Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 03/06

Fri, 14 Jan 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15333/ https://edoc.ub.uni-muenchen.de/15333/1/Haag_Benjamin.pdf Haag, Benjamin

Tue, 11 Jan 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12547/ https://edoc.ub.uni-muenchen.de/12547/1/Liu_Yinghao.pdf Liu, Yinghao ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 10 Jan 2011 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13859/ https://edoc.ub.uni-muenchen.de/13859/1/Murachelli_Andrea_G.pdf Murachelli, Andrea Giovanni ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 21 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12479/ https://edoc.ub.uni-muenchen.de/12479/1/Breugst_Martin.pdf Breugst, Martin ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 21 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/17848/ https://edoc.ub.uni-muenchen.de/17848/1/Freitag_Klaus.pdf Freitag, Klaus ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 20 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/14028/ https://edoc.ub.uni-muenchen.de/14028/1/Saint_Paul_Veronica_2010.pdf Saint Paul, Veronica von ddc

Fri, 17 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15186/ https://edoc.ub.uni-muenchen.de/15186/1/Malakhov_Vladimir.pdf Malakhov, Vladimir

Aromatic amines are known to be strong carcinogens. After metabolic activation, they react as electrophilic arylnitrenium ions with nucleophilic functionalities of the DNA duplex interfering and disrupting DNA and RNA synthesis and leading to mutations. Preferred reaction sites are the amino groups of adenine and guanine and particularly the C8-position of guanine. During metabolic activation, aromatic amines are enzymatically acetylated at N8 position. The non-acetylated lesions reduce the replication efficiency, but are in general faithfully bypassed by high fidelity polymerases. In contrast, the acetylated derivatives block replicative polymerases but can be bypassed with special low-fidelity polymerases. The translesion synthesis DNA polymerase η, for instance, is able to bypass C8 bulky adduct lesions such as the widely studied 2-aminofluorene-dG (AF-dG) and its acetylated analogue (AAF-dG) mainly in an error-free manner. The distinct mutagenic properties of the acetylated and non-acetylated aromatic amine lesions are presumably caused by their different conformational preferences. While the non-acetylated lesions exist in both syn and anti conformation, the corresponding acetylated lesion seems to adopt the syn-conformation with high preference. The mechanism that allows low-fidelity polymerases such as Pol η to replicate past acetylated AAF-dG lesions is still unknown. In this thesis work, the mechanism of the error-free bypass of acetylated aromatic amine dG adducts such as the acetylaminofluorene-dG (AAF-dG) by Pol η is investigated. For that reason, AAF-dG as well as the guanine adducts of other aromatic amines (aniline, 2-aminonaphthalene, 2-aminoanthracene and 1-aminopyrene) were synthesized and successfully incorporated into various oligonucleotides via automated solid-phase DNA synthesis. The synthesized bulky adduct containing oligonucleotides were used for crystallization and for primer extension studies with the translesion synthesis (TLS) polymerase η from S. cerevisiae in order to get insights into the bypass mechanism of bulky adducts by this enzyme. In the present work, structural evidence is provided that yeast Pol η bypasses the bulky adducts AAF-dG and 2-acetylaminoanthracene-dG (AAA-dG) by rotation of the DNA around the bulky moiety, while keeping the AAF-dG in syn conformation.

Thu, 16 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13573/ https://edoc.ub.uni-muenchen.de/13573/1/Muschielok_Adam.pdf Muschielok, Adam

