Apoptosis rate and transcriptional response of pancreatic islets exposed to the PPAR gamma agonist Pioglitazone

To explore the molecular pathways underlying thiazolidinediones effects on pancreatic islets in conditions mimicking normo- and hyperglycemia, apoptosis rate and transcriptional response to Pioglitazone at both physiological and supraphysiological glucose concentrations were evaluated. Adult rat islets were cultured at physiological (5.6 mM) and supraphysiological (23 mM) glucose concentrations in presence of 10 μM Pioglitazone or vehicle. RNA expression profiling was evaluated with the PancChip 13k cDNA microarray after 24-h, and expression results for some selected genes were validated by qRT-PCR. The effects of Pioglitazone were investigated regarding apoptosis rate after 24-, 48- and 72-h. At 5.6 mM glucose, 101 genes were modulated by Pioglitazone, while 1,235 genes were affected at 23 mM glucose. Gene networks related to lipid metabolism were identified as altered by Pioglitazone at both glucose concentrations. At 23 mM glucose, cell cycle and cell death pathways were significantly regulated as well. At 5.6 mM glucose, Pioglitazone elicited a transient reduction in islets apoptosis rate while at 23 mM, Bcl2 expression was reduced and apoptosis rate was increased by Pioglitazone. Our data demonstrate that the effect of Pioglitazone on gene expression profile and apoptosis rate depends on the glucose concentration. The modulation of genes related to cell death and the increased apoptosis rate observed at supraphysiological glucose concentration raise concerns about Pioglitazone’s direct effects in conditions of hyperglycemia and reinforce the necessity of additional studies designed to evaluate TZDs effects on the preservation of β-cell function in situations where glucotoxicity might be more relevant than lipotoxicity.

Two sequential PCR amplifications for detection of Schistosoma mansoni in stool samples with low parasite load

Schistosomiasis constitutes a major public health problem, with an estimated 200 million individuals infected worldwide and 700 million people living in risk areas. In Brazil there are areas of high, medium and low endemicity. Studies have shown that in endemic areas with a low prevalence of Schistosoma infection the sensitivity of parasitological methods is clearly reduced. Consequently diagnosis is often impeded due to the presence of false-negative results. The aim of this study is to present the PCR reamplification (Re-PCR) protocol for the detection of Schistosoma mansoni in samples with low parasite load (with less than 100 eggs per gram (epg) of feces). Three methods were used for the lysis of the envelopes of the S. mansoni eggs and two techniques of DNA extraction were carried out. Extracted DNA was quantified, and the results suggested that the extraction technique, which mixed glass beads with a guanidine isothiocyanate/phenol/chloroform (GT) solution, produced good results. PCR reamplification was conducted and detection sensitivity was found to be five eggs per 500 mg of artificially marked feces. The results achieved using these methods suggest that they are potentially viable for the detection of Schistosoma infection with low parasite load.

Design and development of new green catalytic methodologies for the synthesis of enantiomerically enriched molecules

The following Ph.D work was mainly focused on catalysis, as a key technology, to achieve the objectives of sustainable (green) chemistry. After introducing the concepts of sustainable (green) chemistry and an assessment of new sustainable chemical technologies, the relationship between catalysis and sustainable (green) chemistry was briefly discussed and illustrated via an analysis of some selected and relevant examples. Afterwards, as a continuation of the ongoing interest in Dr. Marco Bandini’s group on organometallic and organocatalytic processes, I addressed my efforts to the design and development of novel catalytic green methodologies for the synthesis of enantiomerically enriched molecules. In the first two projects the attention was focused on the employment of solid supports to carry out reactions that still remain a prerogative of omogeneous catalysis. Firstly, particular emphasis was addressed to the discovery of catalytic enantioselective variants of nitroaldol condensation (commonly termed Henry reaction), using a complex consisting in a polyethylene supported diamino thiopene (DATx) ligands and copper as active species. In the second project, a new class of electrochemically modified surfaces with DATx palladium complexes was presented. The DATx-graphite system proved to be efficient in promoting the Suzuki reaction. Moreover, in collaboration with Prof. Wolf at the University of British Columbia (Vancouver), cyclic voltammetry studies were reported. This study disclosed new opportunities for carbon–carbon forming processes by using heterogeneous, electrodeposited catalyst films. A straightforward metal-free catalysis allowed the exploration around the world of organocatalysis. In fact, three different and novel methodologies, using Cinchona, Guanidine and Phosphine derivatives, were envisioned in the three following projects. An interesting variant of nitroaldol condensation with simple trifluoromethyl ketones and also their application in a non-conventional activation of indolyl cores by Friedel-Crafts-functionalization, led to two novel synthetic protocols. These approaches allowed the preparation of synthetically useful trifluoromethyl derivatives bearing quaternary stereocenters. Lastly, in the sixth project the first γ-alkylation of allenoates with conjugated carbonyl compounds was envisioned. In the last part of this Ph.D thesis bases on an extra-ordinary collaboration with Prof. Balzani and Prof. Gigli, I was involved in the synthesis and characterization of a new type of heteroleptic cyclometaled-Ir(III) complexes, bearing bis-oxazolines (BOXs) as ancillary ligands. The new heteroleptic complexes were fully characterized and in order to examine the electroluminescent properties of FIrBOX(CH2), an Organic Light Emitting Device was realized.

