Purification and characterization of a surfactin-like molecule produced by Bacillus sp. H2O-1 and its antagonistic effect against sulfate reducing bacteria

Abstract Background Bacillus sp. H2O-1, isolated from the connate water of a Brazilian reservoir, produces an antimicrobial substance (denoted as AMS H2O-1) that is active against sulfate reducing bacteria, which are the major bacterial group responsible for biogenic souring and biocorrosion in petroleum reservoirs. Thus, the use of AMS H2O-1 for sulfate reducing bacteria control in the petroleum industry is a promising alternative to chemical biocides. However, prior to the large-scale production of AMS H2O-1 for industrial applications, its chemical structure must be elucidated. This study also analyzed the changes in the wetting properties of different surfaces conditioned with AMS H2O-1 and demonstrated the effect of AMS H2O-1 on sulfate reducing bacteria cells. Results A lipopeptide mixture from AMS H2O-1 was partially purified on a silica gel column and identified via mass spectrometry (ESI-MS). It comprises four major components that range in size from 1007 to 1049 Da. The lipid moiety contains linear and branched β-hydroxy fatty acids that range in length from C13 to C16. The peptide moiety contains seven amino acids identified as Glu-Leu-Leu-Val-Asp-Leu-Leu. Transmission electron microscopy revealed cell membrane alteration of sulfate reducing bacteria after AMS H2O-1 treatment at the minimum inhibitory concentration (5 μg/ml). Cytoplasmic electron dense inclusions were observed in treated cells but not in untreated cells. AMS H2O-1 enhanced the osmosis of sulfate reducing bacteria cells and caused the leakage of the intracellular contents. In addition, contact angle measurements indicated that different surfaces conditioned by AMS H2O-1 were less hydrophobic and more electron-donor than untreated surfaces. Conclusion AMS H2O-1 is a mixture of four surfactin-like homologues, and its biocidal activity and surfactant properties suggest that this compound may be a good candidate for sulfate reducing bacteria control. Thus, it is a potential alternative to the chemical biocides or surface coating agents currently used to prevent SRB growth in petroleum industries.

Deposizione elettrochimica di film polimerici foto-attivi per la realizzazione di celle solari

Plastic solar cells bear the potential for large-scale power generation based on flexible, lightweight, inexpensive materials. Since the discovery of the photo-induced electron transfer from a conjugated polymer (electron-donor) to fullerene or its derivatives molecules (electron-acceptors), followed by the introduction of the bulk heterojunction concept which means donors and acceptors blended together to realize the fotoactive layer, materials and deposition techniques have been extensively studied. In this work, electrochemical-deposition methods of polymeric conductive films were studied in order to realize bulk heterojunction solar cells. Indium Tin Oxide (ITO) glass electrodes modified with a thin layer of poly(3,4-ethylenedioxythiophene) (PEDOT) were electrochemically prepared under potentiodynamic and potentiostatic conditions; then those techniques were applied for the electrochemical co-deposition of donor and acceptor on modified ITO electrode to produce the active layer (blend). For the deposition of the electron-donor polymer the electropolymerization of many functionalized thiophene monomers was investigated while, as regards acceptors, fullerene was used first, then the study was focused on its derivative PCBM ([6,6]-phenyl-C61-butyric acid methyl ester). The polymeric films obtained (PEDOT and blend) were electrochemically and spectrophotometrically characterized and the film thicknesses were evaluated by atomic force microscopy (AFM). Finally, to check the performances and the efficiency of the realized solar cells, tests were carried out under standard conditions. Nowadays bulk heterojunction solar cells are still poorly efficient to be competitively commercialized. A challenge will be to find new materials and better deposition techniques in order to obtain better performances. The research has led to several breakthroughs in efficiency, with a power conversion efficiency approaching 5 %. The efficiency of the solar cells produced in this work is even lower (lower than 1 %). Despite all, solar cells of this type are interesting and may represent a cheaper and easier alternative to traditional silicon-based solar panels.