Host-Guest chemistry based on mesoporous silica materials has attracted increasing attention in the past two decades. Potential applications for these functionalized materials are in the fields of controlled drug delivery, catalysis, separation or encapsulation of functional biomolecules. The present work is focused on the synthesis of nanosized, mesoporous drug delivery devices, which are able to release a preloaded drug as a result of a certain trigger action, e.g. during the endocytosis in a cancer cell. For this purpose, several different synthesis strategies had to be developed in order to incorporate the different required functional groups within one mesoporous silica nanoparticle. A spatial separation of two different functionalities was achieved by the development of a sequential co-condensation approach. With this approach, core-shell bifunctionalized colloidal mesoporous silica could be synthesized. The obtained particles are an important prerequisite for other systems presented in this work. The applicability of the copper-(I)-catalyzed Huisgen reaction (click reaction) as mild synthetic tool for the immobilization of biomolecules in the channels of mesoporous silica was investigated. In this joint project between our group and the research group of Prof. Ernst Wagner (LMU), it was shown that a sensitive enzyme can be immobilized with this strategy in the pores of SBA-15. It was demonstrated that the recoverability and long-term stability of the active enzyme benefits from the encapsulation in the host. The well-known strong biotin-avidin interaction was used for the construction of a protease-responsive cap system for controlling the release from colloidal mesoporous silica. Fluorescein was released from the nanoparticles as a model compound for small drug molecules. In order to monitor the release, a custom-made two-compartment fluorescence cuvette was designed. Thermoresponsive opening through protein denaturation was demonstrated for temperatures higher than 90 °C. A programmable opening temperature for this concept became possible by using DNA-linkers between the silica surface and the avidin cap. It was demonstrated that the length of the double-stranded DNA controls the opening temperature of the avidin cap. This work was carried out as a join project between our group, the research group of Prof. Thomas Carell (LMU) and the baseclick GmbH. Redox-responsive drug delivery was investigated in living cells. In this context, the release of disulfide-linked, dye-labeled cystein from the core of colloidal mesoporous silica was monitored by confocal fluorescence microscopy at a single cell level, in collaboration with the research group of Prof. Christoph Bräuchle. It was shown by photoinduced endosomal rupture that the endosomal escape is a bottleneck in redox-based drug delivery. This concept was extended through the synthesis of photosensitizer-functionalized, PEGylated colloidal mesoporous silica. It was demonstrated that particle-loaded endosomes collapse under irradiation with 405 nm light and release the particles into the cytosol. In another joint project between the groups of Prof. Bein, Prof. Bräuchle, Prof. Rädler and Prof. Leonhardt together with Dr. Ulrich Rothbauer (all LMU), the novel photosensitizer-functionalized porous nanoparticles were used as carriers for the delivery of small GFP-binding antibodies from Camelidae sp. into GFP-tubulin expressing HuH7 cancer cells. Additionally, the particles were encapsulated by a supported lipid bilayer. The attached photosensitizer was shown to play a key role in the delivery mechanism. Light-irradiation was used to destroy both surrounding membranes (supported lipid bilayer and endosomal membrane). Finally, the pH-responsive release of the membrane-intercalating peptide mellitin from a mesoporous SBA-15 host was demonstrated. This was possible through the use of pH-sensitive acetal linkers. The release of the peptide was shown by the lysis of mouse erythrocytes. This work was carried out in collaboration with the group of Prof. Ernst Wagner. To summarize, mesoporous silica materials were functionalized with different biomolecules in order to generate novel materials for potential applications in drug delivery or other controlled release applications. The newly developed concepts provide a basis for future work on mesoporous silica as a powerful and versatile drug delivery platform.

Tue, 14 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12450/ https://edoc.ub.uni-muenchen.de/12450/1/Sinha_Jyoti.pdf Sinha, Jyoti ddc:540, ddc:500, Fakultät für Chemie und Phar

Mon, 13 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12761/ https://edoc.ub.uni-muenchen.de/12761/1/Hammelmann_Verena.pdf Hammelmann, Verena ddc:540, ddc:500, Fakultät für Chemie und Pharmazi

Mon, 13 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/16691/ https://edoc.ub.uni-muenchen.de/16691/1/Biegert_Andreas.pdf Biegert, Andreas ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 10 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12416/ https://edoc.ub.uni-muenchen.de/12416/1/Frohnapfel_Ronald.pdf Frohnapfel, Ronald

Fri, 10 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13146/ https://edoc.ub.uni-muenchen.de/13146/1/Yin_Ruohe.pdf Yin, Ruohe ddc:540, ddc:500, Fakultät für

Thu, 2 Dec 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12372/ https://edoc.ub.uni-muenchen.de/12372/4/Gupta_Rashmi.pdf Gupta, Rashmi ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 26 Nov 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12356/ https://edoc.ub.uni-muenchen.de/12356/1/Hoeller_Christoph.pdf Höller, Christoph Josef

Plasmid based gene therapy approaches often lack long term transgene expression in vivo due to silencing or loss of the vector. One way to overcome these limitations is to combine non-silenced promoters with strong viral enhancers. Here we combine cytomegalovirus (CMV) derived enhancer elements with the strong, human elongation factor 1 alpha (EF1a) promoter in a plasmid backbone devoid of potentially immunostimulating CpG sequences. The transgene expression of plasmids containing either the murine or human immediate early enhancer were monitored in vivo. The human CMV enhancer led to an enhanced and prolonged transgene signal compared to the murine enhancer. The elevated expression in the case of the human enhancer correlated with a higher plasmid copy number found in the liver two months after gene delivery. The transgene expression could be even further increased by using a new synthetic promoter, SCEP (shuffled CMV EF1 promoter) instead of the EF1 promoter, in combination with the human CMV enhancer. Secondly, to reach a tissue specific and high expression in liver carcinoma a plasmid with the AFP promoter was combined with the human CMV enhancer.