Stereoselektive Synthese von Stickstoffheterocyclen an Glycosylaminen als chiralen Auxiliaren

Alkaloide, im allgemeinen Stickstoffheterocyclen, sind wichtige Vorläuferverbindungen von pharmakologisch aktiven Substanzen. Die stereoselektive Synthese von Stickstoffheterocyclen ist von großem Interesse für die Entdeckung und Entwicklung von Arzneistoffen.In der Arbeit wurden Glycosylamine vom Typ des 2,3,4,6-Tetra-O-pivaloyl-?-D-galactosylamins bzw. des 2,3,4-Tri-O-pivaloyl-?-D-arabinosylamins zur diastereoselektiven Synthese mehrfach substituierter Stickstoffheterocyclen eingesetzt. In einer Tandem-Mannich-Michael-Reaktion eines Glycosylimins mit dem Danishefsky-Dien wurden die in Position 6 substituierten Dehydropiperidinone aufgebaut. In einer mehrstufigen Synthesesequenz konnte das 4a-Epimere des natürlichen Pumiliotoxin C als Hydrochlorid dargestellt werden.Mittels der Tandem-Mannich-Michael-Reaktion wurden auch 6,6`-disubstituierte Dehydropiperidinone dargestellt. Die Darstellung zweier Aza-spiro-Verbindungen gelang erstmals ausgehend von den Ketonen Cyclohexanon und 3-Methyl-cyclohexanon über die Glycosylketimine. Das in dieser Reaktion gefundene Nebenprodukt N-Glycosyl-6-(2´-oxo-propyl)-2,3 dehydropiperidin-4-on diente als Ausgangssubstanz für die Pinidinolsynthese.In der angewendeten Weise eignen sich Glycosylamine sehr gut für die stereoselektive Synthese von Stickstoffheterocyclen. Meistens werden die chirale Piperidinalkaloidvorläufer in hohen Ausbeuten und Diastereoselektivitäten erhalten.

Synthesis, characterization and chemical modification of a cationic polyelectrolyte poly(methylene amine)

Polyamine polymers have attracted attention due to their ability to demonstrate pH dependent cationic nature and presence of highly reactive pendant amino groups. These amino groups make them suitable for a host of applications through cross-linking and derivatization. As a result the end use application of a polyamine is largely driven by the number of amino groups and the way they are attached to the polymer backbone. Thus, this piece of work describes the synthesis and investigation of properties of a novel aliphatic polyamine, poly(methylene amine); that carries maximum number of amino group on its backbone. The target polymer, poly(methylene amine); was synthesized via two major steps viz.1.synthesis of precursor polymers of poly(methylene amine) and 2. Hydrolysis of the precursor polymers to obtain poly(methylene amine). The precursor polymers poly (1,3-diacetylimidazole-2-one)(6) and poly(1,3-diformyldihydroimidazol-2-one)(7) were synthesized via radical polymerization of their respective monomers. The monomers were polymerized in bulk as well as in solution at different reaction conditions. The maximum molecular weights were achieved by polymerizing the monomers in bulk (Mn = 6.5 x 104 g/mol and Mw = 2.13 x 105 g/mol) of 6. The precursor polymers were hydrolyzed under strong reaction conditions in ethanol in presence of NaOH, LiCl at 170°C to yield poly(methylene amine). The process of hydrolysis was monitored by IR spectroscopy. The solution properties of poly(methylene amine) and its hydrochloride were investigated by viscosimetry and light scattering. The reduced viscosity of poly (methylene amine) hydrochloride as a function of polymer concentration demonstrated a behavior typical of cationic polyelectrolyte. With decrease in polymer concentration the reduced viscosity of poly(methylene amine) hydrochloride increased gradually. The dynamic light scattering studies also revealed behaviors of a polyelectrolyte. Poly(methylene amine) was reacted with electrophiles to yield novel materials. While the attachment of alkyl group onto the nitrogen would increase nucleophilicity, it would also impose steric hindrance. As a result the degree of substitution on poly(methylene amine) would be governed by both the factors. Therefore, few model reactions with electrophiles were performed on polvinylamine under similar reaction conditions in order to make a comparative evaluation. It was found that under similar reaction conditions the degree of substitution was higher in case of polyvinylamine in comparison with poly (methylene amine).This shows that the steric hindrance outweighs nucleophilicity while deciding degree of substitution of electrophiles on poly(methylene amine). The modification was further extended to its use as an initiator for ring opening polymerization of benzyloxy protected N-carboxyanhydride of z-Lysine. The resulting polymer had an interesting brush like architecture. The solid state morphology of this polymer was investigated by SAXS. The 2D-WAXS diffractograms revealed hexagonal morphology of peptide segments without formation of alpha helices.