Vibrational spectroscopic and electrochemical investigations of multi-centered heme proteins in biomimetic membrane architectures

Membrane proteins play a major role in every living cell. They are the key factors in the cell’s metabolism and in other functions, for example in cell-cell interaction, signal transduction, and transport of ions and nutrients. Cytochrome c oxidase (CcO), as one of the membrane proteins of the respiratory chain, plays a significant role in the energy transformation of higher organisms. CcO is a multi centered heme protein, utilizing redox energy to actively transport protons across the mitochondrial membrane. One aim of this dissertation is to investigate single steps in the mechanism of the ion transfer process coupled to electron transfer, which are not fully understood. The protein-tethered bilayer lipid membrane is a general approach to immobilize membrane proteins in an oriented fashion on a planar electrode embedded in a biomimetic membrane. This system enables the combination of electrochemical techniques with surface enhanced resonance Raman (SERRS), surface enhanced reflection absorption infrared (SEIRAS), and surface plasmon spectroscopy to study protein mediated electron and ion transport processes. The orientation of the enzymes within the surface confined architecture can be controlled by specific site-mutations, i.e. the insertion of a poly-histidine tag to different subunits of the enzyme. CcO can, thus, be oriented uniformly with its natural electron pathway entry pointing either towards or away from the electrode surface. The first orientation allows an ultra-fast direct electron transfer(ET) into the protein, not provided by conventional systems, which can be leveraged to study intrinsic charge transfer processes. The second orientation permits to study the interaction with its natural electron donor cytochrome c. Electrochemical and SERR measurements show conclusively that the redox site structure and the activity of the surface confined enzyme are preserved. Therefore, this biomimetic system offers a unique platform to study the kinetics of the ET processes in order to clarify mechanistic properties of the enzyme. Highly sensitive and ultra fast electrochemical techniques allow the separation of ET steps between all four redox centres including the determination of ET rates. Furthermore, proton transfer coupled to ET could be directly measured and discriminated from other ion transfer processes, revealing novel mechanistic information of the proton transfer mechanism of cytochrome c oxidase. In order to study the kinetics of the ET inside the protein, including the catalytic center, time resolved SEIRAS and SERRS measurements were performed to gain more insight into the structural and coordination changes of the heme environment. The electrical behaviour of tethered membrane systems and membrane intrinsic proteins as well as related charge transfer processes were simulated by solving the respective sets of differential equations, utilizing a software package called SPICE. This helps to understand charge transfer processes across membranes and to develop models that can help to elucidate mechanisms of complex enzymatic processes.

Funktionelle Blockcopolymere für photovoltaische Anwendungen

Zusammenfassung der Dissertation von Pablo A. Walter Thema der Arbeit: Funktionelle Blockcopolymere für photovoltaische Anwendungen Im Zentrum dieser Dissertation stand die Erarbeitung eines Konzepts zum Bau neuartiger Solarzellen auf der Basis von Blockcopolymeren aus Elektronendonatorund Akzeptorblöcken. Dabei soll die Nanophasenseparation in Blockcopolymeren zur Kontrolle und Beeinflussung der Morphologie der Akzeptor- und Donatorkomponenten benutzt werden. Die Blockcopolymere müssen damit alle Materialeigenschaften vereinen, die für den Betrieb einer Solarzelle notwendig sind. Um diesem Ziel gerecht zu werden, war es notwendig, sowohl Monomere als auch neuartige monofunktionale Farbstoffe herzustellen. Es wurden gezielt funktionale Homopolymere und Diblockcopolymeren hergestellt. Es gelang ferner, sowohl die Homopolymere als auch die Blockcopolymere mittels Farbstoffen zu funktionalisieren. Im Zuge der Charakterisierung war es möglich, im Bereich der Polymeranalyse die Glastemperaturen, die Molekulargewichte und die Polydispersitäten zu bestimmen. Des Weiteren galt es, im Rahmen der polymeranalogen Umsetzungen, Möglichkeiten zur gezielten Bestimmung der angebundenen Farbstoffmenge zu entwickeln. Bezüglich der Materialeigenschaften wurden, die HOMOs und die LUMOs der funktionalen Block- und Homopolymere bestimmt. Mittels TEM war es auch erstmals möglich, die Phasenseparation der beiden Blöcke deutlich zu machen. Somit ist es gelungen, neue funktionale Blockcopolymere herzustellen und diese bei dem Bau von neuen polymerbasierten Solarzellen einzusetzen. Es konnte gezeigt werden, dass das Konzept prinzipiell funktioniert.