Research applications and cell therapies involving genetically modified cells require reliable, standardized and cost-effective methods for cell manipulation. The goal of this work is to provide a novel methodology that produces, in a single standardized techonology, genetic modification and cell isolation. We have named this novel procedure ―Magselectofection”. The approach is based on magnetic cell separation and magnetically-guided gene delivery (magnetofection). Optimized gene vectors associated with novel magnetic nanoparticles were formulated to transfect/transduce target cells while they are passaged and separated through a high gradient magnetic field cell separation column. Magnetofection of the Jurkat T cells using selected vector formulations resulted in a significant (up to 4.5-fold) enhancement in both luciferase reporter gene expression and the percentage of cells expressing eGFP, as compared to lipofection. A procedure for vector loading on LS Miltenyi columns was developed that enables up to 100% retention for both non-viral and viral magnetic complexes. We demonstrate, using a model cell mixture of K562 and Jurkat T cells, that the integrated method is highly efficient and specific for the target cell population. This was not only true for the model Jurkat/K562 mixture, but also for Sca-1+ mouse hematopoietic stem cells. With human umbilical cord mesenchymal stem cells (hUC-MSCs), we achieve up to 30% transfected cells with non-viral vector doses as low as 8 pg plasmid DNA per cell and up to 100% transduced cells with a multiplicity of infection of 0.5 TU/cell using lentivirus. Similarly, we obtain 22% eGFP-positive human cord blood hematopoietic stem cells (hCB-HSCs) upon lentiviral magselectofection compared to 0.15% eGFP-positive cells post-standard infection. We achieve up to 50% transduced Sca-1+ mouse stem cells at a lentiviral MOI of 1-3. Up to 5-15% and 20% genetic modified PBMC were found using non-viral and viral magselectofection, respectively. After genetic modification using magselectofection differentiation potential of hCB-HSCs and hUC-MSCs was maintained. Magselectofection requires a minimal number of manipulation steps and results in efficient and specific gene delivery to target cells. This minimizes the necessary vector material while maintaining the cellular differentiation potential of modified stem cells. Magselectofection may become a useful tool for nucleic acid therapy approaches involving ex-vivo genetically modified cells.

Fri, 29 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12221/ https://edoc.ub.uni-muenchen.de/12221/1/Hoffmann_Ruth.pdf Hoffmann, Ruth ddc:540, ddc:500, Fakultät für Chemie un

Fri, 29 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12627/ https://edoc.ub.uni-muenchen.de/12627/1/Schwarz_Thomas.pdf Schwarz, Thomas ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 28 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13695/ https://edoc.ub.uni-muenchen.de/13695/1/Klutz_Kathrin.pdf Klutz, Kathrin

Structural Maintenance of Chromosomes (SMC) proteins are vital for a wide range of cellular processes including chromosome structure and dynamics, gene regulation, and DNA repair. Whereas prokaryotic genomes encode for only one SMC protein that exists as a homodimer, eukaryotes possess six different SMC proteins that form three distinct heterodimeric complexes, with the holocomplexes additionally containing several specific regulatory subunits. The prokaryotic SMC complex is required for chromosome condensation and segregation. In eukaryotes, this function is carried out by the condensin complex with SMC2 and SMC4 at its core. The complex containing SMC1 and SMC3, named cohesin, is responsible for sister chromatid cohesion during mitosis and meiosis. Cohesin is also employed in DNA double-strand break repair, whereas condensin participates in single-strand break repair. The as yet unnamed SMC5-SMC6 complex is involved in several DNA repair pathways as well as homologous recombination in meiosis. SMC proteins consist of N and C-terminal domains that fold back onto each other to create an ATPase “head” domain, connected to a central “hinge” domain via long antiparallel coiled-coils. The hinge domain mediates dimerisation of SMC proteins and binds DNA, but it is not clear to what purpose this activity serves. The aim of this work was therefore to characterise the structure and function of the SMC hinge domain in more detail. Specifically, the hinge domains of the Pyrococcus furiosus SMC protein and of mouse condensin were studied. Both their high-resolution crystal structures as well as low-resolution solution envelopes were determined, and their DNA-binding activity was analysed qualitatively and quantitatively. While the SMC hinge domain fold is largely conserved from prokaryotes to eukaryotes, functionally relevant structural differences can be observed. Most importantly, the surface charge has been almost reversed throughout evolution. The data obtained confirm that of all three eukaryotic SMC complexes, condensin is most closely related to prokaryotic SMC proteins. Both the P. furiosus and the mouse condensin hinge domain preferentially bind single-stranded DNA, but the mouse condensin hinge displays a much higher affinity than its prokaryotic counterpart, suggesting that this function has been enhanced during the course of evolution. The single-stranded DNA-binding activity might be important for the function of the condensin complex in single-strand break repair, but probably plays a different role in prokaryotes, possibly in the DNA-loading process of the prokaryotic SMC complex during replication.

