigment molecule, the absorbed energy is transferred to the so-called reaction center (RC) and induces a charge separation and finally a synthesis of ATP. The transport of the absorbed light energy to the RC happen with almost 100̃\% quantum efficiency. To this end, certain quantum effects seem to play an important role but are not yet completely understood, for example the recently experimentally observed long-lived quantum coherences in the energy transport. The availability of high-resolution X-ray crystal structures of pigment-protein complexes provides the opportunity to investigate the excitation energy transfer and the related quantum effects on the atomic level. To that purpose, this thesis comprises a multi-scale approach using classical molecular dynamics simulations with subsequently applied electronic structure calculations to compute optical properties and excited state dynamics of light-harvesting (LH) systems without additional parameters. This multi-scale approach was applied to two LH systems: the LH2 complex of a purple bacterium and the FMO complex of a green-sulfur bacterium. Consecutive excitation energies for the individual pigments and the electronic couplings between the pigments were obtained for each of the two complexes. These quantities were combined to a time-dependent Hamiltonian which were subsequently used to calculate optical properties and excitation energy transfer dynamics. Based on the fluctuations of the excited state energies, spectral densities were obtained which serve as necessary input quantities in dissipative quantum dynamics calculations. Additionally, spatial correlations of excitation energies, couplings and atomic motions were determined. Such correlations have been proposed as possible origin of the long-lived quantum coherences in the energy transport but found to be very small
On Thurston's characterization theorem for branched covers
No full textLet f be a postcritically finite branched self-cover of a 2-dimensional topological sphere. Such a map induces an analytic self-map f of a finite-dimensional Teichmuller space. We prove that this map extends continuously to the augmented Teichmuller space and give an explicit construction for this extension. This allows us to characterize the dynamics of Thurston's pullback map near invariant strata of the boundary of the augmented Teichmuller space. The resulting classification of invariant boundary strata is used to prove a conjecture by Pilgrim and to infer further properties of Thurston's pullback map. We obtain a complete topological description of canonical obstructions. Our approach also yields new proofs of Thurston's theorem and Pilgrim's Canonical Obstruction theorem
Bacterial Resistance via Multidrug Efflux Pumps A Computational Study
No full textMultidrug resistance against antibiotics is a common problem of treating bacterial infections. One of the major contributors are efflux pumps, that reduce the periplasmic concentration of antibiotics and other toxic compounds by pumping them out into the extracellular space. This thesis comprises several computational approaches to investigate the functions of the individual proteins of Escherichia coli's major efflux pump AcrAB-TolC. In the first part, the opening of the periplasmic tip of TolC, an outer-membrane channel, was investigated by mutating specific residues in this region as it was shown in X-ray crystal structures. Upon docking to one of several possible inner-membrane transporters, TolC is supposed to open iris-likely. In the present simulations, this opening was accomplished to a similar extent as in experiments. In the second part, the dynamics of AcrB's pumping process have been investigated by mimicking conformational changes which have been proposed by X-ray crystallographers. The outcome has been described by using a drug molecule as an unbiased measure. During this investigation, the binding pocket has been found to shrink peristaltically, which forced the drug to leave the binding pocket. In a subsequent work, the influence of its environment has been examined. Especially the interaction with the surrounding water and amino acids revealed several essential features of the extrusion by AcrB. One interesting aspect was the stream of water which slightly assisted the extrusion of the drug and also shielded the electrostatic interaction of the protein and the substrate. This disfavored a strong binding of the drug to the amino acids of the exit channel. Furthermore, the inter- and intramonomeric interactions have been examined and crucial conformational changes of the transition along the cycle have been highlighted. The effect of one particular mutation on the extrusion has been investigated in an additional work. The information presented in this thesis enhances the atomistic and functional understanding of E. coli's major efflux pump. By combining these data with experimental results, the mechanism of extrusion can be interpreted more clearly, which might also improve the chances to develop new antibiotics and inhibitors with a higher efficacy in the future
Advancements of the Sequence Saturation Mutagenesis (SeSaM) Method to Efficiently Explore Protein Sequence Space
No full textElucidating structure-function relationships in proteins and adapting biocatalysts to non-conventional substrates and production conditions are of high academic and economical interest. Independent from any structural information, directed evolution can steer protein properties in iterative cycles of diversity generation on the gene level and screening for improved variants on the protein level. Homogeneous distribution of mutations, consecutive nucleotide exchanges and transversion mutations are the keys to achieve chemically diverse amino acid substitutions. However, commonly used random mutagenesis methods can only generate point mutations and achieve chemically conserved amino acid substitutions due to transition biased mutational spectra or cannot give homogeneous distribution of mutations thus severely hampering the diversity. Sequence Saturation Mutagenesis (SeSaM-Tv+) is a novel random mutagenesis method which can construct transversion biased mutant library and consecutive mutations. Four steps involved for construction of mutant libraries of the gene are (i) Generation of DNA fragments by random incorporation of cleavable dNTPαS in the gene by PCR followed by cleavage and purification of the fragments, (ii) Enzymatic addition of universal base/s, (iii) Elongation of fragments (iv) Replacement of universal bases by standard nucleotides. Choice of dNTPαS used to generate pool of DNA fragments and bias of replacing universal base is responsible for mutational flexibility in SeSaM and consecutive mutations can be created by addition of more than one universal base. SeSaM-Tv+ was reported to achieve enrichment of desired transversions