Aufbau und Simulation des KAOS-Spektrometers für Koinzidenzmessungen in der assoziierten Kaonproduktion

Mit der Erweiterung des Elektronenbeschleunigers MAMI um eine dritte Stufe ist es möglich geworden, am Institut für Kernphysik Teilchen mit offener Strangeness zu produzieren. Für deren Nachweis ist die Drei-Spektrometeranlage der Kollaboration A1 um das von der GSI in Darmstadt übernommene KAOS-Spektrometer erweitert worden. Untersucht wird damit die elementare Reaktion p(e,e' K+)Lambda/Sigma0 wobei das auslaufende Elektron und das Kaon nachgewiesen werden müssen. Wird als Target nicht Wasserstoff verwendet, besteht die Möglichkeit dass sich ein Hyperkern bildet. Spektroskopische Untersuchungen an diesen bieten die Möglichkeit das Potential von Hyperonen in Atomkernen und die Hyperon-Nukleon-Wechselwirkung zu untersuchen. Aufgrund der hervorragenden Strahlqualität bei der Elektroproduktion können hier Massenauflösungen von einigen hundert keV/c² erreicht werden. Mit Hilfe von GEANT4 wurden die Detektoren und die Abbildungseigenschaften des Spektrometers simuliert. Geeignete Ereignisgeneratoren wurden implementiert. Es wurde untersucht, wie mögliche Treffermuster in den Detektoren aussehen, die von einem Trigger auf FPGA-Basis selektiert werden müssen. Ebenso konnte hieraus eine erste Abbildung der Spurkoordinaten auf die Targetkoordinaten und den Teilchenimpuls gewonnen werden. Für das Hyperkernprogramm muss KAOS unter 0° Vorwärtsrichung betrieben werden und der Primärstrahl mit Hilfe einer Schikane durch den Dipol gelenkt werden. Die Simulation zeigt hier eine nur moderate Erhöhung der Strahlenbelastung, vor allem im Bereich des Strahlfängers. Somit ist es möglich, KAOS als doppelseitiges Spektrometer in der Spektrometerhalle zu betreiben. Im Rahmen dieser Arbeit wurden die für sämtliche Detektoren nötige Auslese- und Steuerungselektronik in das vorhandene Datenerfassungssystem und das Steuerungssystem eingebunden. In zwei Strahlzeiten im Herbst 2008 wurden Kaonen im Winkelbereich von 20°-40° mit Impulsen zwischen 400MeV/c und 600MeV/c nachgewiesen. Die aus der Simulation gewonnenen Daten zum Trigger und zur Abbildung kamen zum Einsatz. Es konnte die für eine gute Teilchenidentifikation nötige Zeitauflösung von ca. 1ns FWHM erreicht werden. Die erreichte Winkel- und Impulsauflösung war ausreichend um Lambda und Sigma0-Hyperonen im Spektrum der fehlenden Masse leicht trennen zu können.

Electrical scanning probe microscopy on organic optoelectronic structures

In the field of organic optoelectronics, the nanoscale structure of the materials has huge im-pact on the device performance. Here, scanning force microscopy (SFM) techniques become increasingly important. In addition to topographic information, various surface properties can be recorded on a nanometer length scale, such as electrical conductivity (conductive scanning force microscopy, C-SFM) and surface potential (Kelvin probe force microscopy, KPFM).rnrnIn the context of this work, the electrical SFM modes were applied to study the interplay be-tween morphology and electrical properties in hybrid optoelectronic structures, developed in the group of Prof. J. Gutmann (MPI-P Mainz). In particular, I investigated the working prin-ciple of a novel integrated electron blocking layer system. A structure of electrically conduct-ing pathways along crystalline TiO2 particles in an insulating matrix of a polymer derived ceramic was found and insulating defect structures could be identified. In order to get insights into the internal structure of a device I investigated a working hybrid solar cell by preparing a cross cut with focused ion beam polishing. With C-SFM, the functional layers could be identified and the charge transport properties of the novel active layer composite material could be studied. rnrnIn C-SFM, soft surfaces can be permanently damaged by (i) tip induced forces, (ii) high elec-tric fields and (iii) high current densities close to the SFM-tip. Thus, an alternative operation based on torsion mode topography imaging in combination with current mapping was intro-duced. In torsion mode, the SFM-tip vibrates laterally and in close proximity to the sample surface. Thus, an electrical contact between tip and sample can be established. In a series of reference experiments on standard surfaces, the working mechanism of scanning conductive torsion mode microscopy (SCTMM) was investigated. Moreover, I studied samples covered with free standing semiconducting polymer nano-pillars that were developed in the group of Dr. P. Theato (University Mainz). The application of SCTMM allowed non-destructive imag-ing of the flexible surface at high resolution while measuring the conductance on individual pillarsrnrnIn order to study light induced electrical effects on the level of single nanostructures, a new SFM setup was built. It is equipped with a laser sample illumination and placed in inert at-mosphere. With this photoelectric SFM, I investigated the light induced response in function-alized nanorods that were developed in the group of Prof. R. Zentel (University Mainz). A block-copolymer containing an anchor block and dye moiety and a semiconducting conju-gated polymer moiety was synthesized and covalently bound to ZnO nanorods. This system forms an electron donor/acceptor interface and can thus be seen as a model system of a solar cell on the nanoscale. With a KPFM study on the illuminated samples, the light induced charge separation between the nanorod and the polymeric corona could not only be visualized, but also quantified.rnrnThe results demonstrate that electrical scanning force microscopy can study fundamental processes in nanostructures and give invaluable feedback to the synthetic chemists for the optimization of functional nanomaterials.rn

