Charakterisierung der Vinorin-Synthase aus Rauvolfia serpentina durch Reinigung, Expression, Mutation und Kristallisation

ABSTRACTDie vorliegende Arbeit befasste sich mit der Reinigung,heterologen Expression, Charakterisierung, molekularenAnalyse, Mutation und Kristallisation des EnzymsVinorin-Synthase. Das Enzym spielt eine wichtige Rolle inder Ajmalin-Biosynthese, da es in einerAcetyl-CoA-abhängigen Reaktion die Umwandlung desSarpagan-Alkaloids 16-epi-Vellosimin zu Vinorin unterBildung des Ajmalan-Grundgerüstes katalysiert. Nach der Reinigung der Vinorin-Synthase ausHybrid-Zellkulturen von Rauvolfia serpentina/Rhazya strictamit den fünf chromatographischen TrennmethodenAnionenaustauschchromatographie an SOURCE 30Q, HydrophobeInteraktionen Chromatographie an SOURCE 15PHE,Chromatographie an MacroPrep Ceramic Hydroxyapatit,Anionenaustauschchromatographie an Mono Q undGrößenausschlußchromatographie an Superdex 75 konnte dieVinorin-Synthase aus 2 kg Zellkulturgewebe 991fachangereichert werden.Das nach der Reinigung angefertigte SDS-Gel ermöglichte eineklare Zuordnung der Protein-Bande als Vinorin-Synthase.Der Verdau der Enzymbande mit der Endoproteinase LysC unddie darauffolgende Sequenzierung der Spaltpeptide führte zuvier Peptidsequenzen. Der Datenbankvergleich (SwissProt)zeigte keinerlei Homologien zu Sequenzen bekannterPflanzenenzyme. Mit degenerierten Primern, abgeleitet voneinem der erhaltenen Peptidfragmente und einer konserviertenRegion bekannter Acetyltransferasen gelang es, ein erstescDNA-Fragment der Vinorin-Synthase zu amplifizieren. Mit derMethode der RACE-PCR wurde die Nukleoidsequenzvervollständigt, was zu einem cDNA-Vollängenklon mit einerGröße von 1263 bp führte, der für ein Protein mit 421Aminosäuren (46 kDa) codiert.Das Vinorin-Synthase-Gen wurde in den pQE2-Expressionsvektorligiert, der für einen N-terminalen 6-fachen His-tagcodiert. Anschließend wurde sie erstmals erfolgreich in E.coli im mg-Maßstab exprimiert und bis zur Homogenitätgereinigt. Durch die erfolgreiche Überexpression konnte dieVinorin-Synthase eingehend charakterisiert werden. DerKM-Wert für das Substrat Gardneral wurde mit 20 µM, bzw.41.2 µM bestimmt und Vmax betrug 1 pkat, bzw. 1.71 pkat.Nach erfolgreicher Abspaltung des His-tags wurden diekinetischen Parameter erneut bestimmt (KM- Wert 7.5 µM, bzw.27.52 µM, Vmax 0.7 pkat, bzw. 1.21 pkat). Das Co-Substratzeigt einen KM- Wert von 60.5 µM (Vmax 0.6 pkat). DieVinorin-Synthase besitzt ein Temperatur-Optimum von 35 °Cund ein pH-Optimum bei 7.8.Homologievergleiche mit anderen Enzymen zeigten, dass dieVinorin-Synthase zu einer noch kleinen Familie von bisher 10Acetyltransferasen gehört. Alle Enzyme der Familie haben einHxxxD und ein DFGWG-Motiv zu 100 % konserviert. Basierendauf diesen Homologievergleichen und Inhibitorstudien wurden11 in dieser Proteinfamilie konservierte Aminosäuren gegenAlanin ausgetauscht, um so die Aminosäuren einer in derLiteratur postulierten katalytischen Triade(Ser/Cys-His-Asp) zu identifizieren.Die Mutation aller vorhandenen konservierten Serine undCysteine resultierte in keiner Mutante, die zumvollständigen Aktivitätsverlust des Enzyms führte. Nur dieMutationen H160A und D164A resultierten in einemvollständigen Aktivitätsverlust des Enzyms. Dieses Ergebniswiderlegt die Theorie einer katalytischen Triade und zeigte,dass die Aminosäuren H160A und D164A exklusiv an derkatalytischen Reaktion beteiligt sind.Zur Überprüfung dieser Ergebnisse und zur vollständigenAufklärung des Reaktionsmechanismus wurde dieVinorin-Synthase kristallisiert. Die bis jetzt erhaltenenKristalle (Kristallgröße in µm x: 150, y: 200, z: 200)gehören der Raumgruppe P212121 (orthorhombisch primitiv) anund beugen bis 3.3 Å. Da es bis jetzt keine Kristallstruktureines zur Vinorin-Synthase homologen Proteins gibt, konntedie Struktur noch nicht vollständig aufgeklärt werden. ZurLösung des Phasenproblems wird mit der Methode der multiplenanomalen Dispersion (MAD) jetzt versucht, die ersteKristallstruktur in dieser Enzymfamilie aufzuklären.

