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.

Architettura e verit��. Emil Steffann architetto di chiese

La ricerca pone al suo centro lo studio dell'opera architettonica di Emil Steffann (1899-1968) la cui produzione realizzata consta, nel breve arco temporale che va dal 1950 al 1968, del ragguardevole numero di trentanove chiese, rappresentando un caso emblematico di progettazione e costruzione di edifici per il culto cristiano in grado di raffigurarne concretamente i principi fondativi liturgici, estetici e morfologici. L'architettura di Steffann, profondamente ispirata dallo spirito religioso, legata a figure primigenie che plasmano lo stare-insieme della comunit�� nella qualit�� corporea della materia, dove la presenza liturgica e monumentale si esprime nel silenzio e nella disponibilit�� di uno spazio circoscritto dai muri e direzionato dalla luce, concorre a definire nell'oggettivo amore per il vero la percezione estetico-teologica e la poetica formativa che connaturano, a nostro parere, progetto e segno della chiesa. Il testo concretizza il primo studio monografico completo di questo corpus architettonico e si basa sulla ricognizione diretta delle opere di Steffann; ne �� derivata una narrazione non conseguente a un ordine cronologico o di presupposta importanza degli edifici, bens�� che ricerca ed evidenzia corrispondenze tra nodi di una rete ideativa la quale, con diversi gradi di finitezza, in punti non sempre omogenei del tempo e dello spazio, denota un'esperienza autentica del comporre e del costruire. Il racconto individua gli oggetti architettonici, ne discute la consistenza aprendosi a riferimenti altri (in particolare il pensiero ecclesiologico-liturgico di Romano Guardini e quello estetico-teologico di Hans Urs von Balthasar) in grado di illuminarne la genesi e la manifestazione, li lega infine in sequenze analogiche. Una serie di tavole fotografiche originali, parte ineludibile e integrante della ricerca, testimonia dello stato attuale dei luoghi, connotando ulteriormente l'aspetto info-rappresentativo della loro composizione architettonica. In chiusura, la sintesi architetturale vuole essere uno strumento di verifica e progetto, quindi di trasposizione futura, correlato all'elaborazione documentaria.

Realisierung der IceCube MonteCarlo-Produktion im WLCG und Untersuchung von Auswirkungen meteorologischer Parameter auf die Erzeugung atmosph��rischer Myonen

Das am S��dpol gelegene Neutrinoteleskop IceCube detektiert hochenergetische Neutrinos ��ber die schwache Wechselwirkung geladener und neutraler Str��me. Die Analyse basiert auf einem Vergleich mit Monte-Carlo-Simulationen, deren Produktion global koordiniert wird. In Mainz ist es erstmalig gelungen, Simulationen innerhalb der Architektur des Worldwide LHC Computing Grid (WLCG) zu realisieren, was die M��glichkeit er��ffnet, Monte-Carlo-Berechnungen auch auf andere deutsche Rechnerfarmen (CEs) mit IceCube-Berechtigung zu verteilen. Atmosph��rische Myonen werden mit einer Rate von ��ber 1000 Ereignissen pro Sekunde aufgezeichnet. Eine korrekte Interpretation dieses dominanten Signals, welches um einen Faktor von 10^6 reduziert werden muss um das eigentliche Neutrinosignal zu extrahieren, ist deswegen von gro��er Bedeutung. Eigene Simulationen mit der Software-Umgebung CORSIKA wurden durchgef��hrt um die von Energie und Einfallswinkel abh��ngige Entstehungsh��he atmosph��rischer Myonen zu bestimmen. IceCube Myonraten wurden mit Wetterdaten des European Centre for Medium-Range Weather Forcasts (ECMWF) verglichen und Korrelationen zwischen jahreszeitlichen sowie kurzzeitigen Schwankungen der Atmosph��rentemperatur und Myonraten konnten nachgewiesen werden. Zudem wurde eine Suche nach periodischen Effekten in der Atmosph��re, verursacht durch z.B. meteorologische Schwerewellen, mit Hilfe einer Fourieranalyse anhand der IceCube-Daten durchgef��hrt. Bislang konnte kein signifikanter Nachweis zur Existenz von Schwerewellen am S��dpol erbracht werden.