Cisplatin ist ein weit verbreitetes Zytostatikum, das seine Wirkung durch die Ausbildung von Quervernetzungen innerhalb eines DNA-Stranges entfaltet. Für das Cisplatin 1,2-d(GpG) Dinukleotid-Addukt konnte bereits sowohl in vitro als auch in Zellen die zentrale Bedeutung von DNA Polymerase η (Pol η) während des TLS-Prozesses nachgewiesen werden. In Zusammenarbeit mit der Gruppe Livneh wurde untersucht, ob der 1,3-d(GpTpG) Cisplatin Schaden ebenfalls in Zellen überlesen werden kann. Erstaunlicherweise sind humane Zellen in der Lage auch diesen komplexen Schaden zu überlesen, allerdings nur sehr ineffektiv. Da die Anzahl an Mutationen in Pol η defizienten Zellen deutlich erhöht war, konnte eine Beteiligung von Pol η am TLS-Prozess des 3’dGs des Schadens nachgewiesen werden. Somit konnte erneut die Initiator-Rolle von Pol η im Multi-Polymerasen Modell beim Überlesen von Cisplatin-Schäden bestätigt werden. Um mechanistische Details des Pol η vermittelten TLS-Prozesses zu erhalten, wurde die TLS-Fähigkeit von S. cerevisiae Pol η mittels in vitro Primerverlängerungsstudien untersucht. Dazu wurde DNA, die den 1,3-d(GpTpG) Cisplatin Trinukleotid-Schaden enthielt, hergestellt und mittels enzymatischen Verdaus charakterisiert. Mit dieser Methode konnte die Anwesenheit unerwünschter Nebenprodukte ausgeschlossen und die Entstehung des 1,3 Adduktes eindeutig nachgewiesen werden. Im Gegensatz zum 1,2-d(GpG) Addukt konnte Pol η alleine nicht über den 1,3-d(GpTpG) Cisplatin Schaden replizieren. Einzelnukleotid-Insertionsstudien zeigten, dass gegenüber dem 3’dG fehlerfrei ein dC eingebaut wurde, während der nächste Insertionsschritt nur mit erhöhten Enzymkonzentrationen erfolgte und mutagen war. Der Mechanismus des Replikationsblockes durch den 1,3-d(GpTpG) Cisplatin Schaden wurde mittels Röntgenstrukturanalyse auf atomarer Ebene untersucht. Dazu wurde Cisplatin-geschädigte DNA als Templat in Komplex mit dem katalytischen Fragment von Pol η, einem dATP in der aktiven Tasche und einem Primer mit terminaler 2’,3’-Didesoxyribose im zweiten Schritt der Verlängerung kristallisiert. Die Kristalle beugten die Röntgenstrahlung bis zu einer Auflösung von 2.5 Å. Im Kristall liegt der Komplex in zwei konformationell verschiedenen Formen vor. In beiden Formen (Komplex A und Komplex B) ist eine perfekte Watson-Crick Basenpaarung des 3’dGs (Pt-GTG) mit dem Primer zu erkennen. Dies erklärt die fehlerfreie Replikation des 3’dGs durch Pol η, welche sowohl in Zellen als auch in vitro beobachtet wurde. Das Hauptmerkmal der beschriebenen Struktur ist das zentrale Thymin des Schadens. Es befindet sich nicht zwischen den beiden Guaninen, sondern ist komplett aus dem DNA-Doppelstrang herausgedreht. Folglich ist das zentrale Thymin unfähig, den zweiten Verlängerungsschritt zu dirigieren, der nun gegenüberliegend zum 5’dG (Pt-GTG) erfolgt. Die Struktur beschreibt, wie der 1,3-d(GpTpG) Cisplatin Schaden eine Verlängerung durch Pol η verhindert. Das zentrale Thymin, das durch die Koordination des Platinatoms aus den Trinukleotidschaden herausgedreht ist, ist direkt vor dem Protein positioniert. Es tritt dadurch in sterische Wechselwirkung mit Methionin 74 und verhindert eine weitere Bewegung der Polymerase entlang des DNA-Strangs. Zudem ist zu sehen, warum die Inkorporation gegenüber dem 5’dG des Schadens so ineffizient verläuft. Die Rotation der DNA in die aktive Tasche des Enzyms (Komplex A → Komplex B) wird durch die Ausbildung einer Wasserstoffbrückenbindung zwischen dem dATP in der aktiven Tasche und dem 5’dG des Schadens getrieben. Dabei vergrößert sich aber der Abstand zwischen dem α-Phosphat des dATPs und der „gemodelten“ 3’OH-Gruppe des Primers. Der Abstand von ~8.5 Å ist für einen effektiven Nukleotidyltransfer eindeutig zu groß.

