Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 05/06

Tue, 26 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19146/ https://edoc.ub.uni-muenchen.de/19146/1/Preimesser_Andreas.pdf Preimesser, Andreas ddc:540, ddc:500, Fakultät für Chemie und P

Thu, 14 Jan 2016 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19267/ https://edoc.ub.uni-muenchen.de/19267/2/Gnerlich_Felix.pdf Gnerlich, Felix ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 21 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19020/ https://edoc.ub.uni-muenchen.de/19020/1/Saller_Verena.pdf Saller, Verena ddc:540, ddc:500, Fakultät für

Tue, 15 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19287/ https://edoc.ub.uni-muenchen.de/19287/1/Ranft_Annekathrin.pdf Ranft, Annekathrin ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Die Gegenwart von Kohlenhydraten in biologischen Systemen ist von großer Bedeutung für eine Reihe von zellulären Zyklen, so z.B. die Zell-Zell-Erkennung, die Regulation von Proteinaktivitäten oder die Funktion von Kohlenhydraten als Liganden für die Zelladhäsion. Ferner besitzen eine Reihe von mucinartigen O-Glycanen vielfältige Funktionen in immunologischen Prozessen, welche mit unterschiedlichsten Krankheitsbildern, wie z.B. der Karzinogenese sowie Autoimmun- und Infektionskrankheiten einhergehen. Im Besonderen nehmen viele mucinartige Glycane mit funktionellen Oligosaccharidketten, einschließlich Sialinsäure-tragenden Strukturen wie z.B. Glycophorin, Leukosialin (CD 43 oder auch Sialophorin), Sialyl-Lewis x und Sialyl-Lewis a eine entscheidende Rolle als Liganden in Zell-Zell-Erkennungsmechanismen bei inflammatorischen Prozessen, T-Zell-Differenzierung und Krebsmetastasierung ein. Jüngst wurde eine Reihe innovativer Ansätze für die Immuntherapie von Krebs durch ein besseres Verständnis der abweichenden Glycosylierungsmuster von Glycoproteinen und Glycolipiden auf der Oberfläche von Krebszellen entwickelt, wobei ein Schwerpunkt der Arbeiten auf der Herstellung von kohlenhydrat-basierten Krebsvakzinen ruht. Vor diesem Hintergrund beschäftigt sich die folgende Arbeit mit der Synthese von Epitopen des Sialophorin (CD 43), einem bedeutenden O-Glycan-enthaltenden Sialoglycoprotein, welches auf Leukozyten exprimiert vorliegt und mit einer Reihe von Krankheitsbildern, wie rheumatoider Arthritis, Leukämie, dem Wiskott-Aldrich-Syndrom (WAS) und dem erworbenen Immunschwächesyndrom (AIDS), einhergeht. Der Aufbau komplexer Hexasaccharid-tragender Glycokonjugate konnte hierbei ausgehend von kommerziell erhältlichen Startmaterialien erfolgen. Im Vordergrund stand dabei die Entwicklung eines biomimetischen Synthesewegs, mit dessen Hilfe neben der natürlichen Struktur auch eine Reihe fluorierter Sialophorin-Derivate mit unterschiedlich fluorierten D-Galactose-Bausteinen in den beiden Trisaccharid-Einheiten hergestellt werden können. Dabei sollten sämtliche Glycosylaminosäuren mit einem zur Festphasensynthese kompatiblen Schutzgruppenmuster ausgestattet vorliegen, um einen Einbau in die N-terminale Partialsequenz des Glycoproteins Sialophorin (CD 43) zu erlauben. Beginnend mit der Synthese der unterschiedlich fluorierten D-Galactose-Derivate, der Glucosamin- und der Sialinsäure-Bausteine sollten die regio- und stereoselektiv notwendigen Di- und Trisaccharid-Bausteine chemisch verknüpft werden. Zu diesem Zweck wurden literaturbekannte Königs-Knorr- und Schmidt-Glycosylierungsreaktionen sowie stereoselektive Sialylierungsreaktionen unter Ausnutzung des Nitrileffekts genutzt. Ein weiterer Meilenstein war die regioselektive Glycosylierung der beiden Trisaccharid-Bausteine zu den geschützten Hexasaccharid-Threonin-Konjugaten, die damit für den Einbau in die N-terminale Partialsequenz des Sialoglycoproteins Sialophorin zur Verfügung stehen. Zudem wurden fluorierte sialylierte Trisaccharid-Threonin-Konjugate in die tandem repeat-Sequenz des epithelialen Mucins MUC1 eingebaut, die als potentielle Kandidaten neuer Antitumorvakzine eingesetzt werden können.

Thu, 3 Dec 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19027/ https://edoc.ub.uni-muenchen.de/19027/1/Dittrich_Sebastian.pdf Dittrich, Sebastian ddc:540, ddc:500, Fakultät für Ch

Mon, 30 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18948/ https://edoc.ub.uni-muenchen.de/18948/1/Neuhofer_Christian.pdf Neuhofer, Christian ddc:540, ddc:500, Fakultät für Ch