Tail bridging vs. patchiness

The subject of this thesis are the interactions between nucleosome core particles (NCPs). NCPs are the primary storage units of DNA in eucaryotic cells. Each NCP consists of a core of eight histone proteins and a strand of DNA, which is wrapped around about two times. Each histone protein has a terminal tail passing over and between the superhelix of the wrapped DNA. Special emphasis was placed on the role of the histone tails, since experimental ndings suggest that the tails have a great in uence on the mutual attraction of the NCPs. In those experiments Mangenot et al. observe a dramatic change in the con guration of the tails, which is accompanied by evidence of mutual attraction between NCPs, when a certain salt concentration is reached. Existing models used in the theoretical approaches and in simulations focus on the description of the histone core and the wrapped DNA, but neglect the histone tails. We introduce the multi chain complex as a new simulation model. Here the histone core and the wrapping DNA are modelled via a charged sphere, while the histone tails are represented by oppositely charged chains grafted on the sphere surface. We start by investigating the parameter space describing a single NCP. The Debye-Huckel potential is used to model the electrostatic interactions and to determine the e ective charge of the NCP core. This value is subsequently used for a study of the pairinteraction of two NCPs via an extensive Molecular Dynamics study. The monomer distribution of the full chain model is investigated. The existence of tail bridges between the cores is demonstrated. Finally, by discriminating between bridging and non-bridging con gurations, we can show that the effect of tail bridging between the spheres does indeed account for the observed attraction. The full chain model can serve as a model to study the acetylation of the histone tails of the nucleosome. The reduction of the charge fraction of the tails, that corresponds to the process of acetylation, leads to a reduction or even the disappearance of the attraction. A recent MC study links this e ect to the unfolding of the chromatin ber in the case of acetylated histone tails. In this case the acetylation of the histone tails leads to the formation of heterochromatin, and one could understand how larger regions of the genetic information could be inactivated through this mechanism.

Einfluss von Chromatin-Modulatoren auf Bildung, Reparatur und Mutagenität von DNA-Schäden

Die Entstehung von Mutationen, und somit der erste Schritt der Kanzerogenese, steht in engem Zusammenhang mit der Effektivität der DNA-Reparatur. Werden zur Fehlpaarung neigende (prämutagene) DNA-Schäden, wie z.B. die oxidative Läsion 8-oxoG, zu langsam oder auch fehlerhaft repariert, so führt dies zwangsläufig zu einer Erhöhung der Mutationsrate. Das Zusammenspiel zwischen Schadensentstehung und dessen Reparatur ist somit von großem Interesse. Ein wichtiger Faktor, der dieses Gleichgewicht beeinflussen könnte, ist die Chromatinstruktur, die entscheidend ist für die DNA-Zugänglichkeit.rnrnDie Frage, ob und in welchem Ausmaß der globale Kondensationsgrad des Chromatins die Entstehung und Reparatur von DNA-Schäden und damit die Entstehung von Mutationen beeinflusst, war der Ausgangspunkt für die vorliegende Arbeit. Um die Chromatinstruktur zu modulieren, wurden zum einen Resveratrol, zum anderen die HDAC-Inhibitoren Natriumbutyrat und Trichostatin A eingesetzt. Resveratrol führt, möglicherweise über eine SIRT1-Aktivierung, zu einem kondensierten und schlecht zugänglichen Chromatin. Die HDAC-Inhibitoren hingegen resultieren durch verstärkte Acetylierung von Histonen in einer global dekondensierten, offenen Chromatinstruktur. Mit Hilfe des Photosensibilisators Ro19-8022 in Kombination mit sichtbarem Licht, UV-B-Strahlung und Wasserstoffperoxid wurden in so veränderten Zellen verschiedene Arten von DNA-Schäden induziert, welche jeweils spezifisch sind für unterschiedliche Reparaturwege. Das Ausmaß induzierter Läsionen sowie deren Reparatur wurde mittels Alkalischer Elution und entsprechenden Reparaturendonukleasen bestimmt. rnrnDie Ergebnisse zeigen eine durch Resveratrol unbeeinflusste Schadensinduktion, andererseits jedoch eine deutliche Verlangsamung der Reparatur verschiedener Arten von DNA-Läsionen (oxidative Läsionen, Cyclobutanpyrimidindimere, Einzelstrangbrüche) und somit auch verschiedener Reparaturwege in AS52-Zellen. Die HDAC-Inhibitoren hingegen verursachen ein erhöhtes Ausmaß induzierter Läsionen, jedoch keine Änderung der Reparaturgeschwindigkeit. Die Entstehung spontaner und induzierter Mutationen zeigt sich durch Resveratrol unbeeinflusst, HDAC-Inhibitoren resultieren in signifikant erniedrigten Mutationsraten in AS52-Zellen. Letzterer Effekt ist durch die beobachteten Einflüsse auf Reparatur und Suszeptibilität in den Zellen nicht erklärbar und bedarf einer mechanistischen Aufklärung. Die durch Resveratrol beobachtete Reparatur-Retardierung wurde mechanistisch weiter untersucht. Durch Inhibierung von SIRT1, einer durch Resveratrol aktivierten Deacetylase, konnte dessen Beteiligung an der Reparaturverlangsamung ausgeschlossen werden. Auch eine Beteiligung von oxidativem Stress, dem MAPK-Signalweg (ERK 1/2, p38) oder p53 konnte ausgeschlossen werden. Die Durchführung der Reparaturversuche mit menschlichen HeLa-Zellen zeigten, dass die durch Resveratrol verursachten Effekte quantitativ stark zelltypabhängig sind. Während die Reparatur in HeLa-Zellen deutlich weniger beeinflusst wird, sind dennoch andere Parameter wie Proliferation und Glutathionspiegel eher stärker verändert wie in AS52-Zellen. Der Mechanismus der durch Resveratrol verursachten Reparaturhemmung bedarf somit weiterer Untersuchungen.rn

