Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Archiv
250 Episoden
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Single-molecule methods play a growing role in materials science because they can reveal structural and dynamic features which are obscured by ensemble averaging in conventional spectroscopic techniques. In this work, such methods were used to study the dynamics of single dye molecules (guests) within different surrounding porous matrices (hosts) using wide-field microscopy and single-molecule tracking. A significant amount of tracking data was collected and sophisticated methods to analyse the data according to diffusion theory were developed. A method was established to directly correlate the diffusion information that is provided by single-molecule trajectories with the images of the porous host systems obtained by transmission electron microscopy (TEM). Furthermore, the results from single-molecule tracking experiments were compared with diffusion measurements using pulsed-field gradient NMR in the same samples. The data presented in this thesis thus provide for the first time a detailed picture of the real mesoporous structure and its effects on the dynamic behavior of dye molecules at the nanometre to micron scale, e.g.~information about pore connectivity and accessibility. The methodology established here is expected to provide detailed insights into the dynamics of other important host-guest systems, such as bioactive molecules in porous materials for drug delivery or reactants in porous catalysts.
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Messenger RNA localization occurs in the cytoplasm and allows temporal and spatial regulation of gene expression. In yeast, the localization of ASH1 mRNA to the tip of budding cells allows the asymmetric sorting of Ash1 protein, which has a key function in the regulation of mating-type switching. After cell division, asymmetric distribution of Ash1p restricts mating type switching to only the mother cell. The cytoplasmic transport of ASH1 mRNA to the bud tip depends on the myosin Myo4p, its adaptor She3p, and the specific RNA binding protein She2p. Three additional trans-acting factors Khd1p, Puf6p and Loc1p are involved in this process. All known RNA-binding proteins of ASH1 mRNA have revealed a nuclear connection, when following their cellular distribution by indirect immunofluorescence. Thus, an early step in the localization pathway might be the early recruitment of specific trans-acting factors to the mRNA already in the nucleus. The aim of this thesis was to investigate how nuclear key events such as early binding to localized transcripts and the subsequent assembly into a nuclear RNP can account for effective RNA localization. Following the route of She2p, it was possible to show nucleo-cytoplasmic shuttling of this RNA binding protein. Moreover, ASH1 mRNA and She2p were found accumulated within the nucleolus upon arrest of mRNA export. Interestingly, two additional trans-acting factors, Loc1 and Puf6p, both involved in ASH1 mRNA localization are also nucleolar proteins. Moreover, She2’s nuclear history seems to be important for an effective sorting of Ash1p. When restricting ASH1-She2p association to the cytoplasmic compartment artificially, the ASH1 mRNA was still localized but was prematurely translated during its transport. This suggests that nuclear RNP assembly has an influence on the later stages of cytoplasmic translational control. The nucleolus might represent the appropriate cellular compartment to provide the spatial framework for the assembly of localization competent RNPs since many RNPs are assembled in this region.
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In dieser Dissertation wurden titankatalysierte Hydroaminierungsreaktionen von Alkinen und Alkenen durchgeführt. Außerdem wurde eine Methode für eine indirekte Hydratation von Alkinen mit anti-Markownikow-Regioselektivität entwickelt. Abschließend gelang es Hydroaminierungsreaktionen in der Synthese von biologisch aktiven Heterocyclen, wie Cumaronen, Indolen, Pyrrolen und Pyrrolidinen einzusetzen.
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The study provides a comprehensive overview on different stabilization techniques for liposomal formulations. The selection of the appropriate technology for a particular formulation can thereby be based on several considerations. If free flowable particulate bulk material is desired the spraying-technologies are preferred over lyophilization. Another advantage of spraying-based technologies is the possibility to combine the liposome formation step and the drying step within the same process. It is a prerequisite for the selection of the stabilization technique that the integrity of the liposomes is preserved with the incorporated drug after the process. Spray-drying is related to a thermal stress for the formulation but only for a short time. For heat labile drugs processes with low process temperatures, e.g. freeze-drying, spray freeze-drying, inert-spray drying or supercritical drying are most adequate. However, for technologies with a freezing step a sufficient stabilization against freezing induced stress e.g. by the selection of appropriate cryoprotectors is needed.