In-vitro synthesis and reconstitution of cytochrome bo 3 ubiquinol oxidase in artificial membranes

Forschung ��ber Membranenproteine stellt strenge Hindernisse, seit ruhigem gerade wenige Beispiele der Membranenproteinsorten sind gekennzeichnet worden in den verwendbaren experimentellen Plattformen gegen��ber. Die Hauptherausforderung ist, ihre ausgezeichnete entworfene strukturelle Vollst��ndigkeit zu konservieren, w��hrend die Ausdruck-, Lokalisierungs- und Wiederherstellungprozesse auftreten. In-vitro ��bersetzungssysteme k��nnen Vorteile ��ber auf Zellenbasisgenausdruck zum Beispiel haben, wenn das ��ber-ausgedr��ckte Produkt zur Wirtszelle giftig ist oder wenn fehlende Pfosten-��bersetzungs��nderung in den bakteriellen Ausdrucksystemen die Funktionalit��t der S��ugetier- Proteine oder Mangel an vorhandenem Membranenraum verdirbt, Funktionsausdruck verbieten.rn Der Nachahmer von biologische Membranen wie feste gest��tzte Lipidmembranen sind als Plattform am meisten benutzt, Proteinmembraneninteraktionen nachzuforschen. Wir sind in der Lage, Membranenproteinsorte, da wir eine Plattform f��r Membranenproteinsynthese vorstellen, n��mlich die in-vitrosynthese der Membranenproteine in ein Peptid gest��tztes Membranensystem zu adressieren. Die Wiederherstellung der Membranenproteine in den Lipid bilayers resultiert im Allgemeinen mit verschiedenen Proteinanpassungen. Als Alternative erforschen wir dieses System zum ersten Mal, um genaueres Modell zu den zellularen Membranen zu verursachen und ihre Funktion, wie Proteineinf��gung, Proteinfunktion und Ligandinteraktionen nachzuahmen.rn In dieser Arbeit ist unser Ziel, komplizierte Transmembraneproteine, wie des Cytochrome bo3-ubiquinol Oxydase (Cyt-bo3) direkt innerhalb der biomimetic vorbildlichen Membrane zu synthetisieren. In unserem System wird festes gest��tztes tBLM wie, P19/DMPE/PC als Plattform benutzt. Dieses k��nstliche Membranensystem mimiks die amphiphile Architektur eines Zelle-abgeleiteten Membranensystems.rn

3D-Strukturanalyse von Molluskenh��mocyaninen aus elektronenmikroskopischen Bildern

Diese Arbeit pr��sentiert die bislang h��chst aufgel��sten KryoEM-Strukturen f��r ein Cephalopoden h��mocyanin Dekamer (Nautilus pompilus H��mocyanin, NpH) und ein Gastropoden H��mocyanin Didekamer (keyhole limpet hemocyanin isoform 1). Durch die Methoden des ���molecular modelling��� und ���rigid-body-fiting��� wurde auch eine detaillierte Beschreibung beider Strukturen auf atomarem Niveau erstmalig m��glich. H��mocyanine sind kupferhaltige Sauerstoff-Transportproteine die frei gel��st in Blut zahlreicher Arthropoden und Mollusken vorkommen. Allgemein sind Molluskenh��mocyanine als Dekamere (Hohlzylinder aus 5 Untereinheiten-dimere) oder Didecamere (Zusammenlagerung von zwei Dekameren) zu finden. Durch Anlagerung weiterer Dekamere bilden sich teilweise tubul��re Multidekamere. H��mocyanine der Cephalopoden bestehen ausschlie��lich aus solit��ren Decameren. In Octopus und Nautilus bestehen die 10 Untereinheiten aus 7 funktionellen Einheiten(FU-a bis FU-g), wobei jede FU ein Sauerstoffmolek��l binden kann. FUs a-f bilden die Wand des ringf��rmigen Molek��ls und 10 Kopien der FU-g bilden einen sogenannten ���inneren Kragenkomplex���. Das im Rahmen dieser Arbeit erstelltes molekulares Modell von NpH kl��rt die Struktur des Dekamers vollst��ndig auf. Wir waren zum ersten Mal in der Lage das Untereinheiten-dimer, den Verlauf der Polypeptidkette und 15 unterschiedliche Kontaktstellen zwischen FUs zu identifizieren. Viele der inter-FU-Kontakte weisen Aminos��urenkonstellationen auf, die die Basis f��r die ��bertragung allosterischer Wechselwirkungen zwischen FUs darstellen k��nnten und Hinweise f��r den Aufbau der allosterische Einheit geben. Potentielle Bindungsstellen f��r N-glykosidische Zucker und bivalente Kationen wurden auch identifiziert. Im Gegensatz zu NpH, kommen Gastropoden H��mocyanine (inkl. KLH) haupts��chlich als Didekamere vor und der Kragenkomplex wird in diesem Fall aus 2 FUs gebildet (Fu-g und FU-h). Die zus��tzliche C'-terminale FU-h zeichnet sich durch eine spezielle Verl��ngerung von ~ 100 Aminos��uren aus. KLH stammt aus der kalifornische Schnecke Megathura crenulata und kommt seit mehreren Jahrzehnten als Immunostimulator in der immunologischen Grundlagenforschung und klinischen Anwendung zum Einsatz. KLH weist zwei Isoformen auf, KLH1 und KLH2. Das vorliegende Modell von KLH1 erlaubt die komplexe Architektur dieses riesigen Proteins in allen Details zu verstehen, sowie einen Vergleich zum dem NpH Dekamer auf atomare Ebene. Es wurde gefunden, dass das Untereinheitensegment a-b-c-d-e-f-g, sowie die equivalenten Kontaktstellen zwichen FUs stark konserviert sind. Dies deutet darauf hin, dass in Bezug auf die ��bertragung allosterische Signale zwischen benachbarten FUs, grundlegende Mechanismen in beiden Molek��len beibehalten wurden. Weiterhin, konnten die Verbindungen zwischen den zwei Dekameren ertsmalig identifiziert werden. Schlie��lich, wurde die Topologie der N-glycosidischen Zucker, welche f��r die immunologische Eigenschaften von KLH1 von gro��er Bedeutung sind, auch aufgekl��rt. Somit leistet die vorliegende Arbeit einen wesentlichen Schritt zum Verst��ndnis der Quart��rstruktur und Funktion der Molluskenh��mocyanine.rn