Marine Metagenomics From high-throughput data to ecogenomic interpretation
No full textThe field of marine ecological genomics is evolving at an unprecedented pace. The increasingly cheaper and faster sequencing technologies present new posibilities but also new challegnes. The sheer amound of sequencing data requires elegant solutions for storing, querying and exchanging it. Additonally. sequences alone are not enough to understand the complex interactions between microorganism and the marine environment. Comprehensive environmental and contextual metadata are needed to put the sequences into context. However, the integration of sequence and metadata is not a trivial task. The aim of this thesis was to enhance the field of marine ecological genomics by fulfilling two tasks. Firstly, the technology necessary for data integration, visualization and analysis was set up. Secondly, this technological platform was used to support three ecological analysis of marine microbes. The first task was addressed by improving the existing portal for marine ecological genomics (http://www.megx.net). The main goal of megx.net is to integrate sequences and their metadata based on geographic location. The focus lies on environmental parameters such as temperature, salinity and nutrients. As a result of this thesis, megx.net pofits from a new data model, a net web interface, improved and analysis tools. For the second task, the integrated resources offered by megx.net were used. The effect of environmental stability on the transcription factors content of microbial comunities was quantified using interpolated environmental data. In a second study, interpolations were used to give environmental context to new hypothesis for domains of unknown function in the marine metagenomes. Last but not least, a study of the community structures of high and low DNA content marine microbes was complemented with integrated metadata. The extended technological platform megx.net was sucessfully used to gain new insights into the ecology of marine microbes
Applications of phase registration in robotic mapping
No full textRegistration of sensor data has been an active area of research in many fields from video data to the processing of range information. This thesis presents spectral based registration solutions for a wide variety of applications. The matching itself is based on phase registrations of shifted structures which allows a robust processing with respect to occlusion, interference and distortions inherent to specific sensor types. One application is the determination of homography parameters within video frames of down-looking monocular cameras. The goal is a generation of photo-maps for local areas. Here the parameter space is restricted to rotation, scaling and translation which correspond to sensor movements as x, y, z - translation and rotation. For this task the well known Fourier Mellin Transform (FMT) is applied with improvements and adaptions concerning the image material which is to be processed. The movement of the sensor platforms is assumed to be mainly in parallel to the observed surface, hence the registration of sensor tilts is not considered but an assessment in which way it affects the FMT processing is given.
A second application in this thesis is the processing of 2D range information as provided from e.g. Laser Range Finders (LRF) and 2D sonar systems. The spectral processing as the FMT depends on decoupling translation from other transformations within the data. The corresponding generation and processing of so-called descriptors is a necessary pre-processing step of the registration process. For the registration of rotated and translated scan data, the Mellin transformation is excluded and the parameter space is therefore reduced from 2D to 1D which makes the algorithm even more robust to interference and occlusion.
In a third application this principle is extended to 3D. Compared to the registration in 2D which are based on already existing approaches like the FMT and the polar resampling approach a novel concept for the determination of 3D rotation is introduced. Since algebraically correct approaches like spherical harmonics will not succeed with the available resolution of the spectral magnitude and approximation is suggested in this work. This approximation allows again a spectral phase matching based on a 3D Cartesian grid. The resulting algorithms show extreme robust registration results on real world acquired scan data. The proposed phase matching with its Dirac response allows furthermore a probabilistic post-processing of the registered parameters
Human Resource Management Systems and Workforce Age - The Macro Leverage of Micro Phenomena
No full textThis thesis focuses on the links between organizations' HR systems and the developmental dynamics of aging workforces and their consequences for behavioral and attitudinal work outcomes, which precede organizational performance. Building on tenets and central findings of lifespan developmental psychology, a research model is conceptually developed, that integrates age-contingent individual-level processes as mediators between HR systems and organizational outcomes. According propositions are derived that address essential facets of the framework for the age-related alignment of HR practices.