Polimeri tiofenici funzionali e loro applicazioni nel fotovoltaico

Recentemente, sempre più attenzione è stata rivolta all' utilizzo di coloranti organici come assorbitori di luce per la preparazione di strati fotoattivi in celle solari organiche (OPV). I coloranti organici presentano un'elevata abilità nella cattura della luce solare grazie all'elevato coefficiente di estinzione molare e buone proprietà fotofisiche. Per questi motivi sono eccellenti candidati per l'incremento della conversione fotoelettrica in OPV. In questa tesi viene descritta una nuova strategia per l'incorporazione di derivati porfirinici in catena laterale a copolimeri tiofenici. Gli studi svolti hanno dimostrato che poli(3-bromoesil)tiofene può essere variamente funzionalizzato con idrossitetrafenilporfirina (TPPOH), per l'ottenimento di copolimeri utilizzabili come materiali p-donatori nella realizzazione di OPV. I copolimeri poli[3-(6-bromoesil)tiofene-co-(3-[5-(4-fenossi)-10,15,20-trifenilporfirinil]esil tiofene] P[T6Br-co-T6TPP] contenenti differenti quantità di porfirina, sono stati sintetizzati sia con metodi non regiospecifici che regiospecifici, con lo scopo di confrontarene le proprietà e di verificare se la strutture macromolecolare che presenta una regiochimica di sostituzione sempre uguale, promuove o meno il trasporto della carica elettrica, migliorando di conseguenza l'efficienza. E' stato inoltre effettuato un ulteriore confronto tra questi derivati e derivati simili P[T6H-co-T6TPP] che non contengono l'atomo di bromo in catena laterale con lo scopo di verificare se l'assenza del gruppo reattivo, migliora o meno la stabilità termica e chimica dei film polimerici, agendo favorevolmete sulle performance dei dispositivi fotovoltaici. Tutti i copolimeri sono stati caratterizzati con differenti tecniche: spettroscopia NMR, FT-IR e UV-Vis, analisi termiche DSC e TGA, e GPC. Le celle solari Bulk Heterojunction, preparate utilizzando PCBM come materiale elettron-accettore e i copolimeri come materilai elettron-donatori, sono state testate utilizzando un multimetro Keithley e il Solar Simulator.

Bioremediation of PCB-contaminated marine sediments: From identification of indigenous dehalorespirers to enhancement of microbial reductive dechlorination

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area.

