57 search hits
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New Applications of Fluorescence Correlation Spectroscopy in Materials Science
(2006)
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Heiko Zettl
- In this work we have developed new concepts for the usage of fluorescence correlation spectroscopy. A classical FCS setup was modified in such a way that fluorescent species in aqueous as well as organic environments can be studied at varying temperature. We have synthesised a set of dye-labelled polymers that served as a well-defined system to study polymer diffusion and that was used to characterise the beam path and focal volume in environments with refractive indices different from that of water. Furthermore a new method for the labelling of ionic species was developed. The adaptation of the microscope optics to non-aqueous environments was done by replacing the present microscope objective by a multi-immersion objective. Secondly, a sample chamber was developed that was not only resistant to organic solvents in all parts but also allowed temperature control of the solution. Determining diffusion coefficients of polymers in solution and their concentrations requires the exact knowledge of shape and size of the observation volume. For this purpose we have synthesised a set of polystyrenes with molecular weights ranging from 4 to 1550 kg/mol each chain being labelled with a single dye molecule. All species were anionically polymerised in order to grant a very low polydispersity and with this a high reliability in the determination of the observation volume. This concept can be transferred to other solvents and, hence, shows an easy way to calibrate fluorescence correlation microscopes to different solvents and to investigate non-aqueous solutions. Furthermore, we have shown exemplarily for polystyrene that FCS is capable of determining the crossover between the dilute and the semi-dilute concentration regime. Dye-labelled polymer chains were mixed with unlabelled polymer chains of the same length and their mobility was measured by FCS for different mixing ratios. The change of the mobilities leads to the respective overlap concentrations, which are shown to follow a scaling law in a range of molecular weights from 4 to 1550 kg/mol. This is in excellent agreement with the predictions made by Flory and Huggins. The data shown demonstrate that FCS can measure diffusion properties in ranges that were not accessible before. Another part of this work focusses on concepts to monitor the aggregation of molecules by FCS. Taking low-molecular-weight surfactants as an example it is shown that with the help of Coulomb interaction cationic surfactants can be labelled with anionic dye molecules and vice versa. Moreover, micelle formation is observed already at concentrations slightly below the critical micelle concentration found with classical methods. This findings are in excellent agreement with the predictions made by Israeliachvili in the 1990ies. Additionally, it was demonstrated that by using insoluble dye molecules, which are incorporated by the forming aggregates, aggregate formation can be followed by FCS on a single-molecule level. This procedure was shown to work in both aqueous and organic polymer solutions. The high sensitivity of FCS permitted to determine the critical aggregation concentration of Janus micelles in THF to the very low value of around 8 mg/L. No other experimental method available today is capable of determining aggregation concentrations in such a low concentration regime. In the same way the critical aggregation concentration of block copolymer polystyrene-Amylose in THF was determined. Finally, temperature-dependent correlation curves allowed the determination of reaction constants and enthalpies. This is of particular interest in biochemical contexts, as the amount of available material can be minute. Exemplarily, the binding enthalpy of an RPA protein to a single-stranded DNA strain is determined by temperature-dependent correlation curves. The modifications made to a classical FCS setup were shown to enhance the spectrum of possible applications to new experimental fields. The methods and concepts developed in the framework of this thesis are expected to play an important role in meeting future challenges of polymer physics and microbiology.