Next, two distinct bundles, i.e. growth and maintenance-enhancing HR practices, are conceptually developed. Combining macro and micro perspectives of strategic human resource management and applied psychology, we draw on social exchange theory and emotion regulation processes to hypothesize (a) a main effect of growth-enhancing practices on affective organizational commitment, (b) main effects of both bundles of HR practices on in-role behavior, and (c) moderating effects of age and maintenance-enhancing practices on work outcomes such that increasing employees' age attenuates the HR practices' positive impact. The results of a multilevel study comprising 600 employees and their direct supervisors of 59 business units provide evidence for the hypothesized main effects on affective commitment and the interaction between age and maintenance-enhancing practices on work outcomes.
Furthermore, this thesis examines the role of employees' future time perspective for the association between HR systems and work-related attitudes. Drawing on socioemotional selectivity theory and emotion regulation processes, indirect effects of bundles of HR practices on job satisfaction and organizational commitment through future time perspective were hypothesized and empirically tested. The results of a multilevel study comprising 1,540 employees of 75 business units provide evidence for (a) direct effects of HR practices on workforce's future time perspective and (b) indirect effects demonstrating the mediation of bundles of HR practices on job satisfaction and organizational commitment through future time perspective.
The results, theoretical contributions, future research directions, and practical implications are discussed
Analysis of nonlinear subdivision and multi-scale transforms
No full textSubdivision is a process of recursively refining discrete data using a set of subdivision rules to generate limits (curves, surfaces, height fields) with desirable properties such as continuity, smoothness, reproduction of shape features, and many more. The wide range of applications as well as the necessity of improving the performance of the existing algorithms lead to the invention of a great variety of subdivision schemes. In many cases, such as preserving the data shape, using normal meshes for better compression rates, removing heavily-tailed noise, working with manifold-valued data, linear multi-scale transforms give unsatisfactory results or cannot be applied at all, and nonlinear alternatives are necessary. There are still very few results about Lipschitz stability and Hölder regularity of nonlinear subdivision schemes and the associated multi-scale transforms, which is a very active research field with many open problems, that is driven by both theory and applications. In this thesis we develop a general stability analysis of both univariate schemes and their associated multi-scale transforms in the nonlinear functional setting. We show that, unlike the linear setting, convergence and stability analysis are no longer equivalent, and we derive numerical criteria for the verification of each of them. We extend the univariate convergence and stability results to the multivariate regular setting via local approximation techniques. We establish a general theory for normal multi-scale transforms for curves, based on approximating prediction operators. We propose a globally-convergent normal multi-scale transform, and build an adaptive algorithm based on it that defines a well-posed transform with smooth limits and high detail decay rates. We investigate several extensions of the classical setup for normal multi-scale transforms, namely we use another subdivision operator to generate the normal directions, the combined action of two different subdivision operators for the prediction step, and nonlinear geometry-based predictors, respectively, and show that the properties of the normal multi-scale transforms improve when such extensions are considered
Synthesis of Chlorogenic Acids and Chlorogenic Acid Lactones
No full textThe chlorogenic acids are a family of esters formed between certain trans-cinnamic acids and quinic acid. Chlorogenic acids are among the most abundant polyphenols, which are naturally occurring in plants, with various biological activities beneficial to human health. Chlorogenic acids are secondary metabolites that are found in plants. As a group they generally contain several subgroups and usually several isomers within each subgroup. The development of methods for the synthesis of existing chlorogenic acids and new series of chlorogenic acids were developed in this thesis in order to have the compounds available as analytical standards, for confirmation of previous and future LC/MS work, and most importantly for individual biological testing in various assays. The target compounds were obtained with two strategies 1.selective synthesis using appropriate protecting groups strategies, 2.nonnselective synthesis producing mixtures of isomers. The general strategy, which was applied through out the thesis in order to synthesise the target compounds was, as follows; - Preperation of acid chlorides of cinnamic acids - Preparation of protected quinic acid derivatives - Acylation - Deprotection A series of mono-acyl chlorogenic acids and esters, in particular of those substituted in the 1-, 3-, 4- and 5- positions of the quinic acid with coumaroyl, feruloyl, caffeoyl and dimethoxy cinnamoyl and cinnamoyl substituents, have been successfully synthesised. As intermediates in these syntheses a series of quinide esters, potential metabolites and degradation products of naturally occurring chlorogenic acids have also been obtained. The first examples of di-acyl chlorogenic acids, have been successfully synthesised. A general protection and deprotection strategy for the synthesis of chlorogenic acids has been established. Poly-acyl chlorogenic acids starting from qunic acid and quinic acid lactones also have been established. The right conditions for the synthesis of certain type of chlorogenic acids and esters have been established in this work