Quantum control spectroscopy denotes the combination of optical quantum coherent control with femtosecond spectroscopy. The molecular response to a photo induced process, controlled by shaped ultrashort light pulses, carries information about the system and the induced chemical reaction not obtainable by unshaped pulses. In this work quantum control spectroscopy is used to investigate the photochemical process of beta-carotene during its first few hundred femtoseconds, which are important in the photosynthesis of light harvesting complexes. A special class of shaped pulses, called pulse trains, are investigated. Pulse trains are obtained from Fourier limited pulses, by modulation with a sinusoidal phase mask $phi(omega) = a sin(bomega_0+c)$, leading to a sequence of three or more phase stabilized Gaussian shaped pulses in the time domain. The intensities of these pulses are defined by a, they are separated by equal interpulse distances b and have a distinct phase relation which is defined by c. In this work it will be shown that it is possible to draw a very unique relation between molecular properties and the molecular response to the electrical field in dependance of these parameters. In terms of quantum coherent control, sinusoidal modulated pulse trains have attracted special attention in the context of mode selectivity. In a series of experiments it was observed that pulse train excitation can suppress spectral features in the detection signal when the interpulse distance is adjusted to molecular characteristics like vibrational frequencies. Furthermore, in many control experiments aiming to steer a chemical reaction, the use of learning loops for field optimization leads to pulse shapes that could be reduced to sequences of pulses, comparable to the pulse trains introduced. Replacement of optimized light fields by appropriate adjusted pulse trains were successful in experiments controlling the energy flow in a light harvesting complex. Control could be obtained by variation of the phase parameter c, suggesting that the achieved effect was of coherent origin. The assumption that the carotene units in LH2 were responsible for the successful control, was the motivation for the presented work of quantum control spectroscopy of beta-carotene. Although many efforts have been made to understand the non-linear effects induced by pulse trains, the underlying mechanism is not yet clear. Neither the background of mode selectivity nor the mechanism of chemical reaction control could be deciphered satisfactorily. For spectroscopical investigations, however, the knowledge of the underlying process and its connection to the molecular response is inevitable and are analyzed in detail. Starting with a simple model of bound states in a diatomic molecule, the induced dynamics of the molecular system and the characteristics of the response field are analyzed. First phenomenological investigations of the pulse train induced wave packet dynamics show dependancies between the populations and coherences of the generated molecular state and the choice of the sinusoidal mask parameters. Further investigations imply a mechanism connecting the outcome of the control experiment with the pulse train parameters and the molecular properties which is confirmed by derivation of a formula based on time dependent perturbation theory. The proposed mechanism leads to results which are in accordance with many experimentally observed effects. It is found that pulse train excitation generates vibrational wave packets that can exhibit symmetric phase space structures. Comparable structures appear during long time evolution after excitation with Fourier limited pulses and are known as partial revival states. Experimentally observed effects, like annihilation of spectral signals, are attributed to temporal interference effects between phase shifted vibrational coherences of these symmetric phase space structures. Contribution of such temporal interference effects are found to be essential for the signal interpretation in the case of time limited detection periods in the femtosecond regime. From a detailed analysis rules are extracted which serve to predict and to interprete the outcome of quantum control experiments using sinusoidally modulated pulse trains. It is found that the degree of rotational symmetry of the generated phase space pattern is determined by the ratio of the classical oscillation period of a vibrational mode to the interpulse distance b. In contrast, at a fixed value of b, the variation of the phase parameter c causes an oscillatory exchange between phase shifted components of the generated phase space structures, leading to an oscillatory disturbance of the phase space symmetry. While the phase space symmetry induced by b leads to destructive interference of spectral signals, this effect can be partially removed by c. The resulting oscillations of the peak amplitudes with c reflect the symmetry of the b-generated phase space structures. In a next step the model is extended towards the description of complex biological systems. Investigated are environmental effects, the model expansion to polyatomic molecules and the influence of electronic coupling elements, leading to the participation of additional electronic states. Using the density matrix description, the influence on the pulse train mechanism of elastic and inelastic environmental processes is investigated. Limits are figured out, defining the scope of the extracted rules for the two mask parameters b and c in dissipative environment. Increasing the dimensionality of the model, it is found that the derived mechanism still holds in polyatomic molecules. In accordance with experimental results, it is possible to damp spectral signals of selective vibrational modes by the mentioned destructive interference effects, adapting the interpulse distance to participating modes. By combination of the effects of b and c it is even possible to selectively damp near resonant modes. To come closer to the description of beta-carotene, the model system is extended by an additional diabatically coupled electronic state. Now the spectroscopic response function after Fourier limited excitation, recording the evolution of the excited state population, comprises information exclusively of the reactive coupling modes. Thus, the electronic coupling process can be traced without disturbance of inreactive spectator modes by detection of the excited state population, acting as a window to coupling modes. Additionally it is shown, that the mechanism of pulse train excitation found for bound state potentials still holds in the presence of electronic coupling. The described interference effects appearing in the spectroscopical signals after pulse train excitation, show that a rethinking is required in the interpretation of pulse train control experiments. On the other hand, the different aspects of pulse train control offer a manifold of new applications in various fields of spectroscopy. Parallels to experiments, applying pulse trains under different conditions, like for example nonresonant excitation, lead to the assumption, that the introduced effects are more general. Pulse trains in spectroscopy may enhance the sensitivity and the selectivity of spectral features and could be applied to achieve higher contrast in coherent microscopy. By selective damping of near-resonant modes, application of pulse trains in combination with transient spectroscopy could provide access to the direct observation of dynamical processes. Furthermore, the characteristic response to parameter variations under pulse train excitation can serve to differentiate between vibrational and electronic origins of spectral features. It is this method, that is used in the present work to apply quantum control spectroscopy to the early steps of the photochemical process in beta-carotene, i.e. the energy loss channel due to quenching via a conical intersection. Based on experimental observations, by the described modular construction a model system for beta-carotene is proposed, comprising the key components of the induced photochemical energy transfer process during the first few hundred femtoseconds. The outcome of quantum control experiments of beta-carotene could be predicted and interpreted. By comparison with results of quantum control experiments on beta-carotene, performed in the group of M.~Motzkus (Heidelberg University), it is possible to verify the key assumptions made for the construction of the model system. Observed spectral features in dependance of the parameters b and c can be definitely assigned to vibrational coherences, indicating that a low frequency mode is responsible for the electronic coupling between the excited states S2 and S1 of beta-carotene. The achieved agreement between simulations and experimental results allow to conclude that the process of investigation is described well within the constructed beta-carotene model. The photochemical quenching process takes place on solely two excited states and no further electronic state plays a mentionable role.