Most renewable energy sources suffer from intermittency and have to be coupled with sophisticated energy conversion and storage technologies. An elegant solution is offered by photoelectrochemical water splitting, where solar energy is directly converted into chemical energy by splitting water into oxygen and the energy carrier hydrogen. Photoelectrochemical water splitting requires two photoelectrodes which are immersed in an aqueous electrolyte. These photoelectrodes are semiconductors with valence and conduction bands straddling the redox potential of water. Upon illumination, electrons and holes are produced, separated and transferred to the electrolyte, leading to the evolution of oxygen at the photoanode and the evolution of hydrogen at the photocathode. The resulting hydrogen can be stored, transported and then either burnt in fuel cells to regain electrical energy or used for industrial applications like the Haber-Bosch process. The photoelectrodes are often nanostructured to increase the surface area, at which the reaction takes place. This strategy has been realized with several morphologies such as nanotubes, inverse opals, etc. and has often lead to performance increases of several hundred percent. Therefore, detailed knowledge of the morphology is important and can be obtained by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). TEM is a powerful technique that allows imaging samples with a resolution down to the sub-Ångstrom scale. In addition, TEM can be combined with spectroscopic methods such as electron energyloss spectroscopy (EELS) and energy-dispersive X-ray spectroscopy (EDX) to quantify the chemical composition. In this thesis, three different materials systems were studied by TEM: noble metal nanoparticles on TiO2 for hydrogen evolution with the sacrificial agent MeOH, Fe2O3/WO3 dual absorber photoanodes and photocathodes out of the novel material FeCrAl oxide. Titania is one of the most researched photoanode materials. However, it only absorbs UV light. Au and Au/Ag core-shell nanoparticles were deposited by the project partners Michael Karnahl and Sandra Peglow of the LIKAT and the INP Greifswald, respectively, on anatase thin films by photodeposition and radio frequency magnetron sputtering. These noble metal nanoparticles absorb visible light by surface plasmon resonance and also act as co-catalysts for electrons excited in the titania and injected into them. Cross-section were prepared for a detailed TEM investigation of the microstructure. The distribution of the nanoparticles varied greatly with the synthesis method, as photodeposited particles grew in and on top of the titania, whereas the plasma-deposited nanoparticles only grew on top. Different growth and coarsening mechanisms could be identified and correlated to the synthesis conditions by careful particle size distribution determination. In addition to defect-free nanoparticles, several defects such as five-fold twinning, grain boundaries and stacking faults were found. The TEM analysis was complemented by optical absorption and photocatalysis measurements, and the synthesis as well as the properties could be correlated to microstructural features. Due to its narrow band gap, hematite is a popular photoanode material. However, it also has several disadvantages, which were addressed by several studies. Tin-doping increased the transfer efficiency and therefore the photocurrent, with the tin being enriched at the surface of the hematite nanoparticles and hinting at a structure-function relationship. Deposition of a Co3O4 co-catalyst and the introduction of a conductive scaffold all further increased the photocurrent. Another performance-increasing approach, combining multiple photocatalytically active materials, was tested with Fe2O3/WO3 dual absorbers prepared by Ilina Kondofersky of the group of Prof. Thomas Bein. WO3 was systematically applied as a scaffold and/or as a surface treatment. The arrangement of the different materials and the interfaces between them was studied in detail by TEM. Both the host-guest approach and the surface treatment strongly increased the performance compared to the pure materials and several beneficial interactions could be identified. For example, WO3 strongly scatters visible light, resulting in increased absorption by Fe2O3 and higher current densities. We also determined a cathodic shift in the onset potential to 0.8 V and, compared to pure Fe2O3, increased transfer rates of up to 88 %, and can therefore conclude that the Fe2O3/WO3 dual absorbers are a very promising system. In spite of all the different performance-enhancing strategies developed so far, it is becoming apparent that all currently available materials, regardless of how heavily they are improved, will not reach sufficient performances. This has led to the search for novel materials and in this thesis, meso- and macroporous photocathodes with the overall stoichiometry Fe0.84Cr1.0Al0.16O3 were investigated in close cooperation with Ilina Kondofersky. Using TEM cross-sections, a phase separation into Fe- and Cr-rich phases was observed for both morphologies and could be correlated to the precursor stabilities. In comparison to the mesoporous layer, the macroporous photocathode had a significantly increased charge collection efficiency and therefore performance, proving the benefits of tuning the morphology. In all studies, performance-increasing strategies were successfully applied and we found the performance to depend heavily on the morphologies. By combining the results of all techniques, insight into the complex interplay between synthesis conditions, morphology and properties could be achieved and the gained knowledge is expected to benefit future work.

Fri, 13 Nov 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18951/ https://edoc.ub.uni-muenchen.de/18951/1/Matthias_Philipp.pdf Matthias, Philipp ddc:540, ddc:500, Fakultät für Chemie und Ph

Mon, 26 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18820/ https://edoc.ub.uni-muenchen.de/18820/1/Anamur_Cihad.pdf Anamur, Cihad ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Tue, 20 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18862/ https://edoc.ub.uni-muenchen.de/18862/1/Birk_Gudrun.pdf Birk, Gudrun ddc:540, ddc:500, Fakultät für Chemie und Pharma

Today’s carbon-based economy will not be sustainable in the future. Not only will the known reserves of fossil fuels, like oil, natural gas or coal, be significantly reduced within the next 100 years, but the continued burning of fossil fuels also emits greenhouse gases, which have led to a global increase in temperature, called global warming. To preserve the environment for future generations and to prepare for the time when we will inevitably run out of fossil fuel, we have to change the way we produce our primary energy and focus research and investments on renewable energy sources. While energy from wind and water is already harvested with very high efficiencies, the utilization of solar energy still offers big room for improvements. Although conventional crystalline silicon cells achieve efficiencies around 25 %, their production is very energy intensive and relies on advanced production technologies, which makes them still rather expensive. To make photovoltaics a major part of our energy landscape, an easily prepared type of solar cell consisting of cheap and abundant materials is required. Novel organometal halide perovskite-type materials fulfill these requirements and have proven to be serious competitors for conventional photovoltaics. After only four years of research they already achieve power conversion efficiencies above 20 %. This thesis introduces a fast and easy way to prepare planar heterojunction solar cells based on methylammonium lead iodide (MAPbI3). The photoactive layer is deposited in a 2-step deposition approach, where a thin film of the lead precursor is converted into the final perovskite simply by immersing it into a solution of the other component. The resulting films consist of individual crystals sizes a few 100 nm and covering the whole substrate without significant gaps or holes. Solar cells prepared by this method achieve power conversion efficiencies of 15 %. Furthermore, by adjusting the temperature of the immersion bath, the orientation of the perovskite crystals can be controlled. The orientation, together with the resulting change in efficiency and resistance, gives interesting insights into the anisotropic charge transport properties of this class of materials. Additionally, the conventionally used hole blocking layer, titanium dioxide, was replaced by one made of fullerene molecules. The efficiencies achieved by solar cells employing this kind of electron selective contact reached almost 10 %, although the reproducibility was initially very low. This was attributed to a partial dissolution of the fullerene film during the subsequent preparation steps. To increase the stability of the layer, it was photo-polymerized using UV radiation. This not only reduces the solubility and therefore increases the fraction of solar cells achieving high efficiencies; it also changed the energy levels close to the bandgap. The bandgap energy of organic lead halide perovskite materials is strongly dependent on the composition. By exchanging some or all of the iodide in MAPbI3 with bromide, the difference between valence and conduction band can be changed from 1.5 eV (pure iodide) to 2.25 eV (pure bromide). This substitution can be performed gradually, so that phase pure materials with properties in between the two extremes are obtained. The pure bromide MAPbBr3 perovskite, however, does not perform efficiently in a planar heterojunction solar cell. Its close relative based on formamidinium FAPbBr3 has also been investigated for its suitability as active solar cell material. Although it is structurally very similar to MAPbBr3, with equivalent light absorption and emission properties, a 10 fold higher efficiency was observed for the FA-based compound. This striking difference is mainly attributed to an increased photoluminescence lifetime, resulting in an increased diffusion length of the free charge carriers. Apart from their application as light absorbing materials in solar cells, perovskites have also been investigated for their application as light emitters. Depending on the perovskite used, it was possible to demonstrate red light emission (MAPbI3) or green emission (MAPbBr3).