Solid-state NMR on columnar supramolecular structures

The present work deals with the characterisation of three columnar self-assembled systems, that is, benzene-1,3,5-tricarboxamides, a peripherally thioalkyl-substituted phthalocyanine, and several oligo-(p-phenylenevinylene)s. In order to probe the supramolecular organisation solid-state NMR has been used as the main technique, supported by X-ray measurements, theoretical methods, and thermal analysis. rnrnBenzene-1,3,5-tricarboxamides (BTAs) turned out to be well suited model compounds to study various fundamental supramolecular interactions, such as ��-��-interactions, hydrogen bonding, as well as dynamic and steric effects of attached side chains. Six BTAs have been investigated in total, five with a CO-centred amide group bearing different side chains and one with an inverted N-centred amide group. The physical properties of these BTAs have been investigated as a function of temperature. The results indicated that in case of the CO-centred BTAs the stability of the columnar mesophase depends strongly on the nature of the side chains. Further experiments revealed a coplanar orientation of adjacent BTA molecules in the columnar assembly of CO-centred BTAs, whereas the N-centred BTA, showed a deviating not fully coplanar arrangement. These differences were ascribed to distinct hydrogen bonding schemes, involving a parallel alignment of hydrogen bonds in case of CO-centred BTAs and an antiparallel alignment in case of the N-centred counterpart.rnrn The fundamental insights of the supramolecular organisation of BTAs could be partially adapted to an octa-substituted phthalocyanine with thiododecyl moieties. Solid-state NMR in combination with chemical shift calculations determined a tilted herringbone arrangement of phthalocyanine rings in the crystalline phase as well as in the mesophase. Moreover, 1H NMR measurements in the mesophase of this compound suggested an axial rotation of molecules, which is inhibited in the crystalline phase.rnrnAs a third task, the supramolecular assembly of oligo-(p-phenylenevinylene)s of varying length and with different polar head groups have been investigated by a combined X-ray and solid-state NMR study. The results revealed a columnar structure formation of these compounds, being promoted by phase separation of alkyl side chains and aromatic rigid rods. In this system solid-state NMR yielded meaningful insight into the isotropisation process of butoxy and 2-S-methylbutoxy substituted oligo-(p-phenylenevinylene) rods.rn

Comparative analysis of midline guidance and axon pathfinding in the chelicerates Achaearanea tepidariorum and Cupiennius salei