New synthetic strategies towards indolizines and pyrroles

Indolizines and pyrroles are considered as “privileged” structures since their skeletons were found in many biologically active natural products and they possess a wide range of pharmaceutical properties. Syntheses of these small drug-like molecules are very important in medicinal chemistry. However, most existent methodologies are usually limited to specific substitution patterns or require impractical starting materials or expensive catalysts. Therefore, developing new methodologies for the synthesis of indolizines and pyrroles from commercially available or readily accessible sources is highly desirable.rnIn this PhD thesis, several methods has been described for the synthesis of indolizines and pyrroles. In the first part, indolizines carrying substituents in positions 1-3 were synthesized via a formal [3+2]-cycloaddition of pyridinium ylides and nitroalkenes. Pyridinium salts were prepared by N-alkylation of pyridines with cyanohydrin triflates which could be prepared from corresponding aldehydes via a Strecker reaction followed by O-triflylation. Nitroalkenes were simply prepared from the corresponding aldehydes and nitroalkanes in a nitroaldol condensation. Overall, this modular approach allows to construct the indolizine framework with various substitution patterns starting from a pyridine, two different aldehydes and a nitroalkane. In contrast to reported methods, the produced indolizines do not have to contain an electron-withdrawing group.rnIt has also been found that nitrile-stabilized 2-alkylpyridinium ylides cyclize to unstable 2-aminoindolizines via an intramolecular 5-exo-dig cyclization. Using an in situ acetylation of the amino group, N-protected 2-aminoindolizines could be synthesized. As a less common substitution pattern, indolizines carrying substituents in positions 5–8 were synthesized from enones and 2-(1H-pyrrol-1-yl)nitriles obtained from α-aminonitriles using a modified Paal-Knorr pyrrole synthesis. The decoration of the pyridine unit in the indolizine skeleton has been achieved by a one-pot conjugate addition/cycloaromatization sequence.rnIn the second part of the thesis, the diversity-oriented synthesis of pyrroles from 3,5-diaryl substituted 2H-pyrrole-2-carbonitriles (cyanopyrrolines) obtained in a cyclocondensation of enones with aminoacetonitrile hydrochloride is being discussed. 2,4-Di-, 2,3,5-trisubstituted pyrroles, pyrrole-2-carbonitriles and 2,2’-bipyrroles were synthesized in a one- or two-step protocol. While the microwave-assisted thermal elimination of HCN from cyanopyrrolines gave 2,4-disubstituted pyrroles, DDQ-oxidation of the same intermediates furnished pyrrole-2-carbonitriles. Furthermore, 2,3,5-trisubstituted pyrroles were obtained via a C-2-alkylation of the deprotonated cyanopyrrolines followed by the elimination of HCN. Finally, it has also been found that tetraaryl substituted 2,2’-bipyrroles could be synthesized by the oxidative dimerization of cyanopyrrolines using copper (II) acetate at 100 °C.rn

Synthesis and characterization of polymers containing oligothiophenes

In the present work a series of thiophene oligomers of three and six thiophene units were synthesized, starting from thiophene, and characterized. Polymers containing these electroative side groups were then prepared by two strategies. The oligomers were attached to existing polymer systems and were connected to a polymerizable unit leading to monomer containing the oligothiophenes as side groups. Subsequently the properties of the monomers and the polymers were investigated. A butylcellulose derivative carrying terthienyl side chains (BCTTE, 26) was synthesized starting from cellulose acetate and 5-(2-chloroethyl)-2,2':5',2'-terthiophene (4). The polymer had a degree of substitution (DS) of the butyl and terthienyl side chains of DSbutyl = 1.9 and DSterth = 0.35, respectively. It was successfully spread on a Langmuir-Blodgett (LB) trough and then transferred to several solid substrates. X-rays reflectometry showed an ordered architecture of the cellulose backbones. However, the terthiophene side groups were found as isotropically aligned by polarized UV-Vis spectroscopy. When used as anode material in the electropolymerization of 3-pentylthiophene (28), polythiophene was grafted onto the cellulose backbone through the terthienyl side groups. The polythiophene chains showed an average anisotropic alignment of 20 % along the LB dipping direction, calculated by means of polarized UV-Vis spectroscopy. A second butylcellulose derivative carrying sexithienyl side chains (BCST) was synthesized and investigated, starting from butylcellulose and 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl p-toluensulfonate (7). The polymer showed formation of stable LB monolayers at the air-water interface, but its transfer onto solid substrates was not successful. A poly(p-phenylene-ethynylene) bearing sexithienyl side chains (BzAcST, 31) was prepared by reaction of the two monomers 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl 2,5-diiodobenzoate (15) and 2-[b', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2':5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl 2,5-diethynylbenzoate (18). The polymer was obtained as insoluble product. Upon oxidation with FeCl3 (doping) of the polymer suspension, BzAcST showed an electrical conductivity of ó = 2.5 . 10 -6 S/cm, a typical value for semiconductors. The IR spectrum of the doped polymer presented the diagnostic bands of oxidized sexithiophene in good agreement with literature results. Along with the monomer and polymer synthesis, an a,a '-disubstituted sexithiophene, b ', b ''-dipentyl-5,5'''-bis-(2-hydroxyethyl)-2,2':5',2':5',2'':5'',2'':5'',2'''-sexithiophene (6a),was synthesized and characterized. The UV-Vis absorption of the chromophore wasinvestigated as a function of temperature and different solvents, showing a blue-shift of the absorption maximum with increasing temperature and a red-shift changing the solvent from hexane to ethanol to toluene. Monitoring the change of the UV-Vis spectrum upon electrochemical oxidation, the oxidized chromophore showed a new broad absorption band, red shifted with respect to the p -p* transition of the neutral state. Upon reduction, the new band disappeared and the UV-Vis spectrum of the chromophore was restored. Such oxidation-reduction cycles were totally reversible. This feature, together with the absorption maximum falling in the visible region, makes this chromophore a suitable compound for the development of an electrochemical sensor.Attempts to polymerize acrylic monomers carrying sexythienyl side chains both via radical polymerization, as in the case of 2-[b ', b ''-dipentyl-5'''-(2-hydroxyethyl)-2,2': 5',2':5',2'':5'',2'':5'',2'''-sexithiophen-5-yl]-ethyl acrylate (8), and anionic polymerization, as in the case of 2-{b ', b ''-dipentyl-5'''-[2-(tertbutyldimethylsiloxy)ethyl]-2,2':5',2':5',2'':5'',2'': 5'',2''' -sexithiophen-5-yl}-ethylacrylate (29), were not successful, probably due to the steric hindrance of the oligothiophene side group. However, due to the time consuming and therefore restricted availability of the monomers, a screening of the polymerization conditions towards the formation of polymeric material was not possible.

