5 search hits
-
Characterization of Oligonucleotide Microarray Hybridization: Microarray Fabrication by Light-Directed in situ Synthesis – Development of an Automated DNA Microarray Synthesizer, Characterization of Single Base Mismatch Discrimination and the Position-Dependent Influence of Point Defects on Oligonucleotide Duplex Binding Affinities
(2008)
-
Thomas Naiser
- The present thesis focuses on nucleic acid hybridization between free-floating target sequences and complementary end-tethered oligonucleotide probes on the surface of DNA microarrays. Hybridization experiments were performed on oligonucleotide microarrays (DNA Chips) which were fabricated with an automated synthesis apparatus (developed in the framework of the present thesis). The working principle of the microarray synthesizer is based on a photochemically controlled in situ synthesis process. By means of the combinatorial approach up to 25000 different (arbitrary) probe sequences can be fabricated in parallel - starting from nucleotide building blocks (NPPOC-phosphoramidites) - directly on the surface of the microarray. Great flexibility with regard to the choice of probe sequences is achieved by use of virtual photomasks on the basis of a spatial light modulator (Digital Micromirror Device, DMD, Texas Instruments Inc.). A microscope projection photolithography system is employed to project the virtual masks (i.e. the photomask images shown on the DMD) onto the surface of the microarray substrate. Spatially controlled photodeprotection of photolabile NPPOC protective groups (followed by coupling of a further nucleotide building block) enables massively parallel synthesis of DNA probe sequences. In the automated synthesis process microarrays are routinely fabricated over night. Comparable in situ synthesis systems are currently operated only at very few institutions around the world. We first report the application of phosphorus dendrimer substrates in the in situ synthesis of DNA microarrays. With the phosphorus dendrimer functionalization we obtained superior results in regard to sensitivity, surface homogeneity, signal/background-ratio and reusability of the microarrays. We performed microarray hybridization experiments to investigate the impact of single base defects (deliberately introduced single base mismatches and single base bulges) on the binding affinity of oligonucleotide duplexes. This is particularly interesting with regard to genotyping microarrays which are increasingly employed as a molecular diagnostics tool for the detection of single nucleotide polymorphisms (SNPs). In a number of experiments we investigated the large influence of the single-defect position on duplex binding affinity. The origin of this positional dependence - which is apparently not in agreement with the (two-state) nearest-neighbor model - had not been identified so far. We discovered that the influence of the defect position is not restricted to single base mismatches but can also be observed for single base bulge defects. On the basis of the double-ended zipper model (assuming fluctuating end-domain-opening of the oligonucleotide duplex) we could reproduce the experimentally observed positional influence. Moreover, our theoretical investigations on the zipper model indicate a significant positional influence in regard to the contributions of the individual Watson-Crick nearest-neighbor pairs to the Gibbs free energy of oligonucleotide duplex formation. The present work provides for the first time a theoretical approach for the positional-dependent nearest-neighbor model (PDNN) of Zhang et al.. In the in situ synthesis process of DNA microarrays random point-mutations are introduced into the microarray probe sequences. We have shown - experimentally and by means of a numerical model - that synthesis-related defects significantly affect microarray hybridization characteristics. With regard to single base mismatch discrimination, we discovered significant differences between DNA/DNA- and RNA/DNA hybridization: experimental results indicate an improved discrimination of purine-purine mismatch base pairs in RNA/DNA-duplexes. For the experimentally observed, unexpectedly high stability of Group II single bulges we provide an explanatory approach on the basis of the zipper model. The selection of appropriate (specific and sensitive) probe sequences is of crucial importance for successful application of DNA microarray technology. Our experimental results confirm previous results which show that only a small fraction (in piecewise sections about 20-30%) of a long cRNA target sequence is available for hybridization with the complementary microarray probes. Reduced binding affinities are assumed to originate from the influence of target secondary structure. Using software tools for antisense oligonucleotide design (accounting for target accessibility) we were able to predict efficient microarray probes. We discovered evidence that mechanically stable secondary structures (e.g. double-helical sections) interfere with the microarray surface (sterical hindrance) and thus result in reduced microarray binding affinities.