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Multianvil Hochdruck-/Hochtemperatur Untersuchungen an Seltenerd Boraten, Borat Germanaten, Gallium Oxonitriden und Intermetallischen Phasen. Im Speziellen werden die neuen Borate alpha-Nd2B4O9, beta-Nd(BO2)3, delta-La,Ce(BO2)3 und Pr4B10O21 vorgestellt. Die Erweiterung des Systems durch Germanium führte zum neuen Borat Germanat Ce6(BO4)2Ge9O22. Im Bereich der Gallium Oxonitride konnte eine Spinell-Phase realisiert werden. Hochdruck-/Hochtemperatur Untersuchungen zeigten Phasenumwandlungen vom TiNiSi- und NdPtSb-Typ in den ZrNiAl-Typ bei Drücken von 7-14 GPa und 1000-1400 °C.
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Ziel der Arbeit war die Etablierung RNA Interferenz basierender Systeme zur Senkung der Proteinspiegel von b- und g-Catenin und die darauf aufbauende Evaluierung potentieller b-Catenin Zielgene mittels Microarray Technologie. Der Schwerpunkt sollte dabei auf die Untersuchung von im Wnt-Signalweg beteiligten Genen gelegt werden. Wir konnten in den kolorektalen Karzinomzelllinien SW-480, DLD-1 und HT-29 mehrere voneinander unabhängige siRNA sowohl gegen b- als auch g-Catenin etablieren. Dabei stand neben ausreichender Senkung der Protein- und mRNA-Spiegel auch die Vermeidung unerwünschter Effekte auf die Zellen im Mittelpunkt dieser Etablierungsarbeit. Im Rahmen der vorliegenden Arbeit konnten vier neue funktionale Zelllinien etabliert werden. Sowohl in HT-29 als auch in SW-480 konnte ein universell einsetzbarer TET-Repressor stabil integriert werden. Die Linien HT-29TR und SW-480TR können als Basis für induzierbare Expressionssysteme verwendet werden. Auf Grundlage der Linie SW-480TR konnten Zelllinien etabliert werden, die durch Doxycyclin vermittelte Induktion shRNA gegen b-Catenin exprimieren. Mittels dieser Linien können Effekte, die durch die Senkung der b-Catenin Proteinspiegel hervorgerufen werden, einfach, schnell und zuverlässig in einem breiten Spektrum an in vitro und in vivo Assays untersucht werden. Im Rahmen einer Microarray Analyse des Transkriptoms von SW-480 Zellen nach RNA Interferenz basierter Senkung der b-Catenin Proteinspiegel wurde DKK4 als sehr stark reguliertes Gen auffällig. Mittels RT-PCR konnten wir bestätigen, dass die Expression von DKK4 direkt von den vorliegenden b-Catenin Proteinspiegeln abhängig ist. Basierend auf einer in silico Analyse des DKK4 Promoters wurden mit Hilfe von Reportergen Konstrukten der für die Aktivierbarkeit durch b-Catenin verantwortliche Abschnitt des DKK4 Promoters bestimmt. Wir konnten darstellen, dass die Aktivierung des DKK4 Promotors sowohl von der Präsenz von b-Catenin als auch von TCF4 abhängt. Die Expression von DKK4 bedingt die Gegenwart des aus TCF4 und b-Catenin bestehenden Transaktivierungskomplexes, der für die Aktivierung Wnt-abhängiger Transkription verantwortlich zeichnet. Gleichzeitig stellt der DKK4 Promoter durch die Präsenz von TCF-Bindestellen eine Zielstruktur für diesen Transaktivierungskomplex dar. Unsere Daten belegen, dass DKK4 ein b-Catenin Zielgen darstellt. Wir konnten auflerdem zeigen, dass die Aktivierung Wnt-abhängiger Transkription durch Gabe von rekombinantem DKK4 gehemmt werden kann. Es konnte im Rahmen dieser Arbeit dargestellt werden, dass die Expression des Wnt-Antagonisten DKK4 in einen negativen Feedback Mechanismus, der durch b-Catenin autoreguliert wird, eingebunden ist. Der vermutete Feedback Mechanismus stellt sich wie folgt dar. Wnt-Faktoren aktivieren die Transkription b-Catenin abhangiger Zielgene, darunter auch DKK4. DKK4 initiiert durch Bindung an LRP und Kremen den Abbau von b-Catenin und verhindert somit die weiterführende Transkription von Wnt-b-Catenin Zielgenen. DKK4 übernimmt als autokriner Inhibitor des Wnt-Signalwegs feinregulatorische Aufgaben, um bei moglicher Bedarfsdeckung b-Catenin abhängiger Gentranskription die weitere Aktivierung durch Wnt-Faktoren zu verhindern. Fur den Fall, dass DKK4 als parakriner Inhibitor agiert, wäre es denkbar, dass DKK4 von Zellen im unteren Teil der Krypte, die in hohem Maße durch