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Thermodynamische Stabilisierung von Proteinen durch in vitro Evolution und biophysikalische Analyse ihrer molekularen Grundlagen
(2006)
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Michael Wunderlich
- Proteine sind zentrale Bausteine des Lebens und ihre dreidimensionale Struktur ist für die Funktion von entscheidender Bedeutung. Deswegen ist das Verständnis der konformationellen Stabilität von Proteinen von großem Interesse. In der vorliegenden Arbeit wurde das Selektionssystem Proside zur Stabilisierung von Proteinen genutzt. Proside basiert auf der phage display Technologie und verknüpft die thermodynamische Stabilität von Proteinen mit der Infektiosität von filamentösen Phagen. Die bakteriellen Kälteschockproteine werden sowohl von experimenteller, als auch theoretischer Seite als Modellproteine zur Untersuchung der Stabilität von Proteinen genützt. Es ist bekannt, dass elektrostatische Wechselwirkungen auf der Oberfläche von sehr großer Bedeutung für ihre Stabilität sind. Deswegen sollte versucht werden, alternative Ladungsnetzwerke auf der Oberfläche des Kälteschockproteins Bs-CspB aus B. subtilis unter Verwendung von in vitro Evolution zu etablieren. Zu diesem Zweck wurden Reste anhand von Sequenz- und Strukturvergleichen mit homologen Proteinen ausgewählt und diese Positionen partiell randomisiert. In einem zweiten Ansatz wurden zufällig Mutationen in das Gen von Bs-CspB eingeführt. Nach erfolgter in vitro Evolution der jeweiligen Bibliotheken konnten an sechs oberflächenexponierten Positionen stabilisierende Mutationen identifiziert werden. Die Beiträge dieser Mutationen zur thermodynamischen Stabilität von Bs-CspB wurden analysiert. Die beste Kombination war, mit einem Schmelzpunkt von 83,7°C im Vergleich zu 53,8°C vom Wildtypprotein, sogar stabiler war als das hyperthermophile Homologe Tm-Csp aus T. maritima. Einen beachtlichen Beitrag zu dieser deutlichen Stabilisierung liefern dabei verbesserte Coulomb´sche Wechselwirkungen. Die Beiträge sind dabei jedoch sehr stark von der Umgebung abhängig. Vergleiche der experimentellen Daten mit vorhandenen theoretischen Betrachtungen offenbarten die Schwierigkeiten, Coulombsche Wechselwirkungen auf der Proteinoberfläche korrekt zu berechnen und deren Einfluss auf die thermodynamische Proteinstabilität zu bestimmen. Ein theoretischer Ansatz, welcher unter Verwendung eines quasi-elektrischen Dipolmomentes die Einflüsse von Veränderungen der Ladungsverteilung auf die Stabilität vorhersagte, konnte experimentell nicht verifiziert werden. Ebenso konnten keine Korrelationen zwischen Veränderungen der Stabilität und der Nettoladung des Proteins festgestellt werden und auch keine Korrelation mit der Bildung von Ionenpaaren hergestellt werden. Ebenso wie Bs-CspB stellt auch die b1 Domäne des Streptokokkenproteins G ein Modellprotein zur Untersuchung von thermodynamischer Stabilität dar. In der Gruppe von S. Mayo wurde dieses Protein unter Verwendung von computational design untersucht. Dabei zeigte sich, dass die vier teilweise exponierten Reste ein sehr großes Stabilisierungspotential besitzen. Eine in vitro Evolution dieser vier Positionen durch das Selektionssystem Proside bot somit die Möglichkeit, die Leistungsfähigkeit der beiden Methoden direkt zu vergleichen. Die Selektionen lieferten eine Vielzahl von deutlich stabilisierten Proteinvarianten. Für die stabilste Variante war der Mittelpunkt des thermischen Überganges um 24,7°C erhöht. Die beste Variante aus dem computational design lag von allen untersuchten Varianten auf dem dritten Platz. Sieben der zehn berechneten Varianten, welche in den Kalkulationen alle annähernd gleich stabil waren, enthielten einzeln oder in Kombination die Aminosäurereste L18 und K29. Diese beiden Reste erwiesen sich bei der experimentellen Charakterisierung als sehr ungünstig. Um die Ursachen für diese Unterschiede zu ergründen, wurde von zwei Varianten die dreidimensionale Struktur bestimmt. Zusätzlich wurden die enthaltenen Mutationen einzeln und in unterschiedlicher Kombination thermodynamisch charakterisiert. Dabei zeigten sich bei der Analyse die Schwierigkeiten, strukturelles Verhalten in experimentelle energetische Terme zu übersetzen, was der Grund für die unterschiedlichen Ergebnisse bei der Stabilisierung durch in vitro Evolution und computational design sein dürfte. Im Folgenden sollten für beide Schritte auf dem Weg zur Stabilisierung, der Auswahl von Positionen und dem Finden der besten Aminosäurereste, ein experimentelles Herangehen genützt werden. Durch Selektion von Bibliotheken mit zufälligen Mutationen, erstellt mittels fehlerhafter PCR, konnten viel versprechende Positionen identifiziert werden. Diese wurden dann einer Sättigungmutagenese mit anschließender Selektion unterworfen und die gefundenen Mutationen der beiden stabilsten Varianten wurden abschließend manuell kombiniert. Die erhaltene Variante besaß einen um 35,1°C erhöhten Übergangsmittelpunkt. Bei der Analyse der Ursachen zeigte sich, dass in diesem Fall starke hydrophobe Wechselwirkungen zwischen den durch diese Mutationen eingeführten Seitenketten wirken. Dies zeigt, dass zur Stabilisierung von Proteinen vielfältige Wege beschritten werden können.