-
Dielectric Properties of Molecular Glass Formers; from the Liquid State to the Tunneling Regime
(2008)
-
Catalin P. Ganaru
- The main purpose of this Thesis is to extend the dielectric investigations of molecular glass forming systems down to cryogenic temperatures (2 K), but also to complement previous work above the glass transition temperature Tg. The measurements were performed on systems composed of simple, mostly rigid molecules. Having at hand a large collection of data, previously compiled in Bayreuth group, this work starts with analyzing the characteristic relaxation features in molecular systems above Tg. Here, secondary relaxation processes emerge on the high frequency side of the main (alpha) relaxation peak, namely the excess wing (EW) and the beta-process. The EW manifests itself in the dielectric spectra as a power-law, while beta-process as a second relaxation peak. A new approach is introduced to disentangle the different spectral contributions (alpha-process, EW and beta-process). At variance with previous interpretations, the spectral shape of the alpha-process is assumed to be temperature invariant, obeying frequency temperature superposition (FTS) in the full temperature range above Tg. Its corresponding stretching parameter is taken from the high-temperature spectra, where the analysis is not hampered by the appearance of secondary processes. As a result of this constraint, the EW exponent turns out to be not only temperature, but also system independent. Thus, the overall spectral evolution for systems with no beta-peak above Tg (previously called type A glass formers) is simply described by a small variation of the relative weight of the EW with respect to the alpha-peak. This weight grows upon cooling, in contrast to the behavior of a beta-process. These now called “type A characteristics” are always spoiled by a more or less pronounced manifestation of a beta-process. Based on their different temperature changes, the EW and the beta-process contributions are disentangled close to Tg, and aging experiments carried out in this work are interpreted within the new scenario. In the glass, the interplay of both EW and beta-process determines the relaxation pattern. The beta-process appears as the only feature spoiling the universality in the evolution of the dynamics in molecular glass formers, since its relaxation strength does not correlate with the molecular dipole moment. Based on the above scenario, a consistent comparison between the orientational correlation functions of rank l = 1 (probed by dielectric spectroscopy) and l = 2 (probed by field cycling NMR and light scattering) is carried out. As demonstrated for glycerol, the NMR and light scattering spectra above Tg are scaled according to FTS over 15 decades in frequency. Significant differences in the spectral shape of the susceptibilities of different ranks are recognized at the low, as well as at the high frequency side of the scaled relaxation peak. In contrast, the time constants provided by the three techniques turn out to be essentially the same. Regarding the systematic differences observed at high frequencies, they are explained by assuming that the fast dynamics (EW) proceeds via small angles. Below Tg, NMR and dielectric spectroscopy reflect the same dynamics for glycerol, i.e. an experimental temperature dependence of the susceptibility is revealed. The evolution of the secondary processes (EW and beta-process) is monitored for temperatures well below Tg by applying a high-precision bridge. The bridge was employed to investigate extremely low losses (tan(delta) < 10-5), and, for the first time, the frequency dependence (within three decades) of the permittivity down to cryogenic temperatures was accessed for molecular glasses. Two additional relaxation regimes are identified: below 10 K clear indications are found that the tunneling regime is reached. Here the dielectric loss saturates to a plateau when plotted as a function of temperature and the corresponding weak frequency dependence appears as universal, at variance with the standard tunneling model predicting no frequency dependence here. Scaling out the molecular dipole moment collapses the plateau heights to an approximately system independent value, indicating a common density of tunneling centers in molecular glasses. At higher temperatures (10 K > T > 50 K) indications for thermally activated dynamics in asymmetric double well potentials are found for these systems. Here, for some systems, the dielectric loss displays a peak when plotted as a function of temperature but not as a function of frequency. This is in accordance with the Gilroy-Phillips model, previously used to interpolate the data for inorganic glasses in this regime. The distribution of the activation barriers g(V) is directly accessed by scaling the spectra in accordance with this model. However, g(V) extracted for molecular glasses appears as a stretched exponential.