Wnt-Faktoren aktiviert, werden sezerniert wird, um die darüber liegenden Zellen durch Abschirmung von weiteren Wnt-Signalen vor überschieflender Expression Wnt-b-Catenin abhängiger Gene und Proliferation zu schutzen und die Differenzierung dieser Zellen einzuleiten. Die unkontrollierte Expression von Wnt-b-Catenin Zielgenen nimmt bei der Entstehung kolorektaler Karzinome eine entscheidende Schlüsselposition ein. Zur Etablierung neuer Diagnostik- und Therapieoptionen bedarf es daher auch der stetigen Erweiterung des Verständnisses des Wnt-Signalwegs. Diese Arbeit fügt ein weiteres Rädchen in die komplexe Mechanik dieses Signalwegs ein.
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Gene expression encompasses a multitude of different steps, starting with transcription in the nucleus, co-transcriptional processing and packaging of the mRNA into a mature mRNP, export of the mRNP through the nuclear pore and finally the translation of the message in the cytoplasm. The central coordinator for coupling of the nuclear events is the differentially phosphorylated C-terminal domain (CTD) of RNA polymerase II (RNAP II). The phosphorylation pattern of the CTD not only dictates the progression through the transcription cycle but also the recruitment of mRNA processing machineries. Coupling of transcription to mRNA export is achieved by the TREX complex, which consists in the yeast S. cerevisiae of the heterotetrameric THO complex important for transcription elongation, the SR-like proteins Gbp2 and Hrb1, and Tex1 and the mRNA export factors Sub2 and Yra1. By direct interaction with Yra1, the mRNA export receptor Mex67-Mtr2 is then recruited to the mRNP and transports the mRNP through the nuclear pore complex to the cytoplasm. In a genetic screen for factors that are crucial for TREX complex function in the living cell, Ctk1, a cyclin dependent kinase (CDK) that phosphorylates the C-terminal domain (CTD) of RNAP II during transcription elongation, was identified (Hurt et al. 2004). Surprisingly, besides the TREX components Gbp2 and Hrb1, Ctk1 co-purified ribosomal proteins and translation factors. Using sucrose density centrifugation, it could be shown that Ctk1 indeed associates with translating ribosomes in vivo, suggesting a novel function of this protein in translation. This assumption was confirmed by in vitro translation assays: loss of Ctk1 function leads to a reduction in translational activity. More specifically, Ctk1 is important for efficient translation elongation and contributes to the accurate decoding of the message. Cells depleted for Ctk1 are more sensitive towards drugs that impair translational accuracy and show an increase in the frequency of miscoding in vivo. The function of Ctk1 during decoding of the message is most likely direct, as in extracts of cells depleted for Ctk1 the defect in correct decoding of the message can be restored to wild type levels by addition of purified CTDK-I complex. An explanation for the molecular mechanism of Ctk1’s function is provided by the identification of Rps2 as a novel substrate of Ctk1. Rps2 is a protein of the small ribosomal subunit, located at the mRNA entry tunnel and known to be essential for translational accuracy. Importantly, Rps2 is phosphorylated on serine 238 by Ctk1, and cells containing an rps2-S238A mutation show an increased sensitivity towards drugs that affect translational accuracy and an increase in miscoding as determined by in vitro translation extracts. The role of Ctk1 in translation is probably conserved as CDK9, the mammalian homologue of Ctk1, also associates with polysomes. Since Ctk1 interacts with the TREX complex, which functions at the interface of transcription and mRNA export, Ctk1 might bind to the mRNP during transcription and accompany the mRNP to the ribosomes, where Ctk1 enhances efficient and accurate translation of the mRNA. This study could be an example of a novel layer of control in gene expression: the composition of the mRNP determines its translational fate, including efficiency and accuracy of translation.