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Low Temperature Single-Crystal X-ray Diffraction on A(1-x)A'xMnO3 (A=La,Eu and A'=Ba,Sr)
(2006)
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Nicola Rotiroti
- The present thesis is devoted to A(1-x)A'xMnO3 (A=La and Eu, A'=Ba and Sr) compounds with distorted perovkite-type structure commonly denoted as manganites. The interest toward these compounds manifested when colossal magnetoresistance (CMR) was discovered in manganites. We have selected three particular compositions in order to study three different aspects of their structures, that are: the analysis of the structural behavior (and its correlation to magnetic and electronic properties) of rhombohedral La0.815Ba0.185MnO3 and orthorhombic La0.89Sr0.11MnO3 in dependence on temperature, the studies of the monoclinic symmetry of La0.815Ba0.185MnO3 at T=160K and the evidences of the splitting of the A-site in Eu0.60Sr0.40MnO3. We investigated these materials by single-crystal X-ray diffraction. For La0.815Ba0.185MnO3 the structure was analyzed in the temperature range of 188.9-295K. The temperature of 188.9K has been selected as minimum since the crystal undergoes a first-order structural phase transition from rhombohedral R-3c to monoclinic I2/c at TS=187.1K. We found a change in the thermal expansion coefficient at the transition temperature of the paramagnetic (PI) to ferromagnetic (FM) phase transition (TC=251K), with a larger expansion of the FM phase. Progressive charge localization due to strong electron-phonon interactions in the PI phase might be responsible for a smaller thermal expansion coefficient in the PI phase. Precursor effects near the phase transition at TS towards the monoclinic phase occur for Mn-O distances, Mn-O-Mn angles and the volume of the MnO6 polyhedron, providing a picture of how the structure reacts approaching the structure phase transition. Furthermore, our results allow to conclude that the apparent driving force for the structural phase transition is the replacement of shear-type distortions by Jahn-Teller-type distortions of the MnO6 octahedra, together with a stabilization of the La environment. The same crystal as for studies of the rhombohedral phase was used for analysing the monoclinic phase, but what was a single-crystal in the rhombohedral phase becomes twinned in the monoclinic phase. With the aid of omega-theta mapping of reflection profiles we found that this material displays monoclinic symmetry I2/c despite many reports in the literature of an orthorhombic symmetry Pbnm for this compound as well as for related materials A(1-x)A'xMnO3 with x about 0.2. Another member of the family of perovkite-type materials is represented by La0.89Sr0.11MnO3, with orthorhombic symmetry with space group Pbnm at ambient conditions. The crystal structure was investigated in dependence on temperature, with the aim of analyzing structural changes correlated with the magnetic transitions. A complex evolution of the magnetization measurements against temperature was found. The crystal structure remains orthorhombic over the investigated temperature range 110-240K but several extremes are seen for the lattice parameters as well as for structural and thermal parameters at temperatures where also changes in the magnetic state are observed. The lattice parameters have extreme values at the Curie Temperature of TC=150K. The present results show small anomalies in the temperature dependencies of bond lengths and bond angles, that occur at T=170K. This temperature coincides with the onset of magnetic ordering rather than with TC. Less pronounced maxima at 170K are found for the temperature parameters of oxygen, while the shortest La-O bond is virtually independent on temperature. On the basis of these observations we propose that the tilts and deformations of the MnO6 octahedra are determined by the shortest La-O distance as a kind of boundary condition. The thermal expansion is then accounted for by structural rearrangements while keeping the shortest La-O distance constant at the value corresponding to optimal chemical bonding. The coincidence of structural anomalies with the onset of magnetic order strongly suggests that magnetic ordering occurs at temperatures where the crystal structure allows favorable magnetic interactions. The final part of this work concerns the structural investigation by single-crystal X-ray diffraction at room temperature of Eu0.60Sr0.40MnO3. This composition was never studied before and we found that the crystal structure is orthorhombic with space group Pbnm. Initially, the same positional and displacement parameters were refined for both Eu and Sr atoms, but the difference Fourier maps showed that they do not occupy the same position. The refinement of separate positions reduced the R value, and features in the difference Fourier map were considerably reduced, leading to the first observation of a splitting of the A-A' crystallographic site of A(1-x)A'xMnO3 compounds.