Die Neurogenese und axonale Wegfindung sind in den vergangenen Jahrzehnten Thema einer Vielzahl wissenschaftlicher Untersuchungen in den verschiedensten Organismen gewesen. Die zusammengetragenen Daten in Insekten und Crustaceen geben eine gute ��bersicht dar��ber, wie das Nervensystem in Arthropoden aufgebaut wird. Die entwicklungsbiologischen Prozesse, die daran beteiligt sind, sind in den beiden genannten Gruppen sehr gut verstanden. In den Gruppen der Cheliceraten und Myriapoden jedoch wurden ��hnliche Analysen bisher kaum durchgef��hrt. Das Hauptanliegen dieser Arbeit war es daher, Mechanismen in den Spinnen Achaearanea tepidariorum und Cupiennius salei, zwei Vertretern der Cheliceraten, zu untersuchen, die eine Rolle im Leitsystem der ventralen Mittellinie und bei der axonalen Wegfindung spielen. Eine Vorraussetzung hierf��r sind Kenntnisse ��ber die Architektur des Zentralnervensystems. In einem ersten Schritt beschrieb ich daher grundlegend die Morphologie des Nervensystems im Verlauf der gesamten Embryoalentwicklung. Ich konnte zeigen, dass in Spinnen ein f��r Arthropoden typisches Strickleiternervensystem gebildet wird. Dieses wird von segmental angelegten Neuronen geformt, wobei sowohl Gruppen von Zellen als auch einzelne Neurone daran beteiligt sind, die prim��ren axonalen Trakte zu etablieren. Im Besonderen konnte ich eine Zelle identifizieren, die in Position, Projektionsmuster und der Expression des Markergens even-skipped vergleichbar zum PR2 Neuron in Drosophila ist, welches die posteriore Wurzel des Segmentalnervs anlegt.rnrnIn einem zweiten Ansatz untersuchte ich die ventrale Mittellinie in Spinnen im Bezug auf ihre m��gliche Funktion in der axonalen Wegfindung. Es konnte gezeigt werden, dass es sich beim Epithel der Mittellinie, das die L��cke zwischen beiden Keimstreifh��lften w��hrend des gesamten Prozesses der Inversion ��berspannt, um eine transiente Struktur handelt, die keine neuralen Zellen hervorbringt. Es ist daher vergleichbar mit der so genannten Floor plate in Vertebraten, die ebenfalls nur vor��bergehend existiert. Die Untersuchung von single minded (sim) zeigte, dass es, anders als in Drosophila, wo sim ein wichtiges regulatorisches Gen f��r die korrekte Spezifizierung von Mittellinienzellen ist, nicht in den Zellen der Mittellinie, sondern in diesen benachbarten Zellen, exprimiert wird. Das ist vergleichbar mit Vertebraten. Zus��tzlich konnte ich Expression von sim an den Basen der Gliedmassen und im Kopf nachweisen. Wie in Vertebraten k��nnte sim an der Musterbildung dieser Gewebe beteiligt sein. Dennoch spielt die Mittellinie in Spinnen eine wichtige Rolle als Organisator f��r auswachsende, kommissurale Axone. Diese Funktion teilt sie mit anderen Invertebraten und Vertebraten.rnrnDie Signaltransduktionskaskade, die an der axonalen Wegfindung an der Mittellinie beteiligt ist, ist in den verschiedensten Organismen hoch konserviert. In der vorliegenden Arbeit konnte ich sowohl in Achaearanea als auch in Cupiennius ein netrin Homolog identifizieren und eine konservierte Funktion des Wegfindungsmolek��ls w��hrend der Bildung der Kommissuren aufzeigen. RNAi Experimente belegen, dass, wird die Funktion von netrin herunterreguliert, das Strickleiternervensystem nicht korrekt gebildet wird, ins Besondere die kommissuralen Faszikel. Des Weiteren konnte ich eine neue Funktion von netrin, die bisher in anderen Organsimen noch nicht beschrieben wurde, identifizieren. Neben seiner Rolle in der axonalen Wegfindung, scheint netrin auch an der epithelialen Morphogenese im zentralen Nervensystem beteiligt zu sein. In dieser Funktion scheint netrin in Gliazellen, die die epithelialen Vesikel der Invaginationsgruppen umh��llen, wichtig zu sein, um neurale Vorl��uferzellen in einem undifferenzierten Zustand zu halten. Der Abbau von netrin Transkript durch RNA Interferenz f��hrt zu einer verfr��hten Segregation neuraler Vorl��uferzellen aus dem epithelialen Verband der Invaginationsgruppen und zu einer Zunahme an Zellen, die den fr��hen Differenzierungsmarker islet exprimieren.