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Die Existenz eines Zytoskeletts galt lange als charakteristisches Merkmal eukaryotischer Zellen. Obwohl sich mit der Entdeckung der eubakteriellen Zytoskelettproteine MreB, ParM, FtsZ und CreS ein Paradigmenwechsel vollzog, lagen bislang keine Erkenntnisse über das Vorkommen von Zytoskelettproteinen in Archaeae vor. Der erste Teil der Arbeit beschreibt die strukturelle und biochemische Charakterisierung des Aktinhomologen Ta0583 aus dem Archaeon Thermoplasma acidophilum. Die Kristallstruktur von Ta0583 wurde mit der Methode der SAD-Phasierung bei einer Auflösung von 2,1 Å gelöst. Ta0583 gehört zur Aktin/Hsp70 Superfamilie und besteht aus zwei Domänen, die jeweils das Aktin/Hsp70 Kernelement enthalten. Obwohl Aktin und das archaeale Ta0583 kaum Sequenzidentität aufweisen, besteht eine deutliche strukturelle Homologie. Die Struktur von Ta0583 kombiniert strukturelle Eigenheiten sowohl von Aktin, als auch von den eubakteriellen Aktinhomologen MreB und ParM. So konnte beispielsweise die strukturelle Ähnlichkeit der Nukleotidbindungsstellen von Ta0583 und MreB in vitro durch den Effekt des MreB-Inhibitors S-(3,4-Dichlorobenzyl)-isothioharnstoff (A22) nachgewiesen werden, der die ATPase Aktivität von Ta0583 kompetitiv hemmt. Im Kristallgitter sind die Ta0583 Monomere in Filament-ähnlichen Reihen angeordnet, in denen ähnliche longitudinale Gitterabstände wie in den Protofilamenten von MreB, Aktin und ParM vorliegen. In vitro bildet Ta0583 kristalline Schichten, die ähnliche Gitterabstände wie die quasi-Filamente im Kristall aufweisen. Die Bereitwilligkeit von Ta0583 zur Kristallisation und zur Bildung kristalliner Schichten könnte eine intrinsische Neigung des Proteins zur Bildung von filamentartigen Strukturen andeuten. Das Vorkommen eines Aktin-Homologen im Archaeon T. acidophilum gibt erste Hinweise auf das Vorkommen möglicher Zytoskelettstrukturen neben Eukaryoten und Eubakterien auch in der dritten Domäne des Lebens, den Archaeae. Der zweite Teil der Arbeit befasst sich mit der strukturellen und biochemischen Charakterisierung des in S. cerevisiae essentiellen Stoffwechselenzyms Ugp1p, der UDP-Glukose Pyrophosphorylase (UGPase). Die UGPase katalysiert die Synthese von UDP-Glukose, einem zentralen Glykosyldonor im Stoffwechsel aller Organismen. In S. cerevisiae ist die UGPase ein Oktamer aus identischen Untereinheiten. Obwohl oktamere UGPasen schon in den 1960iger Jahren erstmals charakterisiert wurden, blieb die strukturelle Basis für die Assoziation der Monomere im Komplex bis heute unaufgeklärt. In dieser Arbeit wurde die Struktur von Ugp1p durch Molecular Replacement mit der Struktur einer monomeren UGPase aus A. thaliana bei einer Auflösung von 3,1 Å gelöst. Das Ugp1p Monomer besteht aus drei Domänen, einer N-terminalen Domäne, einer katalytischen SGC-Kerndomäne und einer C-terminalen Oligomerisierungsdomäne mit -Helix Motiv. Anhand der Struktur von Ugp1p konnten mehrere Aminosäurereste identifiziert werden, die die Wechselwirkungen zwischen den Untereinheiten im Oktamer vermitteln. Diese vorwiegend hydrophoben Reste sind in den UGPasen von Tieren und Pilzen konserviert, in den UGPasen der Pflanzen jedoch durch polare und geladene Reste ersetzt. Aufgrund der Konservierung der Reste im Bereich der Oligomerisierungs-Schnittstelle ist davon auszugehen, dass alle UGPasen aus Metazoen und Pilzen Ugp1p-ähnliche Oktamere bilden, pflanzliche UGPasen dagegen anders aufgebaut sind. Während die Aktivität pflanzlicher UGPasen über Assoziation und Dissoziation reguliert zu sein scheint (Martz et al., 2002), sind UGPasen aus Metazoen und Pilzen nicht über Oligomerisierung reguliert. So bildet Ugp1p ausschliesslich stabile Oktamere. In Ugp1p scheint vielmehr das flexible N-terminale Segment, das auch an Ser11 phosphoryliert gefunden wurde (Rutter et al., 2002), die Regulation der Enzymaktivität zu vermitteln. Die Struktur von Ugp1p bildet die Grundlage für gezielte Mutagenesestudien an allen UGPasen aus Metazoen und Pilzen.