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The Dynamics of Molecular Glasses Studied by Light Scattering
(2006)
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Sergei Adichtchev
- The present work is devoted to studying the dynamics in molecular glass formers applying the light-scattering (LS) technique, in particular tandem Fabry-Perot interferometry which allows to cover the frequency range from 0.3 GHz to 1000 GHz. Chapters 4 to 7 each present, in a self-contained way, different aspects of the dynamics, as summarized below. In Chapter 4, the results of the study of the molecular glass formers 2-picoline and m-tricresyl phosphate are presented. The LS spectra are analyzed in the frame of the Mode Coupling Theory (MCT). At high temperatures the evolution of the susceptibility minimum is well described by MCT. Below the critical temperature Tc, the asymptotic scaling laws of MCT fail due to the appearance of the excess wing of the alpha-process, which shows a universal evolution as a function of relaxation time, as was demonstrated by dielectric spectroscopy. A phenomenological approach, which allows to separate slow (alpha-process) and fast relaxation processes in the LS spectra is developed. Applying this approach, the temperature dependence of the non-ergodicity parameter f is obtained. The anomaly of f as well as a crossover to "white noise" of the fast dynamics spectra is found. In Chapter 5, the most extensive dielectric data of glycerol compiled by Lunkenheimer et al. [Contemp. Phys. 41, 15 (2000)] are reanalyzed. In contrast to the analysis of Lunkenheimer et al., the normalized susceptibility spectra, i.e, the dielectric loss data normalized by the static susceptibility, including the high temperature data, are analyzed. For this purpose a phenomenological approach, which describes the whole dielectric spectrum including the alpha-peak, its high frequency wing, and fast dynamics, is applied. The crossover temperature extracted from the phenomenological analysis and defined by the emergence of the high frequency wing upon cooling agrees well with the critical temperature extracted from the MCT analysis. The crossover temperature Tc=288 is significantly higher than reported before. Extracting the non-ergodicity parameter f, the characteristic anomaly similar to the one of 2-picoline and m-TCP discussed in Chapter 4, is found. In Chapter 6, the study of the fast relaxation below Tg in the molecular glasses 2-picoline, m-TCP, o-terphenyl (OTP), as well as in ethanol is presented. In addition to the boson peak, the depolarized LS spectra reveal quasi-elastic contributions that we attribute to i) the nearly constant loss (NCL) in the frequency range below 10 GHz and ii) a power law contribution with positive exponent alpha at higher frequencies. In the majority of glasses the latter may be attributed to thermally activated dynamics in asymmetric double well potentials (ADWP), as was previously found for the DLS spectra in silica. Following the Gilroy-Phillips model, the exponent alpha shows a master curve as a function of T /Vo for the various glasses where Vo specifies the width of the exponential distribution of barriers g(V), i.e., g(V) is propotional to the exp(-V/Vo). In Chapter 7, the investigation of the fast relaxation processes in the structural glass (T<Tg) and in the glassy crystal phase of ethanol, as well as in cyano cyclohexane, is presented. Depolarized and polarized LS spectra including the Brillouin lines were measured. It was found that depolarized, polarized LS and internal friction data exhibit fairly similar behavior, and thus reflect the same relaxations. The DLS spectra were described by assuming that the NCL contribution dominates below a few tens GHz, while the fast relaxational dynamics dominates at higher frequencies.