Functional polymer coatings — My-Patterning and My-Analysis

In dieser Arbeit werden Strukturen beschrieben, die mit Polymeren auf Oberflächen erzeugt wurden. Die Anwendungen reichen von PMMA und PNIPAM Polymerbürsten, über die Restrukturierung von Polystyrol durch Lösemittel bis zu 3D-Strukturen, die aus PAH/ PSS Polyelektrolytmultischichten bestehen. Im ersten Teil werden Polymethylmethacrylat (PMMA) Bürsten in der ionischen Flüssigkeit 1-Butyl-3-Methylimidazolium Hexafluorophospat ([Bmim][PF6]) durch kontrollierte radikalische Polymerisation (ATRP) hergestellt. Kinetische Untersuchungen zeigten ein lineares und dichtes Bürstenwachstum mit einer Wachstumsrate von 4600 g/mol pro nm. Die durchschnittliche Pfropfdichte betrug 0.36 µmol/m2. Als Anwendung wurden Mikrotropfen bestehend aus der ionischen Flüssigkeit, Dimethylformamid und dem ATRP-Katalysator benutzt, um in einer definierten Geometrie Polymerbürsten auf Silizium aufzubringen. Auf diese Weise lässt sich eine bis zu 13 nm dicke Beschichtung erzeugen. Dieses Konzept ist durch die Verdampfung des Monomers Methylmethacrylat (MMA) limitiert. Aus einem 1 µl großen Tropfen aus ionischer Flüssigkeit und MMA (1:1) verdampft MMA innerhalb von 100 s. Daher wurde das Monomer sequentiell zugegeben. Der zweite Teil konzentriert sich auf die Strukturierung von Oberflächen mit Hilfe einer neuen Methode: Tintendruck. Ein piezoelektrisch betriebenes „Drop-on-Demand“ Drucksystem wurde verwendet, um Polystyrol mit 0,4 nl Tropfen aus Toluol zu strukturieren. Die auf diese Art und Weise gebildeten Mikrokrater können Anwendung als Mikrolinsen finden. Die Brennweite der Mikrolinsen kann über die Anzahl an Tropfen, die für die Strukturierung verwendet werden, eingestellt werden. Theoretisch und experimentell wurde die Brennweite im Bereich von 4,5 mm bis 0,21 mm ermittelt. Der zweite Strukturierungsprozess nutzt die Polyelektrolyte Polyvinylamin-Hydrochlorid (PAH) und Polystyrolsulfonat (PSS), um 3D-Strukturen wie z.B. Linien, Schachbretter, Ringe, Stapel mit einer Schicht für Schicht Methode herzustellen. Die Schichtdicke für eine Doppelschicht (DS) liegt im Bereich von 0.6 bis 1.1 nm, wenn NaCl als Elektrolyt mit einer Konzentration von 0,5 mol/l eingesetzt wird. Die Breite der Strukturen beträgt im Mittel 230 µm. Der Prozess wurde erweitert, um Nanomechanische Cantilever Sensoren (NCS) zu beschichten. Auf einem Array bestehend aus acht Cantilevern wurden je zwei Cantilever mit fünf Doppelschichten PAH/ PSS und je zwei Cantilever mit zehn Doppelschichten PAH/ PSS schnell und reproduzierbar beschichtet. Die Massenänderung für die individuellen Cantilever war 0,55 ng für fünf Doppelschichten und 1,08 ng für zehn Doppelschichten. Der daraus resultierende Sensor wurde einer Umgebung mit definierter Luftfeuchtigkeit ausgesetzt. Die Cantilever verbiegen sich durch die Ausdehnung der Beschichtung, da Wasser in das Polymer diffundiert. Eine maximale Verbiegung von 442 nm bei 80% Luftfeuchtigkeit wurde für die mit zehn Doppelschichten beschichteten Cantilever gefunden. Dies entspricht einer Wasseraufnahme von 35%. Zusätzlich konnte aus den Verbiegungsdaten geschlossen werden, dass die Elastizität der Polyelektrolytmultischichten zunimmt, wenn das Polymer gequollen ist. Das thermische Verhalten in Wasser wurde im nächsten Teil an nanomechanischen Cantilever Sensoren, die mit Poly(N-isopropylacrylamid)bürsten (PNIPAM) und plasmapolymerisiertem N,N-Diethylacrylamid beschichtet waren, untersucht. Die Verbiegung des Cantilevers zeigte zwei Bereiche: Bei Temperaturen kleiner der niedrigsten kritischen Temperatur (LCST) ist die Verbiegung durch die Dehydration der Polymerschicht dominiert und bei Temperaturen größer der niedrigsten kritischen Temperatur (LCST) reagiert der Cantilever Sensor überwiegend auf Relaxationsprozesse innerhalb der kollabierten Polymerschicht. Es wurde gefunden, dass das Minimum in der differentiellen Verbiegung mit der niedrigsten kritischen Temperatur von 32°C und 44°C der ausgewählten Polymeren übereinstimmt. Im letzten Teil der Arbeit wurden µ-Reflektivitäts- und µ-GISAXS Experimente eingeführt als neue Methoden, um mikrostrukturierte Proben wie NCS oder PEM Linien mit Röntgenstreuung zu untersuchen. Die Dicke von jedem individuell mit PMMA Bürsten beschichtetem NCS ist im Bereich von 32,9 bis 35,2 nm, was mit Hilfe von µ-Reflektivitätsmessungen bestimmt wurde. Dieses Ergebnis kann mit abbildender Ellipsometrie als komplementäre Methode mit einer maximalen Abweichung von 7% bestätigt werden. Als zweites Beispiel wurde eine gedruckte Polyelektrolytmultischicht aus PAH/PSS untersucht. Die Herstellungsprozedur wurde so modifiziert, dass Goldnanopartikel in die Schichtstruktur eingebracht wurden. Durch Auswertung eines µ-GISAXS Experiments konnte der Einbau der Partikel identifiziert werden. Durch eine Anpassung mit einem Unified Fit Modell wurde herausgefunden, dass die Partikel nicht agglomeriert sind und von einer Polymermatrix umgeben sind.