Production, purification, properties and application of the cellulases from a wild type strain of a yeast isolate

Kurzzusammenfassung Produktion, Reinigung, Eigenschaften und Anwendung von Cellulasen eines Wildtyp-Hefeisolates. Die effiziente Verwendung von Cellulose wird in naher Zukonft ein wichtiges Instrument zur Vermeidung einer Nahrungsmittel- und Energieknappheit werden. Deshalb haben wir uns intensiv mit Cellulasen befaßt, die aus Hefestämmen isoliert wurden. Die Fähigkeit der Cellulase-produktion eines Hefe-Stammes der Feuerwanze Pyrrhocoris apterus wurde genauer untersucht. Die systematische Stellung des Hefe-Isolates PAG1 wurde durch Sequenzierung der 18S rDNA bestimmt. Es zeigte eine nahe Verwandtschaft zu einem bereits beschriebenen Stämme der Gattung Trichosporon. Außerdem wurden die Wachstums-bedingungen für eine optimale Cellulase–Produktion bestimmt. Anschließend konnte eine der produzierten Cellulasen mit FPLC aufgereinigt und deren biochemische Eigenschaften (z.B. Substratspezifität, Temperatur optimum, optimaler pH-Wert, Einfluß von Chemikalien) untersucht werden. Eine Analyse der Abbau-Produkte zeigte, daß kristalline Cellulose und CMC zu Cellobiose, Cellulotriose, Cellulotetraose und Cellulopentaose in einem molaren Verhältnis von 32:16:8:1 umgesetezt wurden. Bei Zusatz von ?-Glykosidase aus demselben Hefestamm entstand nur Glucose und Cellobiose in einem molaren Verhältnis von 1:10. Da bisher nur eine Publikation über Cellulase-produzierende Hefe-Stämme erschienen ist, zeigen auch unsere Untersuchungen, daß Wildtyp-Hefestämme Cellulasen mit interessanten Eigenschaften produzieren können.

Investigations of cooperative interactions in template induced crystallization processes and kinetic studies of nucleation and growth by small-angle neutron scattering

Zusammenfassung Um zu einem besseren Verständnis des Prozesses der Biomineralisation zu gelangen, muss das Zusammenwirken der verschiedenen Typen biologischer Makromoleküle, die am Keimbildungs- und Wachstumsprozess der Minerale beteiligt sind, berücksichtigt werden. In dieser Arbeit wird ein neues Modellsystem eingeführt, das aus einem SAM (self-assembled monolayer) mit verschiedenen Funktionalitäten und unterschiedlichen, gelösten Makromolekülen besteht. Es konnte gezeigt werden, dass die Kristallisation von Vaterit (CaCO3) sowie Strontianit (SrCO3) Nanodrähten der Präsenz von Polyacrylat in Kooperation mit einer COOH-funktionalisierten SAM-Oberfläche zugeschrieben werden kann. Die Kombination bestehend aus einer polaren SAM-Oberfläche und Polyacrylat fungiert als Grenzfläche für die Struktur dirigierende Kristallisation von Nanodraht-Kristallen. Weiter konnte gezeigt werden, dass die Phasenselektion von CaCO3 durch die kooperative Wechselwirkung zwischen einer SAM-Oberfläche und einem daran adsorbierten hb-Polyglycerol kontrolliert wird. Auch die Funktionalität einer SAM-Oberfläche in Gegenwart von Carboxymethyl-cellulose übt einen entscheidenden Einfluss auf die Phasenselektion des entstehenden Produktes aus. In der vorliegenden Arbeit wurden Untersuchungen an CaCO3 zur homogenen Keimbildung, zur Nukleation in Gegenwart eines Proteins sowie auf Kolloiden, die als Template fungieren, mittels Kleinwinkel-Neutronenstreuung durchgeführt. Die homogene Kristallisation in wässriger Lösung stellte sich als ein mehrstufiger Prozess heraus. In Gegenwart des Eiweißproteins Ovalbumin konnten drei Phasen identifiziert werden, darunter eine anfänglich vorhandene amorphe sowie zwei kristalline Phasen.

Cellulose based Lithium ion polymer electrolytes for Lithium batteries

The separator membrane in batteries and fuel cells is of crucial importance for the function of these devices. In lithium ion batteries the separator membrane as well as the polymer matrix of the electrodes consists of polymer electrolytes which are lithium ion conductors. To overcome the disadvantage of currently used polymer electrolytes which are highly swollen with liquids and thus mechanically and electrochemically unstable, the goal of this work is a new generation of solid polymer electrolytes with a rigid backbone and a soft side chain structure. Moreover the novel material should be based on cheap substrates and its synthesis should not be complicated aiming at low overall costs. The new materials are based on hydroxypropylcellulose and oligoethyleneoxide derivatives as starting materials. The grafting of the oligoethyleneoxide side chains onto the cellulose was carried out following two synthetic methods. One is based on a bromide derivative and another based on p-toluolsulfonyl as a leaving group. The side chain reagents were prepared form tri(ethylene glycol) monoethyl ether. In order to improve the mechanical properties the materials were crosslinked. Two different conceptions have been engaged based on either urethane chemistry or photosensitive dimethyl-maleinimide derivatives. PEO - graft - cellulose derivatives with a high degree of substitution between 2,9 and 3,0 were blended with lithium trifluoromethane-sulfonate, lithium bis(trifluorosulfone)imide and lithium tetrafluoroborate. The molar ratios were in the range from 0,02 to 0,2 [Li]/[O]. The products have been characterized with nuclear magnetic resonance (NMR), gel permeation chromatography (GPC) and laserlight scattering (LS) with respect to their degree of substitution and molecular weight. The effect of salt concentration on ionic conductivity, thermal behaviour and morphology has been investiga-ted with impedance spectroscopy, differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA). The crosslinking reactions were controlled with dynamic mechanical analysis (DMS). The degree of substitution of our products is varying between 2,8 and 3,0 as determined by NMR. PEO - graft - cellulose derivatives are highly viscous liquids at room temperature with glass transition temperatures around 215 K. The glass transition temperature for the Lithium salt complexes of PEO - graft - cellulose deri-vatives increase with increasing salt content. The maximum conductivity at room temperature is about 10-4 and at 100°C around 10-3 Scm-1. The presence of lithium salt decreases the thermal stability of the complexes in comparison to pure PEO - graft - cellulose derivatives. Complexes heated over 140 – 150°C completely lose their ionic conductivity. The temperature dependence of the conductivity presented as Arrhenius-type plots for all samples is similar in shape and follows a VTF behaviour. This proofs that the ionic transport is closely related to the segmental motions of the polymer chains. Novel cellulose derivatives with grafted oligoethylen-oxide side chains with well-defined chemical structure and high side chain grafting density have been synthesized. Cellulose was chosen as stiff, rod like macromolecule for the backbone while oligoethylen-oxides are chosen as flexible side chains. A maximum grafting density of 3.0 have been obtained. The best conductivity reaches 10-3 Scm-1 at 100°C for a Li-triflate salt complex with a [Li]/[O] ratio of 0.8. The cross-linked complexes containing the lithium salts form elastomeric films with convenient mechanical stability. Our method of cellulose modification is based on relatively cheap and commercially available substrates and as such appears to be a promising alternative for industrial applications.