-
The Rosensweig instability in isotropic magnetic gels
(2008)
-
Stefan Bohlius
- Die vorliegende Arbeit befasst sich mit der nichtlinearen theoretischen Analyse der Rosensweig Instabilität in isotropen magnetischen Gelen. Die Rosensweig Instabilität beschreibt den Übergang einer zunächst flachen Oberfläche zwischen einer magnetischen Flüssigkeit zu einer hexagonal geordneten Stacheloberfläche, sobald ein senkrecht zur flachen Oberfläche angelegtes homogenes Magnetfeld einen bestimmten kritischen Wert überschreitet. Startet man den Vernetzungsprozess in einer Mischung aus Polymeren, Vernetzungsreagenzien und einem Ferrofluid, so erhält man ein isotropes Ferrogel, ein elastisches Medium, welches zusätzlich superparamagnetisches Verhalten aufweist. Theoretisch lässt sich zeigen, dass auch die Oberfläche dieser Medien in einem angelegten Magnetfeld instabil wird, wobei die typische Wellenlänge im Vergleich zu gewöhnlichen Ferrofluiden unverändert bleibt, während die kritische Magnetfeldstärke mit wachsendem elastischen Schermodul steigt. Besondere Aufmerksamkeit kommt in der Diskussion dem stationären Charakter der Rosensweig Instabilität zu. Dieser ist, wie sich herausstellt, als ein Grenzprozess zu interpretieren, bei welchem die Dynamik der charakteristischen Mode mit Annäherung an die Schwelle immer stärker verlangsamt wird und schließlich zu einem statischen Oberflächenmuster führt. Der Grund für dieses Grenzverhalten ist in der deformierbaren Oberfläche und im Besonderen in der daraus resultierenden kinematischen Randbedingung zu sehen. Unter Anwendung der Energiemethode nach Gailitis, wird die Oberflächenenergiedichte bezüglich regulärer Streifen, Quadrate und Hexagone minimiert. Es zeigt sich, dass am Einsatz der Instabilität Hexagone das energetisch favorisierte Oberflächenmuster sind. Für hohe Magnetfeldstärken hingegen bilden Quadrate die bevorzugte Anordnung. Die Energiemethode hat jedoch bedeutende Nachteile, die als Motivation für eine schwach nichtlineare Analyse der fundamentalen hydrodynamischen Gleichungen und der Herleitung einer Amplitudengleichung dienen. Ganz besondere Beachtung verdient dabei die Bestimmung des adjungierten Systems für die Rosensweig Instabilität. Dieses ist zur Befriedigung der Fredholmschen Alternative, die wiederum die Amplitudengleichungen liefert, von zentraler Bedeutung. Zur Herleitung der adjungierten Gleichungen und der dazugehörigen Randbedingungen wird die Erkenntnis aus der Diskussion der linearen Instabilität, dass das System als dynamisch zu betrachten und der statische Grenzfall erst am Ende zu vollziehen ist, benutzt. Des weiteren stellt es sich als wichtig heraus, die Gleichungen zunächst für ein kompressibles Medium zu adjungieren und ebenfalls erst am Ende die Näherung für inkompressible Medien zu bestimmen. Das adjungierte System wird ebenfalls für die Marangoni Instabilität bestimmt. Dort induzieren Temperaturfluktuationen an der Oberfläche eines Fluids Fluktuationen der Oberflächenspannung, die wiederum Konvektion hervorrufen. Mit Hilfe der Lösungen des adjungierten Systems lassen sich nun die Lösbarkeitsbedingungen in der zweiten und dritten Störungsordnung erfüllen und man erhält letztlich die Amplitudengleichung. Im Rahmen unser Näherungen entkoppeln die hydrodynamischen Volumengleichungen von denen des Magnetfeldes. Allerdings müssen die Lösungen auch noch den Randbedingungen genügen und im Besonderen ist die normale Randbedingungen in den höheren Ordnungen nicht trivial erfüllt. Vielmehr liefert sie noch eine zusätzliche Bedingung zur Fredholmschen Alternative. In der Arbeit wird zum ersten Mal der quadratische Koeffizient aus den fundamentalen hydrodynamischen Gleichungen abgeleitet. Dieser garantiert zum einen die Existenz von Hexagonen, zum anderen das Auftreten einer transkritischen Bifurkation. Beides sind experimentell bestätigte Eigenschaften der Rosensweig Instabilität. Zum anderen enthält die Amplitudengleichung für Ferrogele eine zweifache Zeitableitung. Die linearisierte Amplitudengleichung nimmt im Fall der Ferrogele die Gestalt eines gedämpften harmonischen Oszillators an. Im Fall der Rosensweig Instabilität in Ferroflüssigkeiten, deren zugehörige Amplitudengleichung ebenfalls bestimmt wird, tritt diese zweifache Zeitableitung nicht auf. Die Rosensweig Instabilität ist im Rahmen unserer Näherungen rein oberflächengetriebenen. Das motiviert die Frage, inwieweit dünne magnetische Filme oder Membranen instabil werden können. Diese Frage wird in dieser Arbeit ebenfalls diskutiert. Beschränkt man sich in einer linearen Stabilitätsanalyse auf den symmetrischen Fall, das heißt der isotrope Ferrogelfilm ist auf beiden Seiten vom gleichen Medium umgeben, so findet man, dass der Film linear nicht instabil werden kann. Eine Instabilität zeigt sich nur im Fall von anisotropen magnetischen Gelen oder im Fall eines magnetischen Kontrastes zwischen den beiden umgebenden Medien.