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Microporous zeolites and mesoporous periodically silicious materials offer interesting features, like porosity in general, host-guest interactions and among others, sorption phenomena. The application of addressable individual material pixels, pin-printed onto adequate pre-treated supporting surfaces for gas-sensor systems was evaluated. The contact pin-printing technique, well known in bio-science, was adopted and optimized. The successful deposition of colloidal suspensions of zeolite materials on Au-covered glass slides with chemically attached intermediate anchoring molecules was demonstrated on a 100 μm scale (chapter 3). In a collaboration with the physical department of the LMU Munich (Prof. Kotthaus) a gas sensor system, based on surface acoustic wave devices was developed. Thin layers of porous material in the sub-microgram range were applied, in order to record adsorption isotherms and to determine the released heat of adsorption of specific analyte gases. Related to very small sample amounts and short diffusion times the necessary experimental measurement time could be reduced down to several minutes (chapter 9). An existing rudimentary quartz crystal microbalance (QCM) was enhanced and an automated intelligent equilibrium system was developed. Furthermore, the system was equipped with liquid mass controllers in order to measure the sorption properties together with vaporized liquid solvents (chapter 4). The developed QCM measurement setup was applied as advanced research tool in order to investigate sorption properties of various porous samples and to obtain the thermodynamic parameter, the isosteric heat of adsorption. The incorporation of organic moieties into siliceous frameworks leads to a wide variety of adsorbate–adsorbent interactions including weak Van-der-Waals attractions as well as strong interactions such as Coulomb forces. Depending on the desired properties of such substituted highly porous matrix materials, optimized synthesis routes can be established to enhance the desired internal pore surface–affinity towards certain volatile compounds. Based on a fundamental knowledge of the host–guest system, sorption related applications may benefit from individually fine-tuned and modified sample materials. The sorption isotherms and isosteric heat of adsorption for non-modified, phenyl- Summary IIIIIIIII modified, cyano-modified, vinyl-modified and mercapto-functionalized mesoporous material for ethanol and 1-butanol sorption were determined. Additionally, nanosized zeolites, like ZSM-5, Sil-1 and zeolite beta were investigated (chapter 6). Furthermore, sorption isotherms of vaporized toluene on non-modified and phenyl-functionalized mesoporous silica samples were determined using the gravimetric QCM technique at different temperatures. The mesoporous silica was modified by in situ via co-condensation and via post-synthesis grafting approaches, respectively. All samples were thoroughly characterized by several standard techniques and additionally with toluene sorption experiments on the automated QCM setup. The different heats of adsorption of toluene on the various modified silica surfaces obtained by the sorption data made it possible to gain additional information about the degree and type of surface functionalization. It is thus demonstrated that QCM studies can be a powerful and convenient tool for efficient investigations of functionalized mesoporous silica particles that yield valuable quantitative information on molecule-surface interactions (chapter 8).