Wed, 14 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18795/ https://edoc.ub.uni-muenchen.de/18795/1/Rest_Sebastian_F.pdf Rest, Sebastian Franz ddc:540, ddc:500, Fakultät für Chemie und P

Mon, 12 Oct 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18907/ https://edoc.ub.uni-muenchen.de/18907/1/Barth_Florian.pdf Barth, Florian ddc:540, ddc:500, Fakultät für Chemie und Phar

Idiopathic pulmonary fibrosis (IPF) is an irreversible and progressive disease of the lungs, which is characterized by aberrant tissue remodeling and massive deposition of extracellular matrix proteins. This process is mainly conducted by myofibroblasts, an activated fibroblast phenotype. During the pathogenesis of IPF, the fine alveolar structure is destroyed and gas exchange declines, finally resulting in organ failure. So far, pharmacological treatment options are very limited and lung transplantation still remains the only curative therapy. Pathologic tissue remodeling in IPF is closely connected to altered cell and protein homeostasis. The ubiquitin-proteasome system is critical for degradation of polyubiquitinated proteins in a spatially and timely controlled manner, thereby regulating protein levels. The proteasome is a multicatalytic enzyme complex consisting of a barrel shaped 20S catalytic core particle (CP) and one or two 19S regulatory particles (RP), thus forming active 26S/30S proteasomes. Dysregulation of the proteasome has been reported for several chronic diseases of the heart, brain, and also lung. Furthermore, inhibition of the proteasome has been shown to provide antifibrotic effects in different organs, including the lung. As nothing is known about proteasome function in the pathogenesis of IPF, the first aim of the present study was to analyze proteasomal regulation during tissue remodeling and myofibroblast differentiation. For that, lung fibroblasts were treated with transforming growth factor-β (TGF-β) and proteasome activity as well as composition was examined. For in vivo testing, the bleomycin mouse model of lung fibrosis was used and human lung tissue of IPF patients was analyzed. It was found that induction of myofibroblast differentiation by TGF-β mediated assembly of 19S RPs with 20S CPs, thereby forming 26S/30S complexes, which was critically dependent on the regulatory particle non ATPase 6 subunit (Rpn6). In addition, silencing of Rpn6 in primary human lung fibroblasts counteracted TGF β induced myofibroblast differentiation. During bleomycin-induced fibrotic remodeling of mouse lungs, increased formation of 26S/30S proteasomes was accompanied by augmented expression of Rpn6 in fibrotic lungs. Here, Rpn6 was highly expressed in hyperplastic alveolar epithelial cells and Clara cells. Overexpression of Rpn6 was also observed in myofibroblasts and hyperplastic bronchiolar basal cells of fibrotic lung tissue of IPF patients and accompanied by enhanced polyubiquitination of proteins. As therapeutic application of proteasome inhibitors in pulmonary fibrosis showed controversial results including beneficial antifibrotic effects but also toxicity, the second aim of this study was to test whether site specific inhibition of the proteasome, using the novel second generation inhibitor oprozomib, provides antifibrotic effects in the absence of systemic side effects after local pulmonary application. Oprozomib was compared to the FDA-approved proteasome inhibitor bortezomib and tested on the human alveolar epithelial cancer cell line A549 and on primary mouse alveolar epithelial type II cells regarding its cytotoxic effects. Oprozomib was less toxic than bortezomib and provided high selectivity for the chymotrypsin-like active site of the proteasome. In primary mouse lung fibroblasts, oprozomib showed significant antifibrotic effects like reduction of collagen I and α-smooth muscle actin expression at non-toxic doses. When applied locally into the lungs of healthy mice via instillation, oprozomib was well tolerated and effectively reduced pulmonary proteasome activity. In bleomycin-challenged mice, however, locally applied oprozomib resulted in accelerated weight loss and increased mortality. Furthermore, oprozomib failed to reduce fibrosis in these mice, but rather augmented fibrotic lung remodeling in bleomycin-challenged animals. To conclude, this study identified a novel mechanism for fibrotic remodeling of the lungs involving 26S/30S proteasome activation via Rpn6 upon TGF-β-mediated myofibroblast differentiation. Increased levels of Rpn6 and polyubiquitinated proteins in IPF lungs further suggest an important contribution of the ubiquitin-proteasome system to the pathogenesis of this disease. Inhibition of the proteasome with the novel site-specific proteasome inhibitor oprozomib provided low toxicity and antifibrotic effects in alveolar epithelial cells and pulmonary fibroblasts. These results could not be confirmed in pulmonary fibrosis of bleomycin-treated mice, as oprozomib treatment showed high toxicity in fibrotic animals. In light of these data, current proteasome inhibitors, which block the catalytic core, might be too toxic as therapeutic agents for the treatment of fibrotic lung diseases. However, interference with the formation of 26S/30S proteasomes, as shown by Rpn6 knockdown, might provide a novel concept for therapeutic regulation of proteasome activity in lung fibrosis.

Wed, 30 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/19068/ https://edoc.ub.uni-muenchen.de/19068/1/Niesser_Juergen_G.pdf Niesser, Jürgen Gerd ddc:540, ddc:500, Fakult

Fri, 18 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18799/ https://edoc.ub.uni-muenchen.de/18799/1/Moriya_Kohei.pdf Moriya, Kohei ddc:540, ddc:500, Fakultät für Chemie und Phar