-
Accurate charge densities of amino acids and peptides by the Maximum Entropy Method
(2008)
-
Jeanette Netzel
- In the present thesis accurate charge densities of several amino acids and peptides have been reconstructed by the Maximum Entropy Method (MEM) to study chemical bonds. The MEM model-independently determines the most probable electron density, which simultaneously maximizes the information entropy and fits the X-ray diffraction data. The quality of the MEM densities is enhanced by several extensions to the MEM such as the use of a non-uniform prior-density, the employment of the method of prior-derived F-constraint, static weighting and the choice of the optimal stopping criterion for the MEM iterations. The latter is achieved by inspection of difference Fourier maps and dynamic deformation maps. The reconstructed electron densities have been analyzed according to Baders Atoms in Molecules (AIM) theory to derive information about chemical bonds, in particular covalent bonds and hydrogen bonds. Local maxima of the densities, their associated atomic basins and charges, bond critical points (BCPs) and their densities and second derivatives, i.e. the eigenvalues and the Laplacians, have been determined according to the AIM theory. For all studied compounds it is shown that, providing the employment of the extensions to the MEM, the densities obtained from the MEM exhibit similar properties as those obtained from the multipole method. However, it is demonstrated that the features of hydrogen bonds are described more convincingly by the MEM than by the multipole method. By comparison of densities at BCPs from the MEM with the corresponding values from multipole refinement and from quantum chemical calculations it is shown that each method produces similar densities at BCPs. For all studied compounds it is demonstrated that for MEM densities, densities at BCPs of hydrogen bonds possess a larger magnitude than corresponding values from densities by the multipole method, while the opposite is true for covalent bonds. The values of the Laplacians at BCPs, especially of C–O bonds, show larger discrepancies between values from the MEM and from multipole refinement. These differences are caused by thermal motion which is present in dynamic MEM densities, but absent in static densities produced by the multipole method. The results of the extensive study of electron densities of amino acids and tripeptides show that densities and energetic properties at BCPs of covalent bonds and hydrogen bonds depend exponentially on their bond lengths. The functions of the dependencies of the densities on the bond lengths differ from the corresponding functions fitted to values from the multipole method. It is demonstrated that the ratio of the potential and kinetic energy densities at BCPs of hydrogen bonds reveals the possibility to classify them according to the distance between hydrogen atom and acceptor atom. Short hydrogen bonds possess covalent character, hydrogen bonds with intermediate distance have a mixed covalent-ionic character and long hydrogen bonds are ionic. This classification coincides with the usual classification of strong, intermediate and weak hydrogen bonds as proposed in the literature. The studied hydrogen bonds are classified as possessing mainly a mixed covalent-ionic character. However, for covalent bonds a classification according to the bond lengths does not suffice to characterize them. The results of the amino acids and tripeptides indicate that the prior density contributes a large part to the densities at BCPs. However, for the Laplacians and the energy densities at BCPs the differences between MEM and prior densities are larger than for the densities at BCPs. Densities at BCPs of hydrogen bonds from MEM densities show a different trend in their dependence on the bond distance than corresponding trends from prior densities. Thus, it is demonstrated that the analysis of the true density instead of the prior or procrystal density is recommended in order to extract information about chemical bonding. It is concluded from the results of the Accurate Charge Density studies reported in the present thesis, that the MEM allows a good characterization of chemical bonds and describes the electron density of hydrogen bonds more realistic than the multipole model. Chapter 3 of the present thesis has been published in Acta Crystallogr. B, 63, 285–295 (2007) and is reproduced with permission of the International Union of Crystallography (http://journals.iucr.org). Chapter 4 of the present thesis has been published in CrystEngComm, 10, 335–343 (2008) and is reproduced with permission of The Royal Society of Chemistry (RSC) (http://www.rsc.org).