On-surface chemical reactions on an insulating substrate

Diese Arbeit beschreibt zum ersten Mal die kovalente Verknüpfung organischer Moleküle auf einer Isolatoroberfläche, motiviert im Hinblick auf die Nutzung der Synthesemethode für die molekulare Elektronik und verwandte Anwendungen. Durch die Verwendung der Nichtkontakt-Rasterkraftmikroskopie und der Kelvinprobe-Mikroskopie bei Raumtemperatur wurden grundlegende molekulare Prozesse der Wechselwirkungen zwischen Molekülen und der Calcit(10.4) Oberfläche sowie die chemische Reaktivität der Moleküle auf der Oberfläche analysiert. Das Zusammenspiel zwischen intermolekularen und Molekül-Oberfläche Wechselwirkungen zeigt sich für Biphenyl-4,4'-dicarbonsäure (BPDCA) durch die Koexistenz zweier unterschiedlicher molekularer Strukturen, die einen Einblick in die treibenden Kräfte der molekularen Selbstorganisation bieten. Die sehr ausgeprägte Reihenstruktur basiert auf der optimalen geometrischen Struktur der BPDCA Moleküle zu den Abmessungen des Substrats, während die zweite Struktur durch Wasserstoffbrücken zwischen den Molekülen gekennzeichnet ist. Der Deprotonierungsvorgang von 2,5-Dihydroxybenzoesäure (DHBA)-Molekülen auf Calcit wird bei Zimmertemperatur gezeigt. Zwei Phasen werden beobachtet, die nach Aufbringen der Moleküle koexistieren. Mit der Zeit geht eine bulk-ähnliche Phase in eine stabile, dicht gepackte Phase über. Der Übergang wird durch Betrachtung des Protonierungszustands der Moleküle erklärt. Die bulk-ähnliche Phase benötigt Wasserstoffbrückbindungen zur Strukturbildung. Werden die Moleküle deprotoniert, so wird die resultierende dicht gepackte Phase durch die elektrostatische Wechselwirkung der deprotonierten Carboxylatgruppen mit den Oberflächen-Calciumkationen stabilisiert. 4-Iodbenzoesäure (IBA)-Moleküle bilden auf Calcit nur Inseln an Stufenkanten, was auf die schwache Molekül-Oberflächen-Wechselwirkung zurückzuführen ist. Für einen stärkeren Einfluss des Substrats durchlaufen die Moleküle einen kontrollierten Übergangsschritt vom protonierten zum deprotonierten Zustand. Im deprotonierten Zustand nehmen die Moleküle eine wohldefinierte Adsorptionsposition auf dem Substrat ein. Die deprotonierte Säuregruppe wird ausgenutzt, um die Desorption der halogensubstituierten Benzoesäure-Moleküle bei der thermischer Aktivierung für die Vernetzungsreaktion zu vermeiden. Darüber hinaus wird die Carboxylatgruppe als starker Elektronendonor verwendet um die Phenyl-Halogen-Bindung zu schwächen und somit die homolytische Spaltung dieser Bindung auch bei moderaten Temperaturen zu ermöglichen. Diesem Konzept folgend ist die erste erfolgreiche kovalente Verknüpfung von 2,5-Diiod-benzoesäure, 2,5-Dichlorbenzoesäure, 3,5-Diiod Salicylsäure und 4-Iod-benzoesäure zu durchkonjugierten molekularen Drähten, Zick-Zack-Strukturen sowie Dimere gezeigt durch Ausnutzen von unterschiedlichen Substitutionsposition sowie Ändern der Anzahl der substituierten Halogenatome. Aufbauend auf diesem Erfolg, wird eine zweistufige Vernetzungsreaktion vorgestellt. Zum Induzieren der ortsspezifischen und sequentiellen kovalenten Verknüpfung wird ein Ausgangsmolekül gewählt, das sowohl eine Bromphenyl als auch eine Chlorphenyl Gruppe mit unterschiedlichen Dissoziationsenergien für die homolytische Spaltung besitzt. Die Reaktionsstellen und sequentielle Reihenfolge für die Reaktion sind somit in der molekularen Struktur einkodiert und bisher unerreichte Reaktionspfade können mithilfe der kovalente Verknüpfung organischer Moleküle auf einer Isolatoroberfläche beschritten werden.