Structure and properties of nanostructures from polyelectrolytes and porphyrins: Self-Assembly in aqueous solution

In this work self-assembling model systems in aqueous solution were studied. The systems contained charged polymers, polyelectrolytes, that were combined with oppositely charged counterions to build up supramolecular structures. With imaging, scattering and spectroscopic techniques it was investigated how the structure of building units influences the structure of their assemblies. Polyelectrolytes with different chemical structure, molecular weight and morphology were investigated. In addition to linear polyelectrolytes, semi-flexible cylindrical bottle-brush polymers that possess a defined cross-section and a relatively high persistence along the backbone were studied. The polyelectrolytes were combined with structural organic counterions having charge numbers one to four. Especially the self-assembly of polyelectrolytes with different tetravalent water-soluble porphyrins was studied. Porphyrins have a rigid aromatic structure that has a structural effect on their self-assembly behavior and through which porphyrins are capable of self-aggregation via ��-�� interaction. The main focus of the thesis is the self-assembly of cylindrical bottle-brush polyelectrolytes with tetravalent porphyrins. It was shown that the addition of porphyrins to oppositely charged brush molecules induces a hierarchical formation of stable nanoscale brush-porphyrin networks. The networks can be disconnected by addition of salt and single porphyrin-decoratedrncylindrical brush polymers are obtained. These two new morphologies, brush-porphyrin networks and porphyrin-decorated brush polymers, may have potential as functional materials with interesting mechanical and optical properties.

La conservazione viva dell'esistente. Storia, progetto e restauro nell'opera di Josef Wiedemann

La tesi analizza, nel quadro del secondo dopoguerra, quattro casi studio scelti tra le opere di ricostruzione dell���architetto Josef Wiedemann (1910-2001) nel centro di Monaco di Baviera: Odeon (1951-1952), Alte Akademie (1951-1955), Siegestor (1956-1958) e Glyptothek (1961-1972). L���architetto si occupa di opere simbolo della citt�� di Monaco, affrontando la loro ricostruzione come un tema fondante per la storia e l���identit�� del popolo bavarese, ma soprattutto come un���occasione per definire un metodo d���intervento sulle rovine della guerra. Il suo lavoro �� caratterizzato infatti per la ricerca costante di una sintesi tra interesse per la conservazione dell���antico e apertura al nuovo; ispirandosi all���insegnamento del maestro Hans D��llgast, Wiedemann traccia una nuova originale strada per l���intervento sull���antico, segnata da una profonda capacit�� tecnico-progettuale e dall'attenzione alle nuove esigenze a cui deve rispondere un���architettura contemporanea. Partendo dai suoi scritti e dalle sue opere, si pu�� rilevare un percorso coerente che, partendo dalla conoscenza della storia dell'edificio, ripercorrendone l���evoluzione dallo stato che potremmo definire ���originario��� allo stato di rovina, giunge a produrre nel progetto realizzato una sintesi tra il passato e il futuro. L'architetto, nella visione di Wiedemann, �� chiamato a un compito di grande responsabilit��: conoscere per progettare (o ri-progettare) un edificio che porta impressi su di s�� i segni della propria storia. Nel metodo che viene messo progressivamente a punto operando nel corpo vivo dei monumenti feriti dalla guerra, �� percepibile fino a distinguerlo chiaramente l���interesse e l���influenza del dibattito italiano sul restauro. La conservazione ���viva��� dell'esistente, cos�� come viene definita da Wiedemann stesso, si declina in modo diverso per ogni caso particolare, approdando a risultati differenti tra loro, ma che hanno in comune alcuni principi fondamentali: conoscere, ricordare, conservare e innovare.

Multifunctional magnetically loaded polyorganosiloxane nanocomposites for biomedical applications

For the last few decades, the interest in functional nanomaterials is steadily increasing. Especially, in biomedicine the range of possible applications of multifunctional nanoparticles including dye-labeled makers and drug loaded carrier systems is extraordinary large. The incorporation of magnetic nanoparticles allows for an additional magnetic detection and manipulation. One promising system on the way to multifunctional nanomaterials is the polyorganosiloxane system. Via polycondensation of silan monomers in aqueous dispersion polyorganosiloxane nanoparticles with particle diameter between 10 and 150 nm can be synthesized. The versatile silane chemistry allows for the design of multifunctional network structures. In this work, hydrophilic iron oxide nanoparticles could be encapsulated into the polymeric particles in a highly efficient process whereat the superparamagnetic nature of the inorganic particles was restrained. The influence of different sized particles as well as the amount of the incorporated material was investigated. Using a core-shell architecture, controlled core and surface modifications could be achieved. An effective fluorescent labeling was performed via incorporation of dye-labeled monomers. Additionally, a hydrophilic surface modification was carried out via a grafting onto process of poly(ethylene glycol). Individual core and surface functionalization was achieved and the influence of the modification on the efficiency of the magnetic loading was tested. The applicability of the multifunctional particles in biological systems was proved via cellular uptake and toxicity testings. Furthermore, biofunctionalized particles were synthesized by EDC coupling using biotin and insulin.rnrn