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Fused and spiro furanones from tetronic acid synthons: Oxa and azacycles featuring the butenolide ring
(2006)
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Juan-Manuel Urbina-Gonzalez
- Diverse 4-O-allyl tetronates were obtained by reaction of allyl (alpha-hydroxy)carboxylates with keteneylidenetriphenylphosphorane (Ph3PCCO) in a domino reaction (addition – Wittig olefination) and by direct allylation of tetronic acid via Fischer esterification. 3-Allyl tetronic acids were generated by Claisen rearrangement of 4-O-allyl tetronates under microwave conditions; a subsequent Conia reaction produced the corresponding spiro cyclopropane furan-2,4-diones. Nucleophilic ring opening with diverse alcohols converted the spiro cyclopropane furan-2,4-diones to 3-beta-alkyloxypropyltetronic acids. When using O-benzyl isourea for the 4-O-alkylation of 3-allyl tetronic acids, the 3-benzyl derivatives were also formed (five examples were prepared). The Mitsunobu reaction proved to be a better way for the 4-O-alkylation of 3-allyl tetronic acids. 3,3-Diallyldihydrofuran-2,4-diones with two identical allyl residues were obtained by Tsuji-Trost-type Pd-catalysed allylation of either 4-O-allyltetronates or 3-allyltetronic acids. Pd assisted allylation of sodium 3-allyltetronate with a second allyl acetate gave mixed derivatives as did the Claisen rearrangement of 4-O-allyl 3-allyltetronates under microwave conditions. 3,3-Diallyldihydrofuran-2,4-diones were converted to butanolides with 3,3-spirocyclopentenyl or 3,4-cycloalkanyl annulation by ring closing methathesis with Grubbs’ catalysts. Natural product pesthetoxin was prepared via a double Ph3PCCO addition to alpha-hydroxy benzyl octanoate, thus demonstrating the utility of keteneylidenetriphenylphosphorane as a C2O building block. Finally, (-)-3-epi-blastmycinolactol was prepared enantioselectively via double hydrogenation of 3-but-2-enyl tetronic acid.
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Mechanics of living cells: nonlinear viscoelasticity of single fibroblasts and shape instabilities in axons
(2006)
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Pablo Fernandez
- Biomechanics is a field of major biological relevance. In spite of the vast complexity of biological matter, a number of generic features are found to hold in the mechanics of soft tissues throughout all of its length scales. A major goal in biomechanics is to reduce its general features to those of the cytoskeleton, the filamentous scaffold which provides cells with mechanical integrity, architecture and contractility. The first part of this report describes single-cell uniaxial stretching experiments performed on fibroblasts. When placed between fibronectin coated microplates, fibroblasts adopt a regular, symmetrical shape and generate forces. When a constant cell length is imposed, an increase with time of the pulling force can be observed. This active behaviour can be probed in more detail by superimposing small-amplitude oscillations at frequencies in the range 0.1--1 Hz. The response to the superimposed oscillations is then characterised by the viscoelastic moduli. These are seen to be a function of the average force acting on the cell. This master-relation holds for all cells. At low forces, both moduli are constant; beyond a crossover force, power-law stress stiffening is observed, where as a function of the average force both moduli go as a power-law with exponents in the range 1-1.8. The loss factor depends only weakly on the average force. Remarkably, the moduli are a function of the average force but are independent of the cell length. Therefore this mechanical behaviour is not strain stiffening; rather, it is an example of active, intrinsic stress stiffening. The precise way of sweeping force-space is seen to be irrelevant. The stiffening relation shows a striking similarity to rheological measurements performed on purified actin gels, in an unprecedented example of quantitative agreement between living and dead matter. This mechanical response originates in the semiflexible behaviour of biopolymers. The precise mechanism is however at present not fully understood. Here, a simple explanation is proposed. It is shown that stress stiffening in fibroblasts bears a strong resemblance to the nonlinear mechanics of Euler-Bernoulli beams, which also show a linear regime at low forces and a crossover to power-law stiffening. Systematic analysis of the response of fibroblasts to large amplitude deformations reveals a striking similarity to plasticity in metals. Fibroblasts can be described as showing kinematic (or directional) hardening, a hallmark of composite materials. The second part of this report addresses experiments performed on neurites. These comprise axons --the processes extended by neurons-- as well as PC12 neurites, a model system for axons. After a sudden increase in the external osmotic pressure, axons swell and a cylindrical-peristaltic shape transformation sets in. We interprete this transition as a Rayleigh-Plateau-like instability triggered by elastic membrane tension, similar to the pearling instability known in membrane tubes. Microtubuli disruption by nocodazol strongly increases the maximum amplitude of the instability, as well as slightly increases the wavenumber of the fastest mode, showing microtubuli to be the most important cytoskeletal component in stabilising neurites. After a hypoosmotic shock the neurite volume increases, reaches a maximum, and relaxes back close to its initial value. These experiments were performed at different temperatures and initial osmotic pressure differences. The relaxation time as a function of the temperature closely follows an Arrhenius dependence, suggesting the rate-limiting factor of the relaxation to be the movement of ions through channels. Similar experiments were also performed under drug-induced perturbation of actin, myosin and microtubuli. Cytoskeleton perturbation does not have any significant effect on volume relaxation, indicating that it takes place solely by changes in osmolarity, without a significant role for hydrostatic pressures. A clear effect of drugs is seen in the initial swelling phase, especially after microtubuli disruption by nocodazol. The rate and extent of swelling are significantly higher. Taking the effect of drugs on the evolution of neurite volume together with that on the pearling instability, we suggest that hydrostatic pressure is present in the initial swelling phase and determines the swelling rate. In conclusion, reproducible, quantitative experiments at the single-cell level have been developed which address biologically relevant phenomena. Following a time-honoured tradition in physics, both the cell-pulling experiments and the shape transformations in axons address highly symmetric systems, where the geometry does not preclude the understanding. First interpretations of the observed phenomena have been found, in terms of generic behaviours common to all objects under tension.