Development of analytical methodologies for iodine species in gaseous and particulate phases of the coastal atmosphere

It has been demonstrated that iodine does have an important influence on atmospheric chemistry, especially the formation of new particles and the enrichment of iodine in marine aerosols. It was pointed out that the most probable chemical species involved in the production or growth of these particles are iodine oxides, produced photochemically from biogenic halocarbon emissions and/or iodine emission from the sea surface. However, the iodine chemistry from gaseous to particulate phase in the coastal atmosphere and the chemical nature of the condensing iodine species are still not understood. A Tenax / Carbotrap adsorption sampling technique and a thermo-desorption / cryo-trap / GC-MS system has been further developed and improved for the volatile organic iodine species in the gas phase. Several iodo-hydrocarbons such as CH3I, C2H5I, CH2ICl, CH2IBr and CH2I2 etc., have been measured in samples from a calibration test gas source (standards), real air samples and samples from seaweeds / macro-algae emission experiments. A denuder sampling technique has been developed to characterise potential precursor compounds of coastal particle formation processes, such as molecular iodine in the gas phase. Starch, TMAH (TetraMethylAmmonium Hydroxide) and TBAH (TetraButylAmmonium Hydroxide) coated denuders were tested for their efficiencies to collect I2 at the inner surface, followed by a TMAH extraction and ICP/MS determination, adding tellurium as an internal standard. The developed method has been proved to be an effective, accurate and suitable process for I2 measurement in the field, with the estimated detection limit of ~0.10 ng∙L-1 for a sampling volume of 15 L. An H2O/TMAH-Extraction-ICP/MS method has been developed for the accurate and sensitive determination of iodine species in tropospheric aerosol particles. The particle samples were collected on cellulose-nitrate filters using conventional filter holders or on cellulose nitrate/tedlar-foils using a 5-stage Berner impactor for size-segregated particle analysis. The water soluble species as IO3- and I- were separated by anion exchanging process after water extraction. Non-water soluble species including iodine oxide and organic iodine were digested and extracted by TMAH. Afterwards the triple samples were analysed by ICP/MS. The detection limit for particulate iodine was determined to be 0.10~0.20 ng•m-3 for sampling volumes of 40~100 m3. The developed methods have been used in two field measurements in May 2002 and September 2003, at and around the Mace Head Atmospheric Research Station (MHARS) located at the west coast of Ireland. Elemental iodine as a precursor of the iodine chemistry in the coastal atmosphere, was determined in the gas phase at a seaweed hot-spot around the MHARS, showing I2 concentrations were in the range of 0~1.6 ng∙L-1 and indicating a positive correlation with the ozone concentration. A seaweed-chamber experiment performed at the field measurement station showed that the I2 emission rate from macro-algae was in the range of 0.019~0.022 ng•min-1•kg-1. During these experiments, nanometer-particle concentrations were obtained from the Scanning Mobility Particle Sizer (SMPS) measurements. Particle number concentrations were found to have a linear correlation with elemental iodine in the gas phase of the seaweeds chamber, showing that gaseous I2 is one of the important precursors of the new particle formation in the coastal atmosphere. Iodine contents in the particle phase were measured in both field campaigns at and around the field measurement station. Total iodine concentrations were found to be in the range of 1.0 ~ 21.0 ng∙m-3 in the PM2.5 samples. A significant correlation between the total iodine concentrations and the nanometer-particle number concentrations was observed. The particulate iodine species analysis indicated that iodide contents are usually higher than those of iodate in all samples, with ratios in the range of 2~5:1. It is possible that those water soluble iodine species are transferred through the sea-air interface into the particle phase. The ratio of water soluble (iodate + iodide) and non-water soluble species (probably iodine oxide and organic iodine compounds) was observed to be in the range of 1:1 to 1:2. It appears that higher concentrated non-water soluble species, as the products of the photolysis from the gas phase into the particle phase, can be obtained in those samples while the nucleation events occur. That supports the idea that iodine chemistry in the coastal boundary layer is linked with new particle formation events. Furthermore, artificial aerosol particles were formed from gaseous iodine sources (e.g. CH2I2) using a laboratory reaction-chamber experiment, in which the reaction constant of the CH2I2 photolysis was calculated to be based upon the first order reaction kinetic. The end products of iodine chemistry in the particle phase were identified and quantified.