Differentiation and extracellular matrix interactions of chondrocytes in response to signaling molecules and biomaterials for cartilage repair = Differenzierung und Interaktionen von Chondrozyten mit der extrazellul��ren Matrix als Reaktion auf Signalmolek��le und Biomaterialien f��r die Wiederinstandsetzung von Knorpel

Chondrocytes live isolated in the voluminous extracellular matrix of cartilage, which they secrete and is neither vascularized nor innervated. Nutrient and waste exchanges occur through diffusion leading to low oxygen tension around the cells. Consequently even normal cartilage under normal physiological conditions suffers from a poor reparative potential that predisposes to degenerative conditions, such as osteoarthritis of the joints, with significant clinical effects.rnOne of the key challenges in medicine is the structural and functional replacement of lost or damaged tissues. Current therapeutical approaches are to transplant cells, implant bioartificial tissues, and chemically induce regeneration at the site of the injury. None of them reproduces well the biological and biomechanical properties of hyaline cartilage.rnThis thesis investigates the re-differentiation of chondrocytes and the repair of cartilage mediated by signaling molecules, biomaterials, and factors provided in mixed cellular cultures (co-culture systems). As signaling molecules we have applied prostaglandin E2 (PGE2) and bone morphogenetic protein 1 (BMP-1) and we have transfected chondrocytes with BMP-1 expressing vectors. Our biomaterials have been hydrogels of type-I collagen and gelatin-based scaffolds designed to mimic the architecture and biochemistry of native cartilage and provide a suitable three-dimensional environment for the cells. We have brought chondrocytes to interact with osteosarcoma Cal 72 cells or with murine preosteoblastic KS483 cells, either in a cell-to-cell or in a paracrine manner.rnExogenous stimulation with PGE2 or BMP-1 did not improve the differentiation or the proliferation of human articular chondrocytes. BMP-1 induced chondrocytic de-differentiation in a dose-dependent manner. Prostaglandin stimulation from gelatin-based scaffolds (three-dimensional culture) showed a certain degree of chondrocyte re-differentiaton. Murine preosteoblastic KS483 cells had no beneficial effect on human articular chondrocytes jointly cultivated with them in hydrogels of type I collagen. Although the hydrogels provided the chondrocytes with a proper matrix in which the cells adopted their native morphology; additionally, the expression of chondrocytic proteoglycan increased in the co-cultures after two weeks. The co-culture of chondrocytes with osteoblast-like cells (in transwell systems) resulted in suppression of the regular de-differentiation program that passaged chondrocytes undergo when cultured in monolayers. Under these conditions, the extracellular matrix of the chondrocytes, rich in type-II collagen and aggrecan, was not transformed into the extracellular matrix characteristic of de-differentiated human articular chondrocytes, which is rich in type-I collagen and versican.rnThis thesis suggests novel strategies of tissue engineering for clinical attempts to improve cartilage repair. Since implants are prepared in vitro (ex-vivo) by expanding human articular chondrocytes (autologous or allogeneic), we conclude that it will be convenient to provide a proper three-dimensional support to the chondrocytes in culture, to supplement the culture medium with PGE2, and to stimulate chondrocytes with osteoblastic factors by cultivating them with osteoblasts.rn

Radiolabeling of defined polymer architectures with fluorine-18 and iodine-131 for ex vivo and in vivo evaluation : visualization of structure-property relationships