Mon, 7 Sep 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18665/ https://edoc.ub.uni-muenchen.de/18665/1/Stuerzer_Christine.pdf Stürzer, Christine ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Porous materials play an important role in numerous environmental applications including energy storage, energy conversion and environmental remediation systems. Reducing structural features down to the nanoscale drastically alters materials properties and leads to the enhancement of materials performance. The successful fabrication of efficient functional materials requires a high degree of control over their morphology addressing the needs of target applications. The goal of this work was to develop a versatile general approach towards the synthesis of nanoporous metal oxides by using biogenic cellulose nanocrystals. Nanocrystalline cellulose (NCC) is an abundant biological nanomaterial that can be extracted from natural bulk celluloses. The present thesis demonstrates that the unique properties of NCC enable the efficient synthesis of porous titania and iron oxide (hematite) thin films by using sacrificial templating with cellulose nanocrystals. In particular, this study reveals the mechanism of metal oxide formation in the presence of cellulose, as well as the effect of NCC-templated porous scaffolds on titania performance in photocatalysis and dye sensitized solar cells. Chapter 1 provides general information about properties, application areas and common synthesis methods of nanoporous metal oxides, with an emphasis put on titanium oxide materials and biotemplating approaches. Chapter 2 discusses the basic principles of analytical methods employed to characterize porous nanomaterials. Chapters 3‒6 reveal the experimental procedures towards NCC-templated porous titania and hematite thin films, their characterization and their applications. First, the extraction of cellulose crystals from bulk celluloses is discussed. Different cellulose sources, as well as variable hydrolysis parameters have been employed to define the optimal procedure for the NCC preparation. Cotton fibers have provided the best results regarding the crystallinity, purity and shape of extracted cellulose crystals. Furthermore, repeated washings have been shown to narrow down the size distribution and to improve the crystallinity of cotton NCC. Chapter 4 focuses on the synthesis of porous titania thin films assisted by nanocrystalline cellulose. The tunable porosity of titania thin films is a key factor for successful applications in photovoltaics, sensing and photocatalysis. To synthesize NCC-templated titania, the cellulose nanocrystals are introduced to a titania precursor solution. The colloidal mixtures can be directly spin- or dip- coated on glass, silicon and transparent conducting oxide (TCO) substrates and then calcined to remove the template and to crystallize the titania porous network. The obtained structures are highly porous anatase morphologies having well-defined, narrow pore size distribution. We show that by varying the titania-to-template ratio it is possible to tune the surface area, pore size, pore anisotropy and dimensions of titania crystallites in the films. Post-treatment at high humidity and subsequent slow template removal promote pore widening; this treatment is also beneficial for the multilayer deposition of thick films. The NCC-templated mesoporous titania films show very high activity in the photocatalytic NO (nitrogen(II) oxide) conversion and in the degradation of 4-chlorophenol. Furthermore, the films are successfully applied as anodes in dye-sensitized solar cells. Chapter 5 presents a strategy toward enhancement of the photocatalytic activity of NCC-templated titania thin films by introducing solvothermally synthesized preformed anatase nanoparticles into a sol-gel based biotemplated titania scaffold. The synthesis is based on the self-assembly of two types of precursors, namely crystalline and sol-gel titania, directed by the biogenic NCC template. Due to the shape persistence of the template, the NCC-templated titania scaffolds can accommodate large amounts of preformed titania without a significant reduction of the film porosity. The resulting dual source titania thin films containing different amounts of preformed crystalline species were investigated with time resolved microwave conductivity (TRMC) measurements and tested in the photocatalytic conversion of 4-chlorophenol. The gradual addition of preformed nanoparticles leads to a consistent increase of the mean size of titania crystalline domains, whereas the porosity of the composite is well-preserved due to the rigid nature of the NCC template. The microwave conductivity studies establish increased photoconductivity of the films containing preformed anatase nanoparticles, in comparison to that of films made without the nanoparticles. The synergistic features of the dual source titania, namely the improved crystalline properties brought by the preformed nanocrystals in combination with the high surface area provided by the NCC-templated sol-gel titania, result in a very high photocatalytic activity of the films in the photocatalytic decomposition of 4-chlorophenol. In quantitative terms, the dual source titania films prepared with 75% nanoparticles exhibit a first order degradation rate constant of 0.53 h-1, strongly outperforming the activity of commercial P90 nanopowder showing a rate constant of 0.17 h-1 under the same conditions. We have also adapted the NCC templating protocol for the fabrication of porous iron oxide (hematite) thin films. Chapter 6 discusses the formation of porous iron oxide nanostructures via sol-gel transformations of molecular precursors in the confined space of self-organized cellulose nanocrystals used as a shape-persistent template. The obtained structures are highly porous hematite morphologies featuring pronounced anisotropic porosity. The character of the porous nanostructure depends on the iron salt used as precursor and on the heat treatment, respectively. Moreover, a post-synthetic hydrothermal treatment of the NCC/iron salt composites strongly affects the crystal growth, as well as the porous nanomorphology of the obtained hematite scaffolds. We demonstrate that the hydrothermal treatment alters the crystallization mechanism of the molecular iron precursors, which proceeds via the formation of anisotropic iron oxyhydroxide species. The present study reveals that the nanocellulose templating technique enables a straightforward fabrication of a variety of porous crystalline scaffolds with well-defined mesoporous structure. For the first time the NCC has been used for the fabrication of homogeneous porous metal oxide films on different substrates, in contrast to the previously reported powders or free-standing membranes. The versatility and flexibility of the NCC templating approach offers broad perspectives towards the generalization of this method for the fabrication of different types of nanoporous metal oxides.

Thu, 13 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18637/ https://edoc.ub.uni-muenchen.de/18637/1/Sohmen_Daniel.pdf Sohmen, Daniel ddc:540, ddc:500, Fakultät f

Neurodegenerative disorders such as Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, Amyotrophic Lateral Sclerosis or Prion diseases are chronic, incurable and often fatal. A cardinal feature of all neurodegenerative disorders is the accumulation of misfolded and aggregated proteins. Depending on the disease, these aggregated proteins are cell type specific and have distinct cellular localizations, compositions and structures. Despite intensive research, the contribution of protein misfolding and aggregation to the cell type specific toxicity is not completely understood. In recent years, quantitative proteomics has matured into an exceptionally powerful technology providing accurate quantitative information on almost all cellular proteins as well as protein interactions, modifications, and subcellular localizations, which cannot be addressed by other omics technologies. The aim of this thesis is to investigate key features of neurodegeneration such as misfolded proteins and toxic protein aggregates with cutting edge proteomics, presenting a technological “proof of concept” and novel insights into the (patho)physiology of neurodegeneration.