Thu, 21 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12937/ https://edoc.ub.uni-muenchen.de/12937/1/Schwarz_Christoph.pdf Schwarz, Christoph ddc:540, ddc:500, Fakultät für Chemi

Thu, 21 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15168/ https://edoc.ub.uni-muenchen.de/15168/1/Schaffert_David.pdf Schaffert, David Henning ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 19 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13055/ https://edoc.ub.uni-muenchen.de/13055/1/Pippig_Diana_Angela.pdf Pippig, Diana Angela ddc:540, ddc:500, Fak

In dieser Arbeit werden Ester der Borsäure und von Arylboronsäuren mit Diolen, Methylglycosiden und Glycosen vorgestellt. Die Verbindungen werden durch NMR-Spektroskopie und Einkristallröntgenstrukturanalyse charakterisiert. DFT-Rechnungen werden unterstützend eingesetzt.

MicroRNAs (miRNAs) are a conserved class of small non-coding RNA genes with 19 to 25 nucleotides in length that are found in all higher eukaryotes as well as some DNA viruses. MiRNAs have been elucidated to exert important regulatory functions in many biological and pathological processes, by imperfect base pairing to the 3’ untranslated region (UTR) of target mRNAs, leading to translational repression or mRNA degradation. In the recent years, the rapid development of next generation sequencing (NGS) technologies has tremendously enhanced the identification of novel miRNA genes as well as the profiling of miRNA expression levels. In this study, NGS method has been applied to two different gammaherpesviruses (γ-herpesviruses) associated models: Epstein-Barr virus (EBV)-infected nasopharyngeal carcinoma (NPC) in human and murine gammaherpesvirus 68 (MHV-68)-infected cell lines from mouse. As a result, two novel miRNA precursors from EBV and six novel miRNA precursors from MHV-68 have been successfully identified and characterized. In addition, the completion of MHV-68 miRNA set has revealed a unique viral tRNA (vtRNA)-miRNA-miRNA structure in MHV-68 genome. Furthermore, expression levels of the viral miRNAs suggest a distinct pattern during different stages of the viral life cycle. On the other hand, the profiling of cellular miRNAs also defined a number of miRNAs that have been dysregulated in EBV-positive NPC tissues compared to healthy control tissues, and in MHV-68-infected compared to non-infected NIH 3T3 murine fibroblasts. Among them, miR-15 and miR-16 were upregulated upon EBV or MHV-68 infection. The tumor suppressor gene BRCA1 has been revealed to be the repressed target protein of miR-15/16 in both models, implying an interesting role of miRNAs in the pathogenesis of γ-herpesviruses. Meanwhile, to facilitate the analysis of NGS data, an automated software was designed and developed. Additional information gained during the analysis processes revealed the possible mis-annotations existing in the miRNA registry database, suggesting that the definition and characterization of novel miRNA genes has to be performed with much more caution.

Tue, 5 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13413/ https://edoc.ub.uni-muenchen.de/13413/1/Nagaraj_Nagarjuna.pdf Nagaraj, Nagarjuna ddc:540, ddc:500, Fakultät für Chemie und Pharmazi

Mon, 4 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12153/ https://edoc.ub.uni-muenchen.de/12153/1/Winnacker_Malte.pdf Winnacker, Malte ddc:540, ddc:500, Faku