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Part I The exosome is a conserved 3´- 5´ exoribonuclease complex involved in cellular RNA metabolic processes in eukaryotes and archaea. Its involvement in the accurate processing of nuclear RNA precursors and in the degradation of RNA in both nucleus and cytoplasm implies a central function in the eukaryotic RNA surveillance machinery. This widespread function implies the ability of the exosome to distinguish between RNA substrates that should be matured by the removal of nucleotides to a precisely defined end point, and defective RNAs that undergo rapid and complete degradation. However, the structural and molecular mechanisms of processive 3´- 5´ RNA degradation and substrate specificity remain unclear. To obtain insights into the structural and functional organization of the exosome, I determined crystal structures of two 230 kDa nine subunit exosome isoforms from Archaeoglobus fulgidus. Both exosome isoforms contain a hexameric ring of RNase PH-like domain subunits Rrp41 and Rrp42 with a central chamber. A trimer of Rrp4 or Csl4 subunits is situated on one side of the RNase PH domain ring and forms a multidomain macromolecular interaction surface with central S1 domains and peripheral KH and zinc-ribbon domains. Tungstate soaks identified three phosphorolytic active sites inside the central processing chamber. Additional structural and functional results suggest that the S1 domains of Rrp4 or Csl4 subunits and a subsequent neck in the RNase PH domain ring form an RNA entry pore that only allows access of unstructured RNA to the active sites. The structural results presented here can not only mechanistically unify observed features of exosomes, including processive 3´ RNA degradation of unstructured RNA, the requirement for regulatory factors and coactivators to degrade structured RNA, and the precision in processing RNA species to a defined length. But the high conservation of the archaeal exosome to the eukaryotic exosome and its additional high structural similarity to bacterial mRNA-degrading PNPase suggest a common basis for 3´ RNA-degradation in all kingdoms of life. Furthermore, the structure of the archaeal exosome reveals remarkable architectural and functional similarities to the protein degrading proteasome. Part II Adenosine triphosphate (ATP) dependent nucleic acid unwinding by superfamily 2 (SF2) helicases is required for numerous biological processes, including DNA recombination, RNA decay and viral replication. The structural and molecular mechanism for processive duplex unwinding of SF2 helicases is still unclear, in part due to a lack of structural insights into the actual strand separation reaction. Archaeal SF2 helicase Hel308 preferentially unwinds lagging strands at replication forks and is closely sequence related to human PolΘ and Hel308 as well as Drosophila Mus308. Furthermore, the RecA ATPase-core of archaeal Hel308 shares high sequence conservation to the SF2 RNA decay factors Ski2p and Mtr4p. Thus, archaeal Hel308 appears as representative model to understand processive 3´- 5´ DNA unwinding by SF2 helicases. During this PhD thesis crystal structures of Archaeogloubs fulgidus Hel308 (afHel308) in the absence and presence of a 15mer duplex DNA containing a 10mer 3´-overhang were determinded using X-ray crystallography. afHel308 exhibits two typical SF2 RecA-like domains at the N-terminus. The C-terminus comprises a winged-helix (WH) domain, followed by a unique seven-helix-bundle domain and a helix-loop-helix (HLH) domain. The DNA bound structure captures the initial duplex separation and argues that initial strand separation does not require ATP binding. Comparison with ATP bound SF2 enzymes suggests that ATP binding and hydrolysis promotes processive unwinding of one base pair by a ratchet like transport of the 3’ product strand. In addition, the structure suggests that unwinding is promoted by a prominent β-hairpin loop. The identification of similar β-hairpin loops in Hepatitis C virus (HCV) NS3 helicase and RNA decay factors Ski2p and Mtr4p, and consistency of the results with biochemical data on HCV NS3 helicase argue that the observed duplex unwinding mechanism is applicable to a broader subset of processive SF2 helicases. Furthermore, the interaction between afHel308 and its DNA substrate also may explain how afHel308 is targeted to branched nucleic acid substrates.The presented results provide a first structural framework for duplex unwinding by processive SF2 helicases and reveal important mechanistic differences to SF1 helicases and the SF2 helicase RecG.