Chitosan based hydrogels for transmucosal drug delivery

The aim of this thesis was the formulation of new chitosan based delivery systems for transmucosal drug administration. Transmucosal routes, such as buccal, vaginal and nasal routes, allow the circumvention of the hepatic first pass metabolism and avoid the gastrointestinal chemical and enzymatic degradations. Moreover, transmucosal drug administration can allow to avoid pain or discomfort caused by injections, when drugs are administered through parenteral routes, thus increasing patient compliance. On the other side, the major disadvantage of transmucosal drug administration is represented by the presence of biological fluids and mucus that can remove drug systems from the application site, thus reducing the contact time between drug and mucosa and consequently, decreasing drug bioavailability. For this reason, in this study, the investigation of chitosan delivery systems as mucoadhesive formulations able to increase drugs residence time and to improve their bioavailability, was taken into account. In the paper 1, buccal films based on chitosan-gelatin complexes were prepared and loaded with propranolol hydrochloride. The complexes were characterized and studied in order to evaluate their physical- chemical properties and their ability to release the drug and to allow its permeation through buccal mucosa. In the paper 2, vaginal inserts based on chitosan/alginate complexes were formulated for local delivery of chlorhexidine digluconate. Tests to evaluate the interaction between the polymers and to study drug release properties were performed, as well as the determination of antimicrobial activity against the patogens responsible of vaginitis and candidosis. In the project 3, chitosan based nanoparticles containing cyclodextrin and other excipients, with the capacity to modify insulin bioavailabity were formulated for insulin nasal delivery. Nanoparticles were characterized in terms of size, stability and drug release. Moreover, in vivo tests were performed in order to study the hypoglycemic reduction in rats blood samples.