Differential cell type-specific transcriptional regulation of the CYP1A1 gene

Cytochrome P450 1A1 (CYP1A1) monooxygenase plays an important role in the metabolism of environmental pollutants such as polycyclic aromatic hydrocarbons (PAHs) and halogenated polycyclic aromatic hydrocarbons (HAHs). Oxidation of these compounds converts them to the metabolites that subsequently can be conjugated to hydrophilic endogenous entities e.g. glutathione. Derivates generated in this way are water soluble and can be excreted in bile or urine, which is a defense mechanism. Besides detoxification, metabolism by CYP1A1 may lead to deleterious effects since the highly reactive intermediate metabolites are able to react with DNA and thus cause mutagenic effects, as it is in the case of benzo(a) pyrene (B[a]P). CYP1A1 is normally not expressed or expressed at a very low level in the cells but it is inducible by many PAHs and HAHs e.g. by B[a]P or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Transcriptional activation of the CYP1A1 gene is mediated by aryl hydrocarbon receptor (AHR), a basic-helix-loop-helix (bHLH) transcription factor. In the absence of a ligand AHR stays predominantly in the cytoplasm. Ligand binding causes translocation of AHR to the nuclear compartment, its heterodimerization with another bHLH protein, the aryl hydrocarbon nuclear translocator (ARNT) and binding of the AHR/ARNT heterodimer to a DNA motif designated dioxin responsive element (DRE). This process leads to the transcriptional activation of the responsive genes containing DREs in their regulatory regions, e.g. that coding for CYP1A1. TCDD is the most potent known agonist of AHR. Since it is not metabolized by the activated enzymes, exposure to this compound leads to a persisting activation of AHR resulting in diverse toxic effects in the organism. To enlighten the molecular mechanisms that mediate the toxicity of xenobiotics like TCDD and related compounds, the AHR-dependent regulation of the CYP1A1 gene was investigated in two cell lines: human cervix carcinoma (HeLa) and mouse hepatoma (Hepa). Study of AHR activation and its consequence concerning expression of the CYP1A1 enzyme confirmed the TCDD-dependent formation of the AHR/ARNT complex on DRE leading to an increase of the CYP1A1 transcription in Hepa cells. In contrast, in HeLa cells formation of the AHR/ARNT heterodimer and binding of a protein complex containing AHR and ARNT to DRE occurred naturally in the absence of TCDD. Moreover, treatment with TCDD did not affect the AHR/ARNT dimer formation and binding of these proteins to DRE in these cells. Even though the constitutive complex on DRE exists in HeLa, transcription of the CYP1A1 gene was not increased. Furthermore, the CYP1A1 level in HeLa cells remained unchanged in the presence of TCDD suggesting repressional mechanism of the AHR complex function which may hinder the TCDD-dependent mechanisms in these cells. Similar to the native, the mouse CYP1A1-driven reporter constructs containing different regulatory elements were not inducible by TCDD in HeLa cells, which supported a presence of cell type specific trans-acting factor in HeLa cells able to repress both the native CYP1A1 and CYP1A1-driven reporter genes rather than species specific differences between CYP1A1 genes of human and rodent origin. The different regulation of the AHR-mediated transcription of CYP1A1 gene in Hepa and HeLa cells was further explored in order to elucidate two aspects of the AHR function: (I) mechanism involved in the activation of AHR in the absence of exogenous ligand and (II) factor that repress function of the exogenous ligand-independent AHR/ARNT complex. Since preliminary studies revealed that the activation of PKA causes an activation of AHR in Hepa cells in the absence of TCDD, the PKA-dependent signalling pathway was the proposed endogenous mechanism leading to the TCDD-independent activation of AHR in HeLa cells. Activation of PKA by forskolin or db-cAMP as well as inhibition of the kinase by H89 in both HeLa and Hepa cells did not lead to alterations in the AHR interaction with ARNT in the absence of TCDD and had no effect on binding of these proteins to DRE. Moreover, the modulators of PKA did not influence the CYP1A1 activity in these cells in the presence and in the absence of TCDD. Thus, an involvement of PKA in the regulation of the CYP1A1 Gen in HeLa cells was not evaluated in the course of this study. Repression of genes by transcription factors bound to their responsive elements in the absence of ligands has been described for nuclear receptors. These receptors interact with protein complex containing histone deacetylase (HDAC), enzyme responsible for the repressional effect. Thus, a participation of histone deacetylase in the transcriptional modulation of CYP1A1 gene by the constitutively DNA-bound AHR/ARNT complex was supposed. Inhibition of the HDAC activity by trichostatin A (TSA) or sodium butyrate (NaBu) led to an increase of the CYP1A1 transcription in the presence but not in the absence of TCDD in Hepa and HeLa cells. Since amount of the AHR and ARNT proteins remained unchanged upon treatment of the cells with TSA or NaBu, the transcriptional upregulation of CYP1A1 gene was not due to an increased expression of the regulatory proteins. These findings strongly suggest an involvement of HDAC in the repression of the CYP1A1 gene. Similar to the native human CYP1A1 also the mouse CYP1A1-driven reporter gene transfected into HeLa cells was repressed by histone deacetylase since the presence of TSA or NaBu led to an increase in the reporter activity. Induction of reporter gene did not require a presence of the promoter or negative regulatory regions of the CYP1A1 gene. A promoter-distal fragment containing three DREs together with surrounding sequences was sufficient to mediate the effects of the HDAC inhibitors suggesting that the AHR/ARNT binding to its specific DNA recognition site may be important for the CYP1A1 repression. Histone deacetylase is recruited to the specific genes by corepressors, proteins that bind to the transcription factors and interact with other members of the HDAC complex. Western blot analyses revealed a presence of HDAC1 and the corepressors mSin3A (mammalian homolog of yeast Sin3) and SMRT (silencing mediator for retinoid and thyroid hormone receptor) in both cell types, while the corepressor NCoR (nuclear receptor corepressor) was expressed exclusively in HeLa cells. Thus the high inducibility of CYP1A1 in Hepa cells may be due to the absence of NCoR in these cells in contrast to the non-responsive HeLa cells, where the presence of NCoR would support repression of the gene by histone deacetylase. This hypothesis was verified in reporter gene experiments where expression constructs coding for the particular members of the HDAC complex were cotransfected in Hepa cells together with the TCDD-inducible reporter constructs containing the CYP1A1 regulatory sequences. An overexpression of NCoR however did not decrease but instead led to a slight increase of the reporter gene activity in the cells. The expected inhibition was observed solely in the case of SMRT that slightly reduced constitutive and TCDD-induced reporter gene activity. A simultaneous expression of NCoR and SMRT shown no further effects and coexpression of HDAC1 with the two corepressors did not alter this situation. Thus, additional factors that are likely involved in the repression of CYP1A1 gene by HDAC complex remained to be identified. Taking together, characterisation of an exogenous ligand independent AHR/ARNT complex on DRE in HeLa cells that repress transcription of the CYP1A1 gene creates a model system enabling investigation of endogenous processes involved in the regulation of AHR function. This study implicates HDAC-mediated repression of CYP1A1 gene that contributes to the xenobiotic-induced expression in a tissue specific manner. Elucidation of these processes gains an insight into mechanisms leading to deleterious effects of TCDD and related compounds.