Ruthenium-based light harvesting complexes

Polypyridylkomplexe von Ruthenium(II) besitzen eine Vielzahl von Anwendungen, z. B. in Farbstoff-sensibilisierten Solarzellen und als Photokatalysatoren. [Ru(bpy)3]2+ ist einer der prominentesten Ruthenium(II)-Komplexe und besitzt langlebige angeregte 3MLCT-Zustände mit einer Lebensdauer von 1 µs und einer Lumineszenz-Quantenausbeute von 10%. [Ru(bpy)3]2+ ist chiral und kann Stereoisomere bilden, wenn die Liganden unsymmetrisch substituiert sind oder im Falle von oligonuklearen rac/meso-Komplexen. Bis-tridentate Komplexe wie [Ru(tpy)2]2+ sind achiral und umgehen damit unerwünschte Stereoisomere. [Ru(tpy)2]2+ besitzt jedoch enttäuschende photophysikalische Eigenschaften mit einer 3MLCT-Lebensdauer von nur etwa 0.2 ns und einer Quantenausbeute von ≤ 0.0007%. Die Anbringung von Substituenten an [Ru(tpy)2]2+ sowie die Aufweitung der Liganden-Bisswinkel auf 90° bewirken deutlich verbesserte Eigenschaften der emittierenden 3MLCT-Zustände. rnDieser Strategie folgend wurden in der vorliegenden Arbeit neue bis-tridentate Ruthenium(II)-Komplexe entwickelt, synthetisiert und charakterisiert. Durch Anbringen von Ester-Substituenten und Verwenden von Liganden mit erweiterten Bisswinkeln konnten 3MLCT-Lebensdauern von bis zu 841 ns und Quantenausbeuten von bis zu 1.1% erreicht werden. Die neuen bis-tridentaten Komplexe weisen eine deutlich erhöhte Photostabilität im Vergleich zu tris-bidentatem [Ru(bpy)3]2+ auf. rnDie Komplexe wurden als Emitter in Licht-emittierenden elektrochemischen Zellen eingebaut und zeigen Elektrolumineszenz mit einer tiefroten Farbe, die bis ins NIR reicht. Ebenso wurden die Komplexe als Lichtsammler in Farbstoff-sensibilisierten Solarzellen getestet und erreichen Licht-zu-Energie-Effizienzen von bis zu 0.26%. rnDinukleare, stereochemisch einheitliche Ruthenium(II)-Komplexe wurden oxidiert um die Metall-Metall-Wechselwirkung zwischen Ru(II) und Ru(III) in der einfach oxidierten Spezies zu untersuchen. Die unterschiedlichen Redoxeigenschaften der beiden Rutheniumzentren in den verwendeten dinuklearen Verbindungen führt zu einer valenzlokalisierten Situation in der keine Metall-Metall-Wechselwirkung beobachtet wird. Ebenso wurde die Oxidation eines einkernigen Ruthenium(II)-Komplexes sowie dessen spontane Rückreduktion untersucht.rnEnergietransfersysteme wurden mittels Festphasensynthese hergestellt. Dabei ist ein Bis(terpyridin)ruthenium(II)-Komplex als Energie-Akzeptor über eine unterschiedliche Anzahl an Glycineinheiten mit einem Cumarin-Chromophor als Energie-Donor verknüpft. Bei einer kleinen Zahl an Glycineinheiten (0, 1) findet effektiver Energietransfer vom Cumarin- zum Ruthenium-Chromophor statt, wogegen bei zwei Glycineinheiten ein effektiver Energietransfer verhindert ist.rnLicht-induzierte Ladungstrennung wurde erreicht, indem Bis(terpyridin)ruthenium(II)-Komplexe als Chromophore in einem Donor-Chromophor-Akzeptor-Nanokomposit eingesetzt wurden. Dabei wurde ein Triphenylamin-enthaltendes Blockcopolymer als Elektronendonor und ZnO-Nanostäbchen als Elektronenakzeptor verwendet. Bei Bestrahlung des Chromophors werden Elektronen in die ZnO-Nanostäbchen injiziert und die Elektronenlöcher wandern in das Triphenylamin-enthaltende Blockcopolymer. rnrn

Clinical and biochemical consequences of p450 oxidoreductase deficiency

Patients with P450 oxidoreductase (POR) deficiency typically present with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley-Bixler craniosynostosis syndrome. Since our first report in 2004, more than 40 POR mutations have been identified in over 65 patients. POR is the obligate electron donor to all microsomal P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46, XY newborns as well as overvirilization in those who are 46, XX. This may be explained by impaired aromatization of fetal androgens that may cause maternal virilization and low urinary estriol levels during pregnancy. In addition, the alternate 'backdoor' pathway of androgen biosynthesis, which leads to dihydrotestosterone production bypassing androstenedione and testosterone, may also play a role. Functional assays studying the effects of POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. POR variants may also affect skeletal development and drug metabolism. As most drugs are metabolized by hepatic microsomal P450 enzymes, studies of the impact of POR mutations on drug-metabolizing P450s are particularly important.