Wed, 5 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18537/ https://edoc.ub.uni-muenchen.de/18537/1/Kukushkin_Yury.pdf Kukushkin, Yury

Tue, 4 Aug 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18533/ https://edoc.ub.uni-muenchen.de/18533/1/Izsak_Daniel.pdf Izsák, Dániel ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

The cylindrical chaperonin GroEL and its lid-shaped cofactor GroES of Escherichia coli perform an essential role in assisting protein folding by transiently encapsulating non-native substrate in an ATP-regulated mechanism. It remains controversial whether the chaperonin system functions solely as an infinite dilution chamber, preventing off-pathway aggregation, or actively enhances folding kinetics by modulating the folding energy landscape. Here we developed single-molecule approaches to distinguish between passive and active chaperonin mechanisms. Using low protein concentrations to exclude aggregation, in combination with highly sensitive spectroscopic methods, such as single-molecule Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS), we measured the spontaneous and GroEL/ES-assisted folding of double-mutant maltose binding protein (DM-MBP), and a natural GroEL substrate - dihydrodipicolinate synthase (DapA). We show that both proteins form highly flexible, kinetically trapped folding intermediates, when folding in free solution and do not engage in inter-molecular interactions, such as aggregation, at sufficiently low concentration. We find that in the absence of aggregation, GroEL/ES accelerates folding of DM-MBP up to 8-fold over the spontaneous folding rate. The folding of DapA could be measured at physiological temperature and was found to be ~130-fold accelerated by GroEL/ES. As accelerated folding was independent of repetitive cycles of protein binding and release from GroEL, we demonstrate that iterative annealing does not significantly contribute to chaperonin assisted substrate folding. With a single molecule FRET based approach, we show that a given substrate molecule spends most of the time (~80%) during the GroEL reaction cycle inside the GroEL central cavity, in line with the inner GroEL cage being the active principle in folding catalysis. Moreover, photoinduced electron transfer experiments on DM-MBP provided direct experimental evidence that the confining environment of the chaperonin cage restricts polypeptide chain dynamics. This effect is mainly mediated by the net-negatively charged wall of the GroEL/ES cavity, as shown using the GroEL mutant EL(KKK2) in which the net-negative charge is removed. Taken together, we were able to develop novel approaches, based on single molecule spectroscopy and making use of GroEL as a single molecule sorting machine, to measure GroEL substrate folding rates at sub-nanomolar concentrations. We also, for the first time, provide direct experimental evidence of conformational restriction of an encapsulated polypeptide in a chaperonin cage. Our findings suggest that global encapsulation inside the GroEL/ES cavity, not iterative cycles of annealing and forced unfolding, can accelerate substrate folding by reduction of an entropic energy barrier to the folded state, in strong support of an active chaperonin mechanism. Accelerated folding is biologically significant as it adjusts folding rates relative to the rate of protein synthesis.

Thu, 30 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18685/ https://edoc.ub.uni-muenchen.de/18685/1/Liebner_Robert.pdf Liebner, Robert ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 27 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18504/ https://edoc.ub.uni-muenchen.de/18504/1/Patschinski_Pascal.pdf Patschinski, Pascal ddc:540, ddc:500, Fakultät für Chemie und Phar

Wed, 22 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18742/ https://edoc.ub.uni-muenchen.de/18742/1/Hellenbrand_Tim.pdf Hellenbrand, Tim ddc:540, ddc:500, Fak

Tue, 21 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18520/ https://edoc.ub.uni-muenchen.de/18520/1/Muehlbacher_Wolfgang.pdf Mühlbacher, Wolfgang ddc:54

Bond cleavage and formation are key steps in chemistry and biochemistry. The present work investigates the generation of diphenylmethyl cations (Ph2CH+) via photoinduced bond cleavage of diphenylmethyl derivatives with a cationic or neutral leaving group. The resulting Ph2CH+ cations and its numerous derivatives serve as reference electrophiles for one of the most extensive reactivity scales covering 40 orders of magnitude. In chapter 1, the focus is on the initial bond cleavage of diphenylmethyltriphenylphosphonium ions (Ph2CH−PPh3+) exhibiting a cationic leaving group. With the help of state-of-the-art quantum chemical and quantum dynamical methods, the reaction mechanism of the bond cleavage is revealed. Using a reduced model system, the potential energy surfaces can be calculated at the ONIOM level of theory along specially designed reactive coordinates. Two competing reaction channels emerge: a homolytic one in the S1 state and a heterolytic one in the ground state. They are connected via an energetically accessible conical intersection which makes an efficient generation of the observed Ph2CH+ cations feasible. In contradiction with the experiment in polar or moderately polar solvents, quantum dynamical calculations for the isolated molecule reveal the formation of Ph2CH• radicals. While electrostatic solvent effects are negligible in this system, dynamic solvent effects emerge as being essential to explain the molecular mechanism. Two methods with increasing complexity to describe the dynamic impact of the solvent environment are developed. The first approach, the dynamic continuum ansatz, treats the environment implicitly. It uses Stokes’ law and the dynamic viscosity of the solvent in combination with quantum chemically and dynamically evaluated quantities to obtain the decelerating force exerted on the dissociating fragments. The ansatz does not require any fitting of parameters. The second method, the QD/MD approach, is based on an explicit treatment of the solvent surrounding. It combines molecular dynamics (MD) simulations of the reactant in a box of solvent molecules with quantum dynamics (QD) calculations of the reactant’s dynamics. In this way, a more detailed microscopic picture of the molecular process can be derived taking into account individual arrangements of the solvent. Both methods unveil the crucial impact of the solvent cage on the bond cleavage mechanism. It hinders the free dissociation in the S1 state and guides the molecular system to the conical intersection. QD simulations including the non-adiabatic coupling around the conical intersection show the formation of Ph2CH+ within ∼400 fs which compares well with the initial rise of the cation absorption in the experiment. Chapter 2 deals with the position of the counterion X– in the ion pairs Ph2CH−PPh3+ X–, PhCH2−PPh3+ X–, and (p-CF3-C6H4)CH2−PPh3+ X– in solution with X– being Cl–, Br–, BF4–, and SbF6–. These structures are essential to clarify the role of oxidizable counterions like e.g. Cl– during the initial bond cleavage in dichloromethane. The structures determined quantum chemically in dichloromethane show a similar counterion position than in the crystal. They are confirmed by the good accordance of the calculated and measured 1H NMR shifts. The C(α)–H···X– hydrogen bonds account for the pronounced counterion-dependent 1H NMR shifts of the C(α)–H in CD2Cl2. The strong downfield shift of the signals increases according to SbF6– < BF4–

Fri, 17 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18511/ https://edoc.ub.uni-muenchen.de/18511/1/Mathaes_Roman_Willi.pdf Mathäs, Roman Willi ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Thu, 16 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18492/ https://edoc.ub.uni-muenchen.de/18492/1/Hermawan_Adam.pdf Hermawan, Adam ddc:540, ddc:500, Fakultät für Chemie und

Tue, 14 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18439/ https://edoc.ub.uni-muenchen.de/18439/1/Antipova_Anna.pdf Antipova, Anna ddc:54