Strategie formulative per la veicolazione nasale di farmaci

Microparticelle a base di complessi polielettrolitici di Chitosano/Pectina per il rilascio nasale di Tacrina cloridrato. Lo scopo di questo studio è stata la ricerca di nuove formulazioni solide per la somministrazione nasale di Tacrina cloridrato allo scopo di ridurre l’eccessivo effetto di primo passaggio epatico ed aumentarne la biodisponibilità a livello del Sistema Nervoso Centrale. La Tacrina è stata incapsulata in microparticelle mucoadesive a base di complessi elettrolitici di chitosano e pectina. Le microparticelle sono state preparate mediante due diversi approcci tecnologici (spray-drying e spray-drying/liofilizzazione) e analizzate in termini di caratteristiche dimensionali, morfologiche e chimico-fisiche. Nanoparticelle di Chitosano reticolate con Sodio Cromoglicato per il trattamento della rinite allergica. Il Sodio Cromoglicato è uno dei farmaci utilizzati per il trattamento della rinite allergica. Come noto, la clearance mucociliare provoca una rapida rimozione dei farmaci in soluzione dalla cavità nasale, aumentando così il numero di somministrazioni giornaliere e, di conseguenza, riducendo la compliance del paziente. Per ovviare a tale problema, si è pensato di includere il sodio cromoglicato in nanoparticelle di chitosano, un polimero capace di aderire alla mucosa nasale, prolungare il contatto della formulazione con il sito di applicazione e ridurre il numero di somministrazioni giornaliere. Le nanoparticelle ottenute sono state caratterizzate in termini di dimensioni, resa, efficienza di incapsulazione e caricamento del farmaco, potenziale zeta e caratteristiche mucoadesive. Analisi quantitativa di Budesonide amorfa tramite calorimetria a scansione differenziale. È stato sviluppato un nuovo metodo quantitativo allo stato solido basato sulla Calorimetria a Scansione Differenziale (DSC) in grado di quantificare in modo selettivo e accurato la quantità di Budesonide amorfa presente in una miscela solida. Durante lo sviluppo del metodo sono stati affrontati problemi relativi alla convalida di metodi analitici su campioni solidi quali la miscelazione di polveri solide per la preparazione di miscele standard e il calcolo della precisione.

Einfluss von Hilfsstoffen auf die Bioverfügbarkeit von Substanzen der BCS Klasse III

In dieser Arbeit wurde der Effekt verschiedener Hilfsstoffe auf die Permeabilität von Substanzen der BCS Klasse III untersucht. Drei pharmazeutische Hilfsstoffe wurden hinsichtlich der Möglichkeit ihres Einsatzes als Permeationsverbesserer in Arzneistoffformulierungen untersucht. Außerdem wurde die Beteiligung von Gallensalzen an der Nahrungsmittel-Interaktion von Trospium untersucht.rnEs wurden Komplexe aus Trospium und λ-Carrageen hergestellt. Eine verbesserte Permeation, die höchstwahrscheinlich durch Mukoadhäsion zustande kam, war im Ussing-Kammer-Modell sehr gut reproduzierbar. In vivo war der Effekt nur bei einigen Tieren zu sehen und es kam zu hohen Standardabweichungen.rnTrospium bildet Ionenpaare mit Gallensalzen, welche zu einer besseren Permeabilität des Wirkstoffes führten. In Gegenwart von Nahrungsfetten blieb dieser Effekt aus. Eine Beteiligung der Interaktion von Trospium und Gallensalzen am Food-Effekt kann auf Basis dieser Ergebnisse als wahrscheinlich gelten.rnIm Caco-2-Modell konnte bereits eine Verbesserung der Permeabilität von Trospium durch Zusatz von Eudragit E gezeigt werden. Nun konnte gezeigt werden, dass durch den Hilfsstoff auch in vivo in Ratten eine verbesserte Permeation erreicht werden kann.rnDie Permeationsverbesserung von Aciclovir durch Zusatz von Chitosan-HCl sollte untersucht werden. Im Caco-2-Modell kam es zu einer signifikanten Permeationsverbesserung. Im Ussing-Kammer-Modell wurde die Permeation nicht verbessert. In Loop-Studien konnte nur bei hohen Hilfsstoff-Konzentrationen eine Tendenz zur Permeationsverbesserung erkannt werden.rn

Etablierung eines high content imaging-basierten in vitro Testsystems zur Evaluierung genotoxischer Substanzen