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype-phenotype and structure-function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.

Clinical and biochemical description of a novel CYP21A2 gene mutation 962_963insA using a new 3D model for the P450c21 protein

OBJECTIVE: A severely virilized 46, XX newborn girl was referred to our center for evaluation and treatment of congenital adrenal hyperplasia (CAH) because of highly elevated 17alpha-hydroxyprogesterone levels at newborn screening; biochemical tests confirmed the diagnosis of salt-wasting CAH. Genetic analysis revealed that the girl was compound heterozygote for a previously reported Q318X mutation in exon 8 and a novel insertion of an adenine between nucleotides 962 and 963 in exon 4 of the CYP21A2 gene. This 962_963insA mutation created a frameshift leading to a stop codon at amino acid 161 of the P450c21 protein. AIM AND METHODS: To better understand structure-function relationships of mutant P450c21 proteins, we performed multiple sequence alignments of P450c21 with three mammalian P450s (P450 2C8, 2C9 and 2B4) with known structures as well as with human P450c17. Comparative molecular modeling of human P450c21 was then performed by MODELLER using the X-ray crystal structure of rabbit P450 2B4 as a template. RESULTS: The new three dimensional model of human P450c21 and the sequence alignment were found to be helpful in predicting the role of various amino acids in P450c21, especially those involved in heme binding and interaction with P450 oxidoreductase, the obligate electron donor. CONCLUSION: Our model will help in analyzing the genotype-phenotype relationship of P450c21 mutations which have not been tested for their functional activity in an in vitro assay.

P450 oxidoreductase deficiency - a new form of congenital adrenal hyperplasia

Patients with adrenal insufficiency, genital anomalies and bony malformations resembling the Antley- Bixler syndrome (a craniosynostosis syndrome), are likely to have P450 oxidoreductase (POR) deficiency. Since our first report in 2004, about 26 recessive POR mutations have been identified in 50 patients. POR is the obligate electron donor to all microsomal (type II) P450 enzymes, including the steroidogenic enzymes CYP17A1, CYP21A2 and CYP19A1. POR deficiency may cause disordered sexual development manifested as genital undervirilization in 46,XY newborns as well as overvirilization in those who are 46,XX. This may be explained by impaired aromatization of fetal androgens which may also lead to maternal virilization and low urinary estriol levels during pregnancy. A role for the alternate 'backdoor' pathway of androgen biosynthesis, leading to dihydrotestosterone production bypassing androstenedione and testosterone, has been suggested in POR deficiency but remains unclear. POR variants may play an important role in drug metabolism, as most drugs are metabolized by hepatic microsomal P450 enzymes. However, functional assays studying the effects of specific POR mutations on steroidogenesis showed that several POR variants impaired CYP17A1, CYP21A2 and CYP19A1 activities to different degrees, indicating that each POR variant must be studied separately for each potential target P450 enzyme. Thus, the impact of POR mutations on drug metabolism by hepatic P450s requires further investigation.

IN-SITU TEM PLASMA CHIP NANOFABRICATION AND CHARACTERIZATION

A silicon-based microcell was fabricated with the potential for use in in-situ transmission electron microscopy (TEM) of materials under plasma processing. The microcell consisted of 50 nm-thick film of silicon nitride observation window with 60μm distance between two electrodes. E-beam scattering Mont Carlo simulation showed that the silicon nitride thin film would have very low scattering effect on TEM primary electron beam accelerated at 200 keV. Only 4.7% of primary electrons were scattered by silicon nitride thin film and the Ar gas (60 μm thick at 1 atm pressure) filling the space between silicon nitride films. Theoretical calculation also showed low absorption of high-energy e-beam electrons. Because the plasma cell needs to survive the high vacuum TEM chamber while holding 1 atm internal pressure, a finite element analysis was performed to find the maximum stress the low-stress silicon nitride thin film experienced under pressure. Considering the maximum burst stress of low-stress silicon nitride thin film, the simulation results showed that the 50 nm silicon nitride thin film can be used in TEM under 1 atm pressure as the observation window. Ex-situ plasma generation experiment demonstrated that air plasma can be ignited at DC voltage of 570. A Scanning electron microscopy (SEM) analysis showed that etching and deposition occurred during the plasma process and larger dendrites formed on the positive electrode.