Nuclear medicine imaging techniques such as PET are of increasing relevance in pharmaceutical research being valuable (pre)clinical tools to non-invasively assess drug performance in vivo. Therapeutic drugs, e.g. chemotherapeutics, often suffer from a poor balance between their efficacy and toxicity. Here, polymer based drug delivery systems can modulate the pharmacokinetics of low Mw therapeutics (prolonging blood circulation time, reducing toxic side effects, increasing target site accumulation) and therefore leading to a more efficient therapy. In this regard, poly-N-(2-hydroxypropyl)-methacrylamide (HPMA) constitutes a promising biocompatible polymer. Towards the further development of these structures, non-invasive PET imaging allows insight into structure-property relationships in vivo. This performant tool can guide design optimization towards more effective drug delivery. Hence, versatile radiolabeling strategies need to be developed and establishing 18F- as well as 131I-labeling of diverse HPMA architectures forms the basis for short- as well as long-term in vivo evaluations. By means of the prosthetic group [18F]FETos, 18F-labeling of distinct HPMA polymer architectures (homopolymers, amphiphilic copolymers as well as block copolymers) was successfully accomplished enabling their systematic evaluation in tumor bearing rats. These investigations revealed pronounced differences depending on individual polymer characteristics (molecular weight, amphiphilicity due to incorporated hydrophobic laurylmethacrylate (LMA) segments, architecture) as well as on the studied tumor model. Polymers showed higher uptake for up to 4 h p.i. into Walker 256 tumors vs. AT1 tumors (correlating to a higher cellular uptake in vitro). Highest tumor concentrations were found for amphiphilic HPMA-ran-LMA copolymers in comparison to homopolymers and block copolymers. Notably, the random LMA copolymer P4* (Mw=55 kDa, 25% LMA) exhibited most promising in vivo behavior such as highest blood retention as well as tumor uptake. Further studies concentrated on the influence of PEGylation (���stealth effect���) in terms of improving drug delivery properties of defined polymeric micelles. Here, [18F]fluoroethylation of distinct PEGylated block copolymers (0%, 1%, 5%, 7%, 11% of incorporated PEG2kDa) enabled to systematically study the impact of PEG incorporation ratio and respective architecture on the in vivo performance. Most strikingly, higher PEG content caused prolonged blood circulation as well as a linear increase in tumor uptake (Walker 256 carcinoma). Due to the structural diversity of potential polymeric carrier systems, further versatile 18F-labeling strategies are needed. Therefore, a prosthetic 18F-labeling approach based on the Cu(I)-catalyzed click reaction was established for HPMA-based polymers, providing incorporation of fluorine-18 under mild conditions and in high yields. On this basis, a preliminary ��PET study of a HPMA-based polymer ��� radiolabeled via the prosthetic group [18F]F-PEG3-N3 ��� was successfully accomplished. By revealing early pharmacokinetics, 18F-labeling enables to time-efficiently assess the potential of HPMA polymers for efficient drug delivery. Yet, investigating the long-term fate is essential, especially regarding prolonged circulation properties and passive tumor accumulation (EPR effect). Therefore, radiolabeling of diverse HPMA copolymers with the longer-lived isotope iodine-131 was accomplished enabling in vivo evaluation of copolymer P4* over several days. In this study, tumor retention of 131I-P4* could be demonstrated at least over 48h with concurrent blood clearance thereby confirming promising tumor targeting properties of amphiphilic HPMA copolymer systems based on the EPR effect.

On the flavor problem in strongly coupled theories

rnThis thesis is on the flavor problem of Randall Sundrum modelsrnand their strongly coupled dual theories. These models are particularly wellrnmotivated extensions of the Standard Model, because they simultaneously address rntherngauge hierarchy problem and the hierarchies in the quarkrnmasses and mixings. In order to put this into context, special attention is given to concepts underlying therntheories which can explain the hierarchy problem and the flavor structure of the Standard Model (SM). ThernAdS/CFTrnduality is introduced and its implications for the Randall Sundrum model withrnfermions in the bulk andrngeneral bulk gauge groups is investigated. It will be shown that the differentrnterms in the general 5D propagator of a bulk gauge field can be related tornthe corresponding diagrams of the strongly coupled dual, which allows for arndeeperrnunderstanding of the origin of flavor changing neutral currents generated by thernexchange of the Kaluza Klein excitations of these bulk fields.rnIn the numerical analysis, different observables which are sensitive torncorrections from therntree-levelrnexchange of these resonances will be presented on the basis of updatedrnexperimental data from the Tevatron and LHC experiments. This includesrnelectroweak precision observables, namely corrections to the S and Trnparameters followed by corrections to the Zbb vertex, flavor changingrnobservables with flavor changes at one vertex, viz. BR (Bd -> mu+mu-) and BR (Bs -> mu+mu-), and two vertices,rn viz. S_psiphi and |eps_K|, as well as bounds from direct detectionrnexperiments. rnThe analysis will show that all of these bounds can be brought in agreement withrna new physics scale Lambda_NP in the TeV range, except for the CPrnviolating quantity |eps_K|, which requires Lambda_NP= Ord(10) TeVrnin the absencernof fine-tuning. The numerous modifications of the Randall Sundrum modelrnin the literature, which try to attenuate this bound are reviewed andrncategorized.rnrnSubsequently, a novel solution to this flavor problem, based on an extendedrncolor gauge group in the bulk and its thorough implementation inrnthe RS model, will be presented, as well as an analysis of the observablesrnmentioned above in the extended model. This solution is especially motivatedrnfromrnthe point of view of the strongly coupled dual theory and the implications forrnstrongly coupled models of new physics, which do not possess a holographic dual,rnare examined.rnFinally, the top quark plays a special role in models with a geometric explanation ofrnflavor hierarchies and the predictions in the Randall-Sundrum model with andrnwithout the proposed extension for the forward-backward asymmetryrnA_FB^trnin top pair production are computed.