Investigation of different shellac grades and improvement of release from air suspension coated pellets

Shellac is the purified product of the natural polymer Lac. Shellac types, from different origins and with different ages, all purified by the solvent extraction process were compared in this study. Their physicochemical properties acid value, glass transition temperatures, color numbers and molecular sizes were determined. Metoprolol tartrate pellets were coated by air suspension coating with these different grades of shellac. Two coating levels 20% w/w and 25% w/w were applied and then subjected to in vitro dissolution testing. Enteric resistance was achieved for all tested brands for the two coating levels. At pH 6.8, 7.2 and 7.4, significant variations were obvious between the brands. rnMoreover the molecular size of shellac has a pronounced effect in that shellac types with larger molecular size show a higher and faster release than others, while the one with the smaller molecular size show the opposite effect on the release of metoprolol.rnIn this study commercially available ready for use aqueous shellac solutions (SSB AQUAGOLD), which are based on shellac SSB 57 (Dewaxed Orange Shellac, Bysakhi-Ber type refined in a solvent extraction process), with different manufacturing dates were used. rnTo improve the enteric coating properties of films from aqueous shellac solutions, different aqueous polymeric solutions of hydroxypropyl methylcellulose (HPMC), hydroxypropyl cellulose (HPC), carboyxmethyl cellulose (CMC), gum arabic and polysaccharides (Pullulan®) were used. These water soluble polymers will act as pore formers to enhance drug release from pellets coated with the combination of shellac and these polymers. The influence of these polymers on the gloss of the shellac films, mechanical properties of the films and drug release from metoprolol tartrate pellets were studied.rnThe potential of ethanol to alter the rate of drug release from shellac coated pellets was assessed by using a modified in vitro dose dumping in alcohol (DDA) method and the test concluded that shellac coated dosage forms can be co-administered with alcohol beverages containing ≤ 5% with no effect of alcohol on the shellac coat.rnPellets coated with shellac sodium salts, showed higher release rates than pellets coated with shellac as ammonium salt forms. rn

Liquid crystalline cellulose derivatives for mirrorless lasing

In this thesis cholesteric films made of liquid crystalline cellulose derivatives with improved optical properties were prepared. The choice of the solvent, hydrogen bond influencing additives, the synthetic realization of a very high degree of substitution on the cellulosic polymer and the use of mechanical stirring at the upper concentration limit of the liquid crystalline range were the basis for an improved alignment of the applied cellulose tricarbamates. In combination with a tuned substrate treatment and film preparation method, cholesteric films were obtained, with optical properties that were theoretically predicted and only known from low molecular weight liquid crystals so far. Subsequent polymerization allowed a permanent fixing of the alignment and the fabrication of free standing and insensitive films.rnThe incorporation of inorganic nanorods into the cholesteric host material was mediated with tailored block copolymers, available via controlled radical polymerization methods. In addition to the shape match between the rodlike mesogens of the host and the nanorods it was possible to increase the miscibility of both materials. Nevertheless, the size of the nanorods, in comparison to the mesogens, in these densely packed liquid crystalline phases as well as their long equilibration times were the reasons for phase separation. Nanorods are, in principle, valuable substitutes for organics, but their utilization in cellulosic CLC was not to be combined with a high quality alignment of the cholesteric structure.rnA swelling process of polymerized films in a dye solution or dissolving dyes in non-polymerized CLC was used for incorporation of the organic chromophores. With the first method the CLC could be aligned and polymerized without any disturbance due to dye molecules. The optical properties of dye and CLC were matched, with regard to mirrorless lasing devices. The dye was optically excited and laser emission supported by the cholesteric cavity was obtained. The polarization and wavelength of the emitted radiation as well as its bandwidth, the obtained interference pattern and threshold behavior of the emission proofed the feedback mechanism that was not believed to be realizable in liquid crystalline polymers. rnUtilization of a microfluidic co-flow injection device enabled us to transfer the properties of cellulosic CLC from the planar film shape to spherical micrometer sized particles. The pure material yielded particles with distorted mesogen alignment similar to films prepared by capillary flow. Dilution of the CLC with a solvent that migrated into the carrier phase during particle preparation provided the basis for particles with well ordered areas. rnAlthough cellulose derivatives were known for their liquid crystalline behavior for decades and synthesized in mass production, their application as feedback material was affected by bad optical properties. In comparison to low molar mass compounds, the low degree of order in the CLC phase was the cause. With the improved material, defined lasing emission was shown and characterized. Derivatives of cellulose are desirable materials, because, as a renewable resource, they are available in large amounts for a low price and need only simple derivatization reactions. The fabrication of CLC films with tunable lasing emission, for which this thesis can provide a starting point, is in good agreement with today's requirements of modern technology and its miniaturization.rn

Regulation of Nox4 expression by histone deacetylases in human endothelial cells : involvement of epigenetic mechanisms