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NMR Studies on Termination/- Antitermination Complexes of HIV-1 Transcription
(2006)
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Nageswara Rao Jampani
- After HIV enters the host cell the viral RNA is reverse transcribed into double stranded DNA and then integrated into the host genome. At this step, transcription of HIV RNA by Rpol II is tightly regulated by several cellular factors including NELF, DSIF, and pTEFb, and the virus encoded Tat protein. The work presented in this thesis is mainly focussed on understanding the mechanism of termination and antitermination of HIV-1 transcription. Some of the cysteines in the wild type HIV-1 Tat are required for the Zn2+ dependent interaction with pTEFb, but not needed for the binding of TAR RNA in vitro. These cysteines are readily oxidised and leading to the protein aggregation. Thus, a mutant, Tat-Cys-, lacking all cysteines was used for binding studies with TAR RNA. 1H-15N HSQC and 1H-1H TOCSY spectra were measured to monitor the interaction between Tat-Cys- and TAR RNA. The results presented indicate Tat-Cys- is binding to TAR RNA around the bulge region and is able to induce a conformational change in TAR RNA. This is further supported by observing a change in the CD signal at 265 nm upon addition of Tat-Cys- to TAR RNA. {1H}-15N steady state NOE experiments showed that the core region of Tat remains unstructured even in the complex with TAR RNA. Based on the NMR and CD spectroscopic data presented in this thesis, and data published by others it is tempting to speculate that the core and basic regions of Tat-Cys- remain unstructured upon binding to TAR RNA. Furthermore, the solution structure of the RNA binding domain of NELF-E, NELF-E RRM, was determined using multidimensional NMR spectroscopy. The data reveal that the structure of NELF-E RRM exhibits a babbab topology. The atomic coordinates were deposited in the protein data bank (pdb) under the accession code 2BZ2. RNA binding studies were performed with TAR RNA and various oligoribonucleotides. NMR studies with NELF-E RRM:TAR complexes indicate NELF-E RRM binds to various RNAs in a very similar manner but with different affinities. Mapping of chemical shift perturbations on the structure of NELF-E RRM indicate that the RNA binding interface is mainly located on the b-sheet surface. Large chemical shift perturbations are observed for the residues located in the flexible C-terminal region of NELF-E RRM and in the loop between b3 and a2. Among the various RNAs tested, TAR49-57 displayed the highest affinity to NELF-E RRM. Further structural characterisation of the NELF-E RRM:TAR49-57 complex revealed that the C-terminal region of NEFL-E RRM adopts a small 310 helix around the b3-a2 loop and is stabilised by several hydrophic interactions. The C-terminal region of NELF-E RRM in the RNA bound conformation is very close to the RNA binding interface and could be involved in the RNA binding. However, further structural characterisation of NELF-E RRM in the complex with RNA will be needed to fully understand the mechanism of RNA recognition.