In recent years there has been a considerable focus on the development of subunit vaccines, preferred over traditional vaccines for reasons of safety and purity. However, subunit vaccines are less immunogenic than attenuated vaccines and need therefor multiple administrations in combination with immunostimulatory adjuvants, in order to induce immunity. The sustained release of a vaccine together with the release of an adjuvant is a potential alternative to giving multiple doses. The aim of this thesis was to manufacture lipid implants for vaccine delivery by twin-screw (tsc) extrusion and evaluate the potency of these lipid systems to stimulate an immune response in vivo. To accomplish this, lipid implants consisting of cholesterol, soybean lecithin, and Dynasan 114 (D114) were prepared. Different formulations were evaluated for their extrudability before adding the model antigen ovalbumin (OVA) and the adjuvant Quil-A (QA) to the formulation. Investigating the release behaviour of OVA and QA showed that mainly cholesterol influences the release behaviour of OVA, increasing the fraction of cholesterol slows down the release of OVA. To further slow down the release of OVA from the implants, they were cured at different temperature resulting in an even longer OVA release. Furthermore, the addition of QA to the implants influenced the release behaviour of OVA and vice versa. The investigation of the implant polymorphism after the extrusion process as well as during storage showed good stability. To combine the advantage of particulate delivery and sustained release, preformed liposomes were incorporated into the implants prior to extrusion. For the analysis of the immune response, two sets of animal experiments in mice were performed, one evaluating the kinetics of the release of the model antigen in vivo, a second one to evaluate the immune response in vivo. Evaluation of these data indicated a correlation between the in vitro and in vivo release behaviour of OVA. Furthermore, immune responses similar to those induced by two booster injections, consisting of OVA and alum could be achieved using implant formulations containing QA. These results further emphasized the importance of adjuvant in the formulation. The incorporation of preformed liposomes into the implants on the other hand did not lead to an improved outcome. In a second part of this work, an in vivo tumour study was prepared, using the TRP2 peptide as active ingredient. Due to the use of this expensive peptide, a transfer to a different extruder was necessary. The influence that a change of the production device has on the implants characteristics was investigated. Once the formulation was adapted to the new extruder, implants containing TRP2 and QA were produced. The in vitro release of TRP2 proved to be very slow, much different from the OVA release. Furthermore, the preparation of vesicular phospholipid gels (VPGs) as an alternative lipid delivery system for TRP2 was investigated. The TRP2 release from the VPGs was also slow and incomplete. Both formulations were used in an in vivo tumour growth study. Mice were injected with B16F10luc2 melanoma cells, 6 days later formulations were administered. VPGs showed adverse reactions in the mouse and are therefore not s suitable delivery system. TRP2 implants showed a slow delay in the start of tumour growth, but were not more potent that TRP2 in PBS injections given to the mice. The very slow in vitro release data of TRP2 brought up the question about interactions between the lipid implants and the peptide influencing the release. Choosing peptides of different size and hydropathy, an investigation of their release behaviour and interaction with the implants was conducted. In conclusion, lipid implants were well tolerated and offer a great potential as sustained release delivery system for vaccines. They allow releasing the active component and the adjuvant together, enabling to achieve a strong immune response.

Tue, 14 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18623/ https://edoc.ub.uni-muenchen.de/18623/1/Zgela_Dominik.pdf Zgela, Dominik ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 13 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18883/ https://edoc.ub.uni-muenchen.de/18883/1/Karch_Florian.pdf Karch, Florian ddc:540, ddc:

One of the most critical periods in mammalian development is early embryogenesis. Its timing and morphology have been well studied, but molecular processes are still poorly understood. Several transcriptomic studies have addressed these molecular events. However, development of early embryonic stages before activation of the embryonic genome depends on sufficiently stored products of the maternal genome and adequate de/activation and relocation of proteins. Therefore, this thesis addresses early mammalian development, i.e., oocyte maturation and the first steps of embryogenesis, by holistic iTRAQ-based discovery approaches and by a targeted approach based on SRM. Numerous proteins important for oocyte maturation and embryogenesis so far not described in the mammalian system were identified and quantified, and protein profiles for key players previously described in the literature were contributed. In oocyte maturation, bioinformatics analysis revealed proteins increasing in abundance involved in rearrangement of the cytoskeleton, protein transport and cell cycle progression. Supplementation of oocyte maturation media with the gonadotropins follicle stimulation hormone and luteinizing hormone resulted in an increase of developmentally important proteins, but did not change developmental rates. Addition of growth hormone during oocyte maturation led to diverse and opposing effects at the level of proteins and also did not influence subsequent embryo development. During early embryogenesis, a considerable fraction of proteins continuously increased in abundance, despite a strongly attenuated rate of translation reported for this period. Bioinformatics analysis revealed particularly interesting proteins involved in the p53 pathway, lipid metabolism and mitosis. Activation of the unfolded protein response is demonstrated, which may be an indicator of the still lower success rates of in vitro versus in vivo embryo production. Relevant differences between transcript and protein abundance levels were detected, e.g., for WEE2, which highlights the importance of innovative proteomic tools and workflows to complement transcriptome data of early embryogenesis. iTRAQ results of early embryogenesis were successfully verified by targeted 5-plex SRM analysis. By principal component analysis, SRM quantifications comprising a panel of only five proteins were shown to discriminate between all four developmental stages analyzed. Using an expanded 27-plex SRM assay, proteins were quantified in nine developmentally interesting stages and absolute protein contents were established for nine proteins. SRM is a highly sensitive tool for detection of disturbances and disorders of embryonic development at the molecular level, thus complementing morphological analyses by high resolution microscopy.

Wed, 8 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18448/ https://edoc.ub.uni-muenchen.de/18448/1/Weber_Janine.pdf Weber, Janine ddc:540, ddc:500, Fakultät für Chemie und Ph

Tue, 7 Jul 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18509/ https://edoc.ub.uni-muenchen.de/18509/1/Wiesenmayer_Josef_Erwin.pdf Wiesenmayer, Josef Erwin ddc:540, ddc:500, Fakultät

Tue, 30 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18530/ https://edoc.ub.uni-muenchen.de/18530/1/Friederichs_Gina.pdf Friederichs, Gina ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Fri, 26 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18403/ https://edoc.ub.uni-muenchen.de/18403/1/Hettstedt_Christina.pdf Hettstedt, Christina ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Mon, 15 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18564/ https://edoc.ub.uni-muenchen.de/18564/1/Baumann_Dominik.pdf Baumann, Dominik ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