Oswald Mathias Ungers: Belvederestra��e 60. Zu einer neuen Archtektur.

La presente Tesi di Dottorato intende affrontare una lettura critica della Casa in Belvederestra��e 60, realizzata dall���architetto Oswald Mathias Ungers (Kaisersesch, 12 luglio 1926 ��� K��ln, 30 settembre 2007), nel 1958-���59 a K��ln-M��ngersdorf, come studio per s�� ed abitazione per la propria famiglia. Questo primo oggetto della ricerca viene considerato evidente espressione delle convinzioni formali e compositive dell���architetto, negli anni Cinquanta e Sessanta. A differenza di altri progetti residenziali coevi ed antecedenti, frutto di un���elaborazione autonoma, la prima casa che costruisce per s�� riflette una maggiore libert�� di pensiero, dettata dalla coincidenza delle figure di progettista e committente; a ci�� si aggiunge anche una precisa volont�� dichiarativa ed ideologica. Proprio quest���ultimo aspetto permette di introdurre il secondo oggetto della Tesi: il manifesto ���ideologico���, Zu einer neuen Architektur, scritto dallo stesso Oswald Mathias Ungers e da Reinhard Gieselmann, alla fine del 1960; un breve testo che espone, con toni perentori ed inappellabili, il punto di vista dei due architetti nei confronti di un panorama architettonico e critico, caratterizzato da una sterilit�� di pensiero dilagante, a causa dell���egemonia costruttiva funzionalista. La ricerca indaga quindi le forti reciprocit�� delle due opere: casa e testo, viste in chiave di ���manifesto scritto e manifesto costruito���. Il primo legame tra i due soggetti �� senza dubbio la concomitanza temporale, (tra il 1958 ed il 1960) associata ad un rapporto causa-effetto, tale per cui il manifesto viene redatto a difesa delle aspre critiche scaturite dalla pubblicazione della casa sulla rivista Bauwelt. Il secondo nesso �� la possibilit�� di comprendere le accezioni effettive dei termini impiegati nella redazione del testo, attraverso le forme di una delle opere maggiormente personali dell���architetto, estraendone il senso e conferendogli un���immagine architettonica. Si vuole creare cos�� un rapporto biunivoco di traducibilit��, dell���architettura nello scritto e della semantica ungersiana in azioni compositive.

Supramolekulare Affinit��tsmaterialien basierend auf (���)-Isosteviol

Der Einsatz von optisch aktiven Elementen im R��ckgrat von Wirtstrukturen erm��glicht die enantiofaciale Differenzierung von Gastmolek��len. In Vorarbeiten wurde die Synthese eines Triphenylenketals basierend auf dem optisch reinen Naturstoff (���)-Isosteviol entwickelt. Aufbauend darauf wurde im Zuge dieser Dissertation ein para-Toluolsulfonamid-funktionalisierter Rezeptor synthetisiert, welcher exzellentes Verhalten als Affinit��tsmaterial in den im Arbeitskreis eingesetzten Quarzmikrowaagen-basierten Sensoren (QMB) aufweist. Im Zuge der Konstruktion einer (���)-Isosteviol-basierten Architektur mit verkleinerter Kavit��t wurde der F��nfring des (���)-Isosteviols (���D-Ring���) in einem mehrstufigen Prozess zu einem sechsgliedrigen Ring erweitert. Eine Ketalisierung ��ber die Ketofunktion am entsprechenden Ring dieser Verbindung lieferte den Grundbaustein f��r die Konstruktion von Triphenylenketalen. Weiterhin wurde die Synthese von Rezeptorstrukturen mit Triptycen-Ger��st in Verbindung mit (���)-Isosteviol beschrieben. Die Kombination verschiedener Ester des (���)-Isosteviol-Diketons mit Hexaammoniumtriptycen-Hexachlorid f��hrte zum Aufbau der Rezeptorarchitekturen in Ausbeuten von bis zu 95%, die entsprechenden Produkte fielen als all-syn- und anti,anti,syn-Isomere an. Beide Isomere zeigten teils exzellentes Verhalten als Affinit��tsmaterialen. Insbesondere in der Detektion kleinster Mengen an Benzol zeigte sich eins dieser Substrate als f��higer als s��mtliche vorher bekannten und getesteten Substanzen. In einem letzten, abschlie��enden Schritt wurde ��ber eine Olefin-Metathese eine K��figstruktur f��r den Einsatz in der Sensorik dargestellt.