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New Carbazole Based Materials for Optoelectronic Applications
(2006)
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Martin Sonntag
- The motivation for this thesis was the synthesis, characterization and the testing of new, environmentally stable materials based on aromatic amines for OFET and OLED applications. The preparation of high quality thin films from solution as well as from the gas phase was an another important issue. The first part of this thesis deals with star-shaped molecular glasses with triphenylamine as core molecule. Substituted fluorene and carbazole units were attached to the core molecule as side arms via a trifold Suzuki cross coupling reaction. The target compounds were highly purified by medium pressure liquid chromatography (MPLC) as purity is an important prerequisite for organic materials to be used for optoelectronic applications. Before the new materials were finally tested in transistor devices, a suitable surface treatment of the OFET substrates was developed. By introducing self-assembled monolayers, prepared from hexamethyldisilazane (HMDS), on top of the SiO2 insulator layer of the FET substrates, the field-effect mobility was increased by at least one order of magnitude. Furthermore it was possible to improve on/off-ratios as well as turn on voltages. In conclusion hole carrier mobilities up to 3 x10-4 cm2/Vs and on/off-ratios of 10000 were achieved from the new star-shaped compounds. The performance of the devices was not affected by a four month storage period in air and daylight. The second part of this thesis describes the synthesis and characterization of a new class of fused heterocycles based on carbazole units. For this issue a series of bisindenocarbazoles is introduced as a new class of fused heterocycles. A synthetical procedure was designed which allows to tailor the thermal properties of the target compounds by introducing different alkyl substituents in the very last step of the synthesis. Yields up to 50 % can be obtained after six synthetical steps including purification of the intermediates. CV measurements showed the electrochemical stability of the novel compounds. Altogether five bisindenocarbazoles with different alkyl substitution patterns have been prepared and characterized. Their morphology varies from highly crystalline materials with short alkyl side chains to amorphous molecular glasses if longer or branched alkyl groups are attached to the core. As the bisindenocarbazoles exhibit a bright blue fluorescence together with high quantum yields, they were tested as blue emitter for OLED applications. In typical setups for blue light emitting LEDs, the blue emitter is doped into a wide band gap host material in order to avoid quenching of the electroluminescence and to adjust the energy levels of the different materials used in the setup. For this issue CBP, mCP and TCTA were tested as matrix materials together with a bisindenocarbazole as emitter. A combinatorial evaporation setup was used for the preparation of the OLED devices in order to dope the different host systems by co-evaporation of the bisindenocarbazole dye. This deposition method also allows the variation of the film thicknesses of the charge transport layers in a single experiment. By using this device architecture a deep-blue emission from the bisindenocarbazole dye at CIE color coordinates of x = 0.19 and y = 0.17 was obtained at a hole blocking layer thickness of 40 nm. Luminance values up to 200 cd/m2 were achieved with this series of devices. The turn on of the light emission was observed at 5 V. These very first results show that the bisindenocarbazole is a promising new blue fluorescent emitter for organic LEDs. Due to the rigid rod-like core of the bisindenocarbazole it was possible to obtain a novel derivative exhibiting a broad nematic mesophase by extending the core with aromatic side groups. By using a suitable alignment method, it is possible to obtain well ordered LC monodomains from which increased charge carrier mobilities can be obtained. The nematic LC phase was characterized with polarizing microscopy (POM) and small angle X-ray scattering (SAXS).
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Biochemical analysis of the interaction between transfer ribonucleic acid and exportin-t
(2006)
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Sheng Li
- In this thesis the ternary complex of tRNA•exportin-t•Ran•GTP was studied by biochemical and biophysical methods. Firstly, photocrosslinking was used to determine the contact sites between tRNA and exportin-t. 4-Thiouridine (s4U) was introduced into tRNAPheT.th by in vitro transcription with T7 RNA polymerase and crosslinked to exportin-t by irradiation at 312 nm. The crosslinking was Ran•GTP dependent and could be competitively inhibited by other tRNA species, showing that the crosslinking was the consequence of the formation of a tRNA•exportin-t•Ran•GTP complex. The crosslinked complex of (s4U)tRNAPheT.th-exportin-t after proteinase K digestion was incubated with a primer complementary to the 3'end of the tRNAPheT.th in the primer extension reaction. The elongation of the reverse transcriptase was forced to halt at s4Us crosslinked to peptides, namely, U55, U47, U33, U20. Among them, U47 and U55 were shown to be the major crosslinking sites. U47A mutation was introduced into tRNAPheT.th to test the role of U47. The mutant tRNAPheT.th transcript was still able to bind exportin-t, though a little weaker than normal tRNAPheT.th transcript. In contrast, s4U containing mutant tRNAPheT.th U47A exhibited a much poorer capability to crosslink exportin-t. It is concluded that U47 may be a major contact site between tRNAPheT.th and exportin-t. Secondly, the binding abilities of different tRNA species in calf liver tRNABulk to exportin-t was examined by affinity chromatography on immobilized exportin-t. With a stepwise elution of 250 mM and 500 mM KCl, tRNABulk was fractionated into 2 peaks. Therefore, Different tRNAs bound to exportin-t with different affinity. Among 7 tRNAs tested, the relative affinity to exportin-t ranked as tRNALeuCAG > tRNASerGCU, tRNALeuCAA, tRNASerUGA > tRNASerAGA, tRNALeuAAG > tRNAArgACG. To interpret the different affinity of tRNAs to exportin-t, it is proposed that exportin-t preferentially exports tRNAs that are required the stronger by the protein synthesis machine. The extent of requirement of a specific tRNA by cells is supposed as the ratio between the tRNA concentration in a cell and its codon frequency in the genome, if all tRNAs are supposed to be aminoacylated and transported to ribosome equally. It was found that among 7 tRNAs identified on the 2D urea PAGE, the theoretical estimation of 5 tRNA species upon their requirement by cell ranked the same as their relative affinity to exportin-t. Finally, atomic force microscopy was used to observe exportin-t and its interaction with tRNA in a native and undisturbed state directly. Exportin-t-esterase, immobilized to trifluoromethyl ketone (TFK) modified mica surface, showed a diameter of 15 ± 2 nm. After binding tRNA and Ran•GppNHp, the diameter of the complex somewhat increased to 16 ± 2 nm.