RNA interference (RNAi) is a highly conserved process of gene silencing in which Argonaute family proteins are guided by small RNA molecules to complementary targets. In the fission yeast Schizosaccharomyces pombe, RNAi is required for heterochromatin formation at centromeres. Although it seems counterintuitive, pericentromeric heterochromatin in fission yeast is transcribed. The transcripts are processed by RNAi machinery, which is in turn guided back to the pericentromeric repeats by sequence complementarity of the Argonaute-bound small interfering RNA (siRNA) and the nascent transcript. This generates a positive-feedback loop of siRNA amplification that recruits factors required for the assembly of heterochromatin. Previously, it was suggested that a fission yeast class of Dicer-independent small RNAs called primal small RNAs (priRNAs) initiates the positive-feedback loop of siRNA generation and heterochromatin assembly. However, the biogenesis of priRNAs as well as of Dicer-independent small RNAs from other organisms was not well understood. The results presented here identify Triman, a novel 3’-5’ exonuclease that is involved in the final step of biogenesis of both priRNAs and siRNAs in fission yeast. It was observed that Argonaute binds longer priRNA and siRNA precursors from the total RNA fraction. This is followed by the recruitment of Triman to trim 3’ ends of Argonaute-bound small RNAs to the mature size. The final trimming of priRNAs and siRNAs is required for de novo heterochromatin formation at centromeres and the mating-type locus as well as for the maintenance of facultative heterochromatin islands. Furthermore, it was shown that in cells lacking Rrp6, a nuclease subunit of the exosome, RNAi targets various genes across the yeast genome. This demonstrated that the exosome protects the genome against aberrant RNAi. Spurious RNAi targeting in rrp6∆ cells at majority of loci occurs via accumulation of antisense transcripts that are processed into priRNAs in a Triman-dependent manner. These results suggest that Argonaute association with cellular degradation products which are processed into priRNAs might serve as a surveillance mechanism to guard the genome against invading genomic elements (Marasovic et al. 2013).

Fri, 12 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18691/ https://edoc.ub.uni-muenchen.de/18691/1/Butryn_Agata_A.pdf Butryn, Agata Anna ddc:540, ddc:500, Fakultä

Thu, 11 Jun 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18506/ https://edoc.ub.uni-muenchen.de/18506/1/Plodek_Alois.pdf Plodek, Alois ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

The 2-oxoglutarate and Fe (II)-dependent ten-eleven translocation (TET) enzymes can oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxycytosine (5caC). Thus, it was hypothesized that this could be a pathway for active DNA demethylation. However, several studies have shown that 5hmC levels accumulate especially in neurons suggesting a role as an epigenetic mark. Nevertheless, the mechanism controlling TET activity and the role of 5hmC are poorly understood. In particular, it is not known how the neuronal TET3 isoform lacking a DNA binding domain is targeted to the DNA. In this study, the role of 5hmC during retinal maturation by genome-wide mapping of 5hmC in immature (postnatal week 2) and mature mouse retina (postnatal week 3) was studied and correlated with expression data. Furthermore, interaction partners of neu-ronal TET3, the main isoform in retinal neurons, were identified. 5hmC accumulates during retinal maturation especially in retinal and neuronal genes. Furthermore, the accumulation of 5hmC is associated with increased transcription. Among the identified proteins the transcriptional repressor REST was identified as a highly enriched TET3-specific interactor. Interestingly, REST was able to enhance TET3 hydroxylase activity. Furthermore, increased 5hmC levels were detected in REST target genes during retinal maturation and overexpression of TET3 activated transcription of REST-target genes. Moreover, NSD3 and two other histone methyltransferases were found to interact with TET3 that are able to mediate H3K36 trimethylation. Finally, it was shown that TET3 is able to enhance NSD3-mediated H3K36 trimethylation to pro-mote transcriptional activation. In conclusion, the data suggests that 5hmC is a stable epigenetic base in retinal neurons that is involved in transcriptional activation. Furthermore, it was shown that 5hmC is generated by TET3 that is recruited to the DNA by transcriptional regulators such as REST in a context-specific manner.

Even though platelets are the smallest cells in circulating blood, they play an integral role in blood clotting where they are activated, adhere to the vessel wall, and contribute to hemostasis. But over the years it was discovered that those anucleate cells have more extended functions. They organize their cellular vitality similar to nucleated cells and have an active protein metabolism performing protein de novo synthesis as well as protein degradation. One of the main degradation systems in cells is the proteasome. Besides protein quality control, the proteasome is involved in important cellular processes like cell survival, transcription, development, selective elimination of abnormal proteins and antigen processing. A dysregulation of this multicatalytic protein complex leads to various disease developments. Proteasome inhibitors, for instance, have been studied for treating cancer. Platelets like nucleated cells contain a proteasome. However, the impact of the proteasome on platelet functions remains poorly investigated until today. A better knowledge of signaling pathways in platelets aids in understanding how alterations in proteasome functions affect platelet-mediated processes and diseases. This study confirms the existence of a functional proteasome in human platelets and illustrates an important role in platelet biology, as well as sepsis. With this study the role of the proteasome in anucleate platelets is demonstrated in more detail and a signaling pathway regulating its activity was observed. Here, the proteasome in platelets is linked to platelet aggregation. First, proteasome inhibitors epoxomicin and bortezomib reduce ADP- and collagen-induced aggregation. Furthermore, the 26S chymotrypsin-like activity of the proteasome is enhanced when platelets are incubated with the platelet agonist collagen. Additionally, cytoskeletal proteins Filamin A and Talin-1, which are crucial for platelet activation, were identified as proteasome substrates and increased cleavage of these proteins occurs with proteasome activation. To investigate possible mechanisms of regulating the proteasome, the signaling pathway related to NFκB was analyzed under platelet agonist treatment. The NFκB pathway, that mediates aggregation, is initiated when platelets are treated with collagen and the inhibitory protein of NFκB, IκBα, is degraded in collagen-stimulated platelets. More interestingly, NFκB inhibitors prevent collagen-stimulated enhancement of the proteasome activity. In return the connection of the proteasome and the NFκB pathway is further demonstrated as NFκB inhibitors restrict cleavage of the proteasome substrate Talin-1. These results propose a novel pathway that involves the proteasome and that is in return connected with non-genomic functions of NFκB in regulating platelet aggregation. In a second part this work shows for the first time that mitochondrial membrane depolarization in platelets correlates with the disease course and disease severity in patients with sepsis. Additionally, during these studies increased proteasome activity was observed in sepsis patients compared to control patients and pathogenic bacteria intensified the 26S trypsin-like activity of human platelets. Therefore, molecular markers of platelet vitality may be valuable parameters to help evaluating the clinical outcome of sepsis patients. In summary, the study confirms the existence of a functional proteasome in human platelets, contributes to our understanding how the proteasome affects platelet functions such as aggregation and how this may be regulated on a molecular basis. Furthermore, it allows for new insights in the disease course of sepsis and identifies new molecular markers for assessing the disease severity and clinical outcome of sepsis patients.