-
Nonlinear macroscopic description of liquid crystalline elastomers in external fields
(2008)
-
Andreas Menzel
- We concentrate on a continuum characterization of the macroscopic behavior of side-chain liquid single crystal elastomers (SCLSCEs). These materials consist of chemically crosslinked polymer backbones, to which mesogenic units are attached as side-groups. Due to specific routes of synthesis SCLSCEs feature a monodomain of the liquid crystalline order in the ground state. Their macroscopic coupling of liquid crystalline order and elastic mechanical deformations makes them unique. In particular, we investigate the macroscopic behavior of cholesteric and nematic SCLSCEs in external electric and mechanical fields. We characterize the respective liquid crystalline state using the director field and describe the current state of mechanical distortion by a displacement field. The specific coupling between these two components is taken into account explicitly by additional macroscopic variables. These are the relative rotations between the director orientation and the polymer network. Using this kind of description, we first study the influence of an external electric field on the state of a cholesteric SCLSCE. For this purpose, the field direction is chosen to be parallel to the helical axis of the cholesteric mesogen alignment. Director reorientation and mechanical distortions are analyzed to linear order. In the case of low electric field amplitudes, we find an effect that has been termed rotatoelectric. Here, with increasing electric field amplitude, the director arrangement rotates around the helical axis, relative to the polymer network. This effect is specific for cholesteric SCLSCEs. We discuss several aspects important for an experimental observation of this effect. Next, we investigate the dielectric instabilities of a cholesteric SCLSCE in a Frederiks splay geometry at higher electric field amplitudes. On the one hand, we find a scenario that corresponds to the Frederiks transition in conventional low molecular weight liquid crystals. Here, the director reorientation arises in a way that is spatially homogeneous in the directions perpendicular to the cholesteric helical axis. On the other hand, however, we also find a qualitatively different instability. The latter is characterized by spatial undulations of the director reorientation, which occur in at least one direction perpendicular to the cholesteric helical axis. We recover the same results in the case of an external magnetic field. Besides, we discuss elastic mechanical compressions or dilations of a cholesteric SCLSCE in the directions parallel and perpendicular to the cholesteric helical axis. Here, small amplitudes of deformation lead to a distortion of the cholesteric helical structure. In the simplest case, we obtain an elongation or compression of the cholesteric helix along its axis. Furthermore, we propose ways to experimentally access so-far unknown values of the material parameters involved. We proceed by developing a model to characterize the nonlinear macroscopic behavior of the materials. For this purpose, we identify two coupled preferred directions in nematic and cholesteric SCLSCEs. One of them is imprinted into the polymer network during the process of synthesis to align the mesogens in a liquid crystalline monodomain. On the other hand, the actual average mesogen orientation may deviate from this imprinted direction and is described by the director field. We derive expressions characterizing nonlinear relative rotations between these two coupled preferred directions and we include them as macroscopic variables into our description. Using our model, we first investigate the shear deformation of a nematic SCLSCE. If the shear plane contains the director, the latter will be reoriented due to the mechanical deformation. In addition, however, we find as a nonlinear effect that the director reorientation acts back onto the elastic mechanical distortion of the material. This leads to compressive and dilative strain deformations. Finally, we study the specific stress-strain behavior of nematic SCLSCEs. It has been found for nematic SCLSCEs stretched perpendicularly to the initial director orientation that their director reorients towards the stretching direction. This reorientation of the director sets in above a critical threshold strain. In the strain regime where the director reorientation occurs, the slope of the corresponding stress-strain curve is significantly decreased. We demonstrate that our model describes this nonlinear behavior. Furthermore, we compare the predictions of our model with experimental data. As a result, we find that nonlinear relative rotations play the central role in the macroscopic characterization of the behavior of the materials. However, we also conclude that the macroscopic stress-strain behavior can be qualitatively influenced by those contributions to the elastic response that are not connected to the director reorientation and relative rotations.