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Oligosaccharid-gerichtete RNA-Aptamere
(2006)
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Maria Milovnikova
- Im Rahmen dieser Arbeit sollte die SELEX Methode, zur Gewinnung von RNA-Aptameren, die ausgewählte an der Membranoberfläche eukaryotischer Zellen häufig vorhandene Oligosaccharide spezifisch erkennen, etabliert werden. Zum Erreichen dieser Ziele wurden vier Oligosaccharide Gal-1,4-b-GlcNAc-1,2-Man-OR; a-Neu5Ac-2,3-b-Gal-1,4-b-GlcNAc-1,2-Man-OR; 2,4-(b-Gal-1,4-b-GlcNAc)2-a-Man-OR und 2,4-(a-Neu5Ac-2,3-b-Gal-1,4-b-GlcNAc)2-a-Man-OR mittels chemisch-enzymatischer Verfahren mit zwei unterschiedlichen Spacern synthetisiert. Die Spacer ermöglichten eine kovalente Immobilisierung der Oligosaccharide an verschiedenen Oberflächen durch eine Amino- oder Thiolgruppe. Es wurde der Einfluss der Zusammensetzung, der Größe, der Verzweigung und der Ladung der Oligosaccharide auf die Ergebnisse der in vitro Selektion unter verschiedenen Selektionsbedingungen untersucht. Die biochemischen Eigenschaften und die Bindungsspezifität von drei selektierten RNA-Aptameren 5G, 9G und 10G, die bei mehreren unabhängigen Selektionen isoliert wurden, wurden analysiert. Zur qualitativen Analyse der RNA/Oligosacharid-Interaktion wurde auch die Oberflächen-Plasmon-Resonanz- Spektroskopie benutzt. Gleichzeitig wurden die Bindungskonstanten für RNA Aptamere 5G, 9G, 10G und das Trisaccharid Gal-1,4-b-GlcNAc-1,2-Man bestimmt. Um auswertbare Resultate aus den Oberflächen-Plasmon-Resonanz-Spektroskopie-Experimenten zu erhalten, mussten diese für die spezifischen Eigenschaften der Bindungspartner optimiert werden. Wahrscheinlich auch wegen der Tendenz der Aptamere zur Oligomerisierung konnte nicht eindeutig gezeigt werden, ob die selektierten Aptamere die Oligosaccharide, die auf Sepharose 6B-basierende Festphase oder beides erkennen. Dies führte zu weiteren Selektionen mit modifizierten und optimierten Bedingungen. Es hat sich gezeigt, dass man durch gezielte Variationen des Selektionsprozesses die Selektion von RNA-Aptameren gegen Oligosaccharide lenken kann. Die Selektionen lieferten neue Oligosaccharid-bindende RNA-Aptamere. Es zeigt sich, dass die Optimierung der Selektionsbedingungen für eine erfolgreiche in vitro Selektion und Gewinnung von spezifisch bindenden Aptameren wesentlich ist. Den größten Einfluss auf die Ergebnisse der Selektion hatten die Eigenschaften der Festphase, die zu der Immobilisierung der Oligosaccharide benutzt wurde.