Transcription of protein coding genes by RNA polymerase II (RNAPII) is an essential step in gene expression. Transcription elongation is a highly dynamic and discontinuous process that includes frequent pausing of RNAPII, backtracking, and arrest both in vitro and in vivo. Consequently, a multitude of transcription elongation factors are needed for efficient transcription elongation. When transcription elongation factors fail to “restart” RNAPII the persistently stalled RNAPII complex prevents transcription and thus has to be recognized and removed to free the gene for subsequent polymerases. Similarly, DNA damage causes stalling of RNAPII. In this case, the DNA damage is either repaired by Transcription-Coupled Repair (TCR) or RNAPII is degraded as a “last resort” mechanism by the ubiquitin proteasome system. In contrast to RNAPII degradation caused by DNA damage, the cellular pathway for removal of transcriptionally stalled RNAPII complexes has remained largely obscure. However, it was speculated that transcriptionally stalled RNAPII complexes are degraded by the same pathway as RNAPII stalled due to DNA damage. Here, it is shown that the pathway for degradation of transcriptionally stalled RNAPII is distinct from the DNA damage-dependent pathway, providing the first evidence that the cell distinguishes between RNAPII complexes stalled for different reasons. The novel cellular pathway for transcriptional stalling-dependent degradation of RNAPII is termed TRADE. Specifically, in the TRADE pathway a different yet overlapping set of enzymes is responsible for poly- and de-ubiquitylation of transcriptionally stalled RNAPII. Moreover, the catalytic 20S proteasome is recruited to transcribed genes indicating that Rpb1 of transcriptionally stalled RNAPII complexes is degraded at the site of transcription. Importantly, nucleotide starvation and temperature stress which might mimic natural conditions of transcription elongation impairment also lead to RNAPII degradation. Finally, this study provides the first evidence that the mechanism for the controlled degradation of the transcriptionally stalled RNA polymerase complex might also exist for transcription by RNAPI and RNAPIII. Taken together, the TRADE pathway elucidated in this study ensures continued transcription.

Fri, 1 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12158/ https://edoc.ub.uni-muenchen.de/12158/1/Kanzian_Tanja.pdf Kanzian, Tanja ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 23 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12173/ https://edoc.ub.uni-muenchen.de/12173/1/streidl_nicolas.pdf Streidl, Nicolas ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 20 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12398/ https://edoc.ub.uni-muenchen.de/12398/1/Strasser_Ralf.pdf Strasser, Ralf ddc:540, ddc:500, Fakultät für Chemie

Fri, 17 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12126/ https://edoc.ub.uni-muenchen.de/12126/1/Ghaschghaie_Natascha.pdf Ghaschghaie, Natascha ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 10 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13696/ https://edoc.ub.uni-muenchen.de/13696/1/Braun_Cordula.pdf Braun, Cordula ddc:540, ddc:500, Fakultät für Che