Nox4 is a member of the NADPH oxidase family, which represents a major source of reactive oxygen species (ROS) in the vascular wall. Nox4-mediated ROS production mainly depends on the expression levels of the enzyme. The aim of my study was to investigate the mechanisms of Nox4 transcription regulation by histone deacetylases (HDAC). Treatment of human umbilical vein endothelial cells (HUVEC) and HUVEC-derived EA.hy926 cells with the pan-HDAC inhibitor scriptaid led to a marked decrease in Nox4 mRNA expression. A similar down-regulation of Nox4 mRNA expression was observed by siRNA-mediated knockdown of HDAC3. HDAC inhibition in endothelial cells was associated with enhanced histone acetylation, increased chromatin accessibility in the human Nox4 promoter region, with no significant changes in DNA methylation. In addition, the present study provided evidence that c-Jun played an important role in controlling Nox4 transcription. Knockdown of c-Jun with siRNA led to a down-regulation of Nox4 mRNA expression. In response to scriptaid treatment, the binding of c-Jun to the Nox4 promoter region was reduced despite the open chromatin structure. In parallel, the binding of RNA polymerase IIa to the Nox4 promoter was significantly inhibited as well, which may explain the reduction in Nox4 transcription. In conclusion, HDAC inhibition decreases Nox4 transcription in human endothelial cells by preventing the binding of transcription factor(s) and polymerase(s) to the Nox4 promoter, most likely because of a hyperacetylation-mediated steric inhibition. In addition, HDAC inhibition-induced Nox4 downregulation may also involves microRNA-mediated mRNA destabilization, because the effect of the scriptaid could be partially blocked by DICER1 knockdown or by transcription inhibition.

Consequences of DNA damage for gene transcription : direct effects of modified nucleobases and the role of base excision repair = Folgen von DNA-Schäden für die Gentranskription

The presence of damaged nucleobases in DNA can negatively influence transcription of genes. One of the mechanisms by which DNA damage interferes with reading of genetic information is a direct blockage of the elongating RNA polymerase complexes – an effect well described for bulky adducts induced by several chemical substances and UV-irradiation. However, other mechanisms must exist as well because many of the endogenously occurring non-bulky DNA base modifications have transcription-inhibitory properties in cells, whilstrnnot constituting a roadblock for RNA polymerases under cell free conditions. The inhibition of transcription by non-blocking DNA damage was investigated in this work by employing the reporter gene-based assays. Comparison between various types of DNA damage (UV-induced pyrimidine photoproducts, oxidative purine modifications induced by photosensitisation, defined synthetic modified bases such as 8-oxoguanine and uracil, and sequence-specific single-strand breaks) showed that distinct mechanisms of inhibition of transcription can be engaged, and that DNA repair can influence transcription of the affectedrngenes in several different ways.rnQuantitative expression analyses of reporter genes damaged either by the exposure of cells to UV or delivered into cells by transient transfection supported the earlier evidence that transcription arrest at the damage sites is the major mechanism for the inhibition of transcription by this kind of DNA lesions and that recovery of transcription requires a functional nucleotide excision repair gene Csb (ERCC6) in mouse cells. In contrast, oxidisedrnpurines generated by photosensitisation do not cause transcriptional blockage by a direct mechanism, but rather lead to transcriptional repression of the damaged gene which is associated with altered histone acetylation in the promoter region. The whole chain of events leading to transcriptional silencing in response to DNA damage remains to be uncovered. Yet, the data presented here identify repair-induced single-strand breaks – which arise from excision of damaged bases by the DNA repair glycosylases or endonucleases – as arnputative initiatory factor in this process. Such an indirect mechanism was supported by requirement of the 8-oxoguanine DNA glycosylase (OGG1) for the inhibition of transcription by synthetic 8-oxodG incorporated into a reporter gene and by the delays observed for the inhibition of transcription caused by structurally unrelated base modifications (8-oxoguanine and uracil). It is thereby hypothesized that excision of the modified bases could be a generalrnmechanism for inhibition of transcription by DNA damage which is processed by the base excision repair (BER) pathway. Further gene expression analyses of plasmids containing single-strand breaks or abasic sites in the transcribed sequences revealed strong transcription inhibitory potentials of these lesions, in agreement with the presumption that BER intermediates are largely responsible for the observed effects. Experiments with synthetic base modifications positioned within the defined DNA sequences showed thatrninhibition of transcription did not require the localisation of the lesion in the transcribed DNA strand; therefore the damage sensing mechanism has to be different from the direct encounters of transcribing RNA polymerase complexes with DNA damage.rnAltogether, this work provides new evidence that processing of various DNA basernmodifications by BER can perturb transcription of damaged genes by triggering a gene silencing mechanism. As gene expression can be influenced even by a single DNA damage event, this mechanism could have relevance for the endogenous DNA damage induced in cells under normal physiological conditions, with a possible link to gene silencing in general.

Nanoparticle preparation process using novel microjet reactor technology for enhancing dissolution rates of poorly water soluble drugs

In this study a novel method MicroJet reactor technology was developed to enable the custom preparation of nanoparticles. rnDanazol/HPMCP HP50 and Gliclazide/Eudragit S100 nanoparticles were used as model systems for the investigation of effects of process parameters and microjet reactor setup on the nanoparticle properties during the microjet reactor construction. rnFollowing the feasibility study of the microjet reactor system, three different nanoparticle formulations were prepared using fenofibrate as model drug. Fenofibrate nanoparticles stabilized with poloxamer 407 (FN), fenofibrate nanoparticles in hydroxypropyl methyl cellulose phthalate (HPMCP) matrix (FHN) and fenofibrate nanoparticles in HPMCP and chitosan matrix (FHCN) were prepared under controlled precipitation using MicroJet reactor technology. Particle sizes of all the nanoparticle formulations were adjusted to 200-250 nm. rnThe changes in the experimental parameters altered the system thermodynamics resulting in the production of nanoparticles between 20-1000 nm (PDI<0.2) with high drug loading efficiencies (96.5% in 20:1 polymer:drug ratio).rnDrug releases from all nanoparticle formulations were fast and complete after 15 minutes both in FaSSIF and FeSSIF medium whereas in mucodhesiveness tests, only FHCN formulation was found to be mucoadhesive. Results of the Caco-2 studies revealed that % dose absorbed values were significantly higher (p<0.01) for FHCN in both cases where FaSSIF and FeSSIF were used as transport buffer.rn