Epithelial barriers are central to the development of metazoans by compartmentalizing the body in distinct chemical milieus essential for the function of many organs. One such barrier is the blood-brain barrier, which isolates the nervous system from the body fluid to maintain its ionic homeostasis and ensure nerve pulse transmission. In Drosophila, the blood-brain barrier is formed late in embryogenesis by a thin epithelium of subperineurial glia that ensheath the nervous system. Similar to other epithelia, subperineurial glia seal the paracellular space by forming large multiprotein complexes at the lateral membrane, the septate junctions (SJs), which impede free diffusion and mediate barrier function. To identify novel genes required for blood-brain barrier formation, we followed a genome-wide in vivo RNAi approach. We initially screened almost the whole genome for genes required in glia for adult viability and impressively identified 3679 potential candidates. Subsequently, we tested these candidates for requirement in subperineurial glia for adult survival and identified 383 genes. At a last step, we directly asked if blood-brain barrier formation is compromised in the knock-down of the genes by performing the embryonic dye penetration assay in a selection of candidates and identified five genes that play a role during barrier development. Three of these genes, macroglobulin complement-related (mcr) and the previously uncharacterized pasiflora1 and pasiflora2 are further characterized in the context of this thesis. Here we show that all three proteins are novel components of the Drosophila SJ. Pasiflora1 and Pasiflora2 belong to a previously uncharacterized family of tetra-spanning membrane proteins, while Mcr was reported to be a secreted protein in S2 cells required for phagocytosis and clearance of specific pathogens. Through detailed phenotypic analysis we demonstrate that the mutants show leaky blood-brain and tracheal barriers, overelongated tracheal tubes and mislocalization of SJ proteins, phenotypes that are characteristic of SJ mutants. Consistent with the observed phenotypes, the genes are co-expressed in SJ-forming embryonic epithelia and glia and are required cell-autonomously to exert their function. In columnar epithelia, the proteins localize at the apicolateral membrane compartment, where they colocalize with other SJ proteins, and similar to known SJ components, their restricted localization depends on other complex members. Using fluorescence recovery after photobleaching experiments, we demonstrate for Pasiflora proteins that they are core SJ components, as they are required for complex formation and themselves show restricted mobility within the membrane of wild-type epithelial cells, but fast diffusion in cells with disrupted SJs. Taken together, our results show that Pasiflora1 and Pasiflora2 are novel integral SJ components and implicate a new family of tetraspan proteins in the development of cell junctions. In addition, we find a new unexpected role for Mcr as a transmembrane SJ protein, which raises questions about a potential intriguing link between epithelial barrier function, phagocytosis and innate immunity and has potential implications for the function of occluding junctions.

Tue, 19 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18308/ https://edoc.ub.uni-muenchen.de/18308/1/Achrainer_Florian.pdf Achrainer, Florian ddc:540, ddc:500, Fakultät für Chemie und Pha

Mon, 11 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18289/ https://edoc.ub.uni-muenchen.de/18289/1/Kettner_Marcos.pdf Kettner, Marcos Adrian ddc:540, ddc:500, Fakultät für Chemie

Peroxisomes are highly dynamic organelles of eukaryotic cells, carrying out essential oxidative metabolic processes. These organelles scavenge reactive oxygen species such as hydrogen peroxide (H2O2) and catabolise fatty acids, which are particular hallmarks and highly conserved features of peroxisomes among different species. Peroxisomal proteins and enzymes are encoded by nuclear DNA and therefore, targeted post-translationally into the peroxisomal matrix. A special class of proteins, collectively called peroxins, perform certain cellular tasks, such as peroxisomal matrix protein import or membrane development in order to maintain peroxisome biogenesis as well as a constant flux of matrix proteins into peroxisomes. The type II AAA+ peroxins Pex1/Pex6 are a core component of the peroxisomal matrix protein import system. ATPases of the AAA+ family of proteins generally assemble into large, macromolecular machines, structurally remodelling their substrate protein, which is driven by the hydrolysis of ATP. The main function of Pex1/6 complexes is to release the receptor Pex5 from peroxisomal membranes after matrix protein import. This relocation of Pex5 into the cytosol ensures a constant pool of available receptor molecules for subsequent cycles of protein import into peroxisomes. Accordingly, certain mutations in mammalian Pex1/Pex6 proteins compromise peroxisome biogenesis and thus, lipid metabolism, causing severe genetic Zellweger diseases in humans. In collaboration with Professor Ralf Erdmann and colleagues at the Ruhr-Universität Bochum, we characterize the structure and function of the AAA+ Pex1/6 complex from yeast Saccharomyces cerevisiae. Single particle electron microscopy (EM) in combination with biochemical assays allows us to analyze how ATP turnover is related to the biological function of the Pex1/6 complex. This study presents EM structures of Pex1/6 complexes assembled in the presence of ADP, ATP, ADP-AlFx and ATPγS, providing a comprehensive structural characterization of the heterohexameric type II AAA+ complex in different nucleotide states. Our EM reconstructions reveal an unexpected triangular overall shape, different than observed for the closely related and well-characterized homohexameric AAA+ protein p97. We show that the heterohexameric Pex1/6 complex is composed of a trimer of heterodimers with alternating subunit arrangement of Pex1 and Pex6 moieties. Furthermore, our results suggest that conserved aromatic residues, lining the central pore of the Pex1/6 D2 ring mediate substrate interactions. These residues correspond to substrate interaction regions in related AAA+ proteins. Comparing Pex1/6 EM reconstructions in different nucleotide states implicates that the mechanical function of Pex1/6 involves an N- to C-terminal protein translocation mechanism along the central pore. The Pex1/6 EM structures resolve symmetric and asymmetric large-scale domain motions, which likely create a power stroke during cycles of ATP binding and hydrolysis. We conclude that Pex5 is probably partially or completely unfolded while it is threaded through the central pore of Pex1/6 complexes. In addition, ATP hydrolysis assays of Pex1/Pex6 complexes containing single amino acid exchanges in individual Walker B motifs reveal that not all active sites are functionally equivalent. In isolated complexes, ATP turnover mainly occurs in Pex6 D2 domains, while Pex1 subunits sustain the structural integrity of the complex. We further resolve the structures of Pex1/6 Walker B variants and observe mutually exclusive protomer-protomer communication. In the Pex1/6 complex, a Walker B mutation induces ATP hydrolysis in the adjacent D2 domain, presenting a structural framework of protomer-protomer communication in the AAA+ heterohexamer.

Thu, 7 May 2015 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18376/ https://edoc.ub.uni-muenchen.de/18376/1/Grimm_Stefanie_Heidrun.pdf Grimm, Stefanie Heidrun ddc:540, ddc:500, F