Thu, 9 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12430/ https://edoc.ub.uni-muenchen.de/12430/1/Lorenz_Phillipp.pdf Lorenz, Phillipp ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Ein lebender Organismus ist unter anderem durch seine Fähigkeit zum präzisen Auf- und Zusammenbau höherer molekularer Strukturen charakterisiert, wobei die Faltung und Assemblierung von Proteinen eine bedeutende Rolle spielt. Die Proteinfaltung wird durch molekulare Chaperone unterstützt und optimiert, bis ein Protein seine native, biologisch funktionelle Struktur eingenommen hat. Durch exogene Einflüsse oder endogene Veränderungen eines Proteins, z.B. bei neurodegenerativen Erkrankungen wie M. Alzheimer, M. Parkinson oder Chorea Huntington, oder des gesamten Proteinnetzwerkes, kann Proteinfehlfaltung, Aggregation und die Ausbildung amyloider Strukturen, verbunden mit Zytotoxizität, auftreten. Die zur Fehlfaltung und Bildung ähnlicher amyloider Aggregate führenden strukturellen Determinanten der Zytotoxizität, verursacht durch Proteine unterschiedlicher Primärstruktur und Länge, sind nur unzureichend erforscht. Eine Hypothese besagt, dass lösliche intermediäre Oligomere der aggregierenden Proteine die toxische Spezies in einem wahrscheinlich multifunktionellen pathogenen Geschehen darstellen. Es gibt Hinweise, dass eine zusammenbrechende Proteostase verbunden mit einer zu geringeren Kapazität molekularer Chaperone zu den deletären Effekten führt. Auch ist nicht abschließend geklärt, ob und zu welchem Anteil die Toxizität durch Aggregation des Proteins und damit verbundener erhöhter Pathogenität bedingt ist, oder inwieweit durch einen Funktionsverlust des fehlgefalteten Proteins selbst. Um zytotoxische Effekte in humanen Zellen zu analysieren, wurden de novo generierte beta-Faltblattproteine untersucht, welche durch Aggregation in der Zelle keine Autofunktionsstörung auslösen sollten. Es wurde gezeigt, dass diese artifiziellen Proteine in HEK293T-Zellen amyloide Aggregate bildeten und zytotoxisch wirkten, im Vergleich zu de novo generierten alpha-helikalen Proteinen, welche löslich und homogen in der Zelle verteilt vorlagen und nahezu keine Zytotoxizität aufwiesen. Drei aus einer kombinatorischen Bibliothek ausgewählte de novo amyloide Proteine, beta4, beta17 und beta23, waren zytotoxisch mit der Gradierung beta4 < beta17 < beta23, sie induzierten Apoptose und veränderten die Zellmorphologie. Die Zytotoxizität korrelierte mit vorhandenen präfibrillären, intermediären Oligomeren. Die Proteine beeinträchtigten die Rückfaltung von GFP-Luciferase in gleicher Abstufung, ebenso eine Induktion der Stressantwort und die Proteinbiogenese. Die Aggregate colokalisierten mit GFP-Luciferase, jedoch nicht mit GFP. Eine massenspektrometrische Untersuchung der Interaktionspartner der drei de novo amyloiden Proteine in Kombination mit SILAC und Co-IP wies Interaktionen mit metastabilen Proteinen essentieller zellulärer Funktionen nach, dabei wurde Hsp110 als stark angereichertes Chaperon unter den Interaktoren identifiziert. Eine Überexpression von Hsp110 verminderte die Zytotoxizität der de novo Proteine beta4 und beta17, jedoch nicht beta23. Hsp110 war ebenfalls in der Lage, Aggregate teilweise zu solubilisieren und eine normalisierte Zellmorphologie wieder herzustellen. Um einen beta-Strang verkürzte oder verlängerte Mutanten der semitoxischen beta-Faltblattproteine beta4 und beta17 wiesen eine erhöhte Zytotoxizität auf, so dass wahrscheinlich generell beta-Faltblattproteine mit einer ungeraden Anzahl an beta-Strängen toxischer sind als ihre Derivate mit gerader Anzahl an beta-Strängen, da ungepaarte reaktive beta-Stränge vorliegen dürften. Zusammenfassend stellen die de novo beta-Faltblattproteine ein attraktives Modell dar, um aggregierende, amyloide Proteine ohne biologische Funktion in vivo zu untersuchen. Inkubation humaner Zellen mit dem Prolin-Analogon Azetidin-2-carbonsäure führte in Anwesenheit eines proteasomalen Inhibitors zur Verstärkung der Zytotoxizität, es entstanden amyloide Aggregate und präfibrilläre Intermediate, so dass die Hypothese der Verstärkung von Funktion und Pathogenität durch Aggregation in diesem System weiter untermauert wurde. Expression von Huntingtin mit expandierter PolyQ-Sequenz und einem angefügten hydrophoben CL1-Degron führte zu einer Erhöhung der Löslichkeit, zu verstärkter Inhibition des Ubiquitin-Proteasom-Systems und zu erhöhter Zytotoxitzität im Vergleich zu expandiertem Huntingtin ohne CL1-Degron. Die Zytotoxizität des mit Degron versehenen Huntingtins konnte mittels Überexpression von expandiertem Huntingtin ohne Degron durch Coaggregation verringert werden. Die Ergebnisse sprechen für die Hypothesen, dass präfibrilläre Intermediate die maßgeblichen zytotoxischen Spezies darstellen, während große Aggregate eine protektive Funktion einnehmen können. Eine Überexpression fehlfaltender Proteine kann in multifaktorieller Weise zur Interaktion mit essentiellen zellulären Proteinen führen und die Funktion metastabiler Proteine beeinträchtigen, was u.a. im Falle der de novo amyloiden Proteine zur Inhibition der Proteinbiogenese und der HSR führt. Akkumulation endogener fehlgefalteter Proteine durch proteasomale Inhibition legt den Mechanismus einer Verstärkung der Zytotoxizität durch amyloide, aggregierende Proteine per se nahe.

Fri, 3 Sep 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13153/ https://edoc.ub.uni-muenchen.de/13153/1/Giglmeier_Helene.pdf Giglmeier, Helene ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 13 Aug 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12105/ https://edoc.ub.uni-muenchen.de/12105/1/Wirth_Stefan.pdf Wirth, Stefan ddc:540, ddc:500, Fakultät für Chemie un

Thu, 12 Aug 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12058/ https://edoc.ub.uni-muenchen.de/12058/1/Trindler_Christian.pdf.pdf Trindler, Christian ddc:540, dd

Thu, 29 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/13692/ https://edoc.ub.uni-muenchen.de/13692/1/Fuchs_Sebastian.pdf Fuchs, Sebastian ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 27 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12125/ https://edoc.ub.uni-muenchen.de/12125/1/Mathes_Johannes.pdf Mathes, Johannes ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 27 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12545/ https://edoc.ub.uni-muenchen.de/12545/1/Schaubhut_Frank.pdf Schaubhut, Frank ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 26 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11933/ https://edoc.ub.uni-muenchen.de/11933/1/Dettmer_Ulf.pdf Dettmer, Ulf d

Mon, 26 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12103/ https://edoc.ub.uni-muenchen.de/12103/1/Fenske_Stefanie.pdf Fenske, Stefanie ddc:540, ddc:500, Fakultät für C

Thu, 22 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/12218/ https://edoc.ub.uni-muenchen.de/12218/1/Lowitzer_Stephan.pdf Lowitzer, Stephan ddc:540, ddc:500, Fakultät für Chemie

Tue, 20 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11819/ https://edoc.ub.uni-muenchen.de/11819/1/Lebold_Timo.pdf Lebold, Timo ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 16 Jul 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/11894/ https://edoc.ub.uni-muenchen.de/11894/1/Tarantik_Karina_R.pdf Tarantik, Karina