Zur Registrierung von Pharmazeutika ist eine umfassende Analyse ihres genotoxischen Potentials von Nöten. Aufgrund der Vielzahl genotoxischer Mechanismen und deren resultierenden Schäden wird ein gestaffeltes Testdesign durch die ICH-Richtlinie S2(R1) „Guidance on genotoxicity testing and data interpretation for pharmaceuticals intended for human use S2(R1)“ definiert, um alle genotoxischen Substanzen zu identifizieren. Die Standardtestbatterie ist in der frühen Phase der Arzneimittelentwicklung aufgrund des geringen Durchsatzes und des Mangels an verfügbarer Substanzmenge vermindert anwendbar. Darüber hinaus verfügen in vitro Genotoxizitätstests in Säugerzellen über eine relativ geringe Spezifität. Für eine vollständige Sicherheitsbeurteilung wird eine in vivo Testung auf Kanzerogenität benötigt. Allerdings sind diese Testsysteme kosten- und zeitintensiv. Aufgrund dessen zielen neue Forschungsansätze auf die Verbesserung der Prädiktivität und die Erfassung des genotoxischen Potentials bereits in der frühen Phase der Arzneimittelentwicklung ab. Die high content imaging (HCI)-Technologie offeriert einen Ansatz zur Verbesserung des Durchsatzes verglichen mit der Standardtestbatterie. Zusätzlich hat ein Zell-basiertes Modell den Vorteil Daten relativ schnell bei gleichzeitig geringem Bedarf an Substanzmenge zu generieren. Demzufolge ermöglichen HCI-basierte Testsysteme eine Prüfung in der frühen Phase der pharmazeutischen Arzneimittelentwicklung. Das Ziel dieser Studie ist die Entwicklung eines neuen, spezifischen und sensitiven HCI-basierten Testsytems für Genotoxine und Progenotoxine in vitro unter Verwendung von HepG2-Zellen gewesen. Aufgrund ihrer begrenzten metabolischen Kapazität wurde ein kombiniertes System bestehend aus HepG2-Zellen und einem metabolischen Aktivierungssystem zur Testung progenotoxischer Substanzen etabliert. Basierend auf einer vorherigen Genomexpressionsprofilierung (Boehme et al., 2011) und einer Literaturrecherche wurden die folgenden neun unterschiedlichen Proteine der DNA-Schadensantwort als putative Marker der Substanz-induzierten Genotoxizität ausgewählt: p-p53 (Ser15), p21, p-H2AX (Ser139), p-Chk1 (Ser345) p-ATM (Ser1981), p-ATR (Ser428), p-CDC2 (Thr14/Tyr15), GADD45A und p-Chk2 (Thr68). Die Expression bzw. Aktivierung dieser Proteine wurde 48 h nach Behandlung mit den (pro-) genotoxischen Substanzen (Cyclophosphamid, 7,12-Dimethylbenz[a]anthracen, Aflatoxin B1, 2-Acetylaminofluoren, Methylmethansulfonat, Actinomycin D, Etoposid) und den nicht-genotoxischen Substanzen (D-Mannitol, Phenforminhydrochlorid, Progesteron) unter Verwendung der HCI-Technologie ermittelt. Die beste Klassifizierung wurde bei Verwendung der folgenden fünf der ursprünglichen neun putativen Markerproteine erreicht: p-p53 (Ser15), p21, p-H2AX (Ser139), p-Chk1 (Ser345) und p-ATM (Ser1981). In einem zweiten Teil dieser Arbeit wurden die fünf ausgewählten Proteine mit Substanzen, welche von dem European Centre for the Validation of Alternative Methods (ECVAM) zur Beurteilung der Leistung neuer oder modifizierter in vitro Genotoxizitätstests empfohlen sind, getestet. Dieses neue Testsystem erzielte eine Sensitivität von 80 % und eine Spezifität von 86 %, was in einer Prädiktivität von 84 % resultierte. Der synergetische Effekt dieser fünf Proteine ermöglicht die Identifizierung von genotoxischen Substanzen, welche DNA-Schädigungen durch eine Vielzahl von unterschiedlichen Mechanismen induzieren, mit einem hohen Erfolg. Zusammenfassend konnte ein hochprädiktives Prüfungssystem mit metabolischer Aktivierung für ein breites Spektrum potenziell genotoxischer Substanzen generiert werden, welches sich aufgrund des hohen Durchsatzes, des geringen Zeitaufwandes und der geringen Menge benötigter Substanz zur Substanzpriorisierung und -selektion in der Phase der Leitstrukturoptimierung eignet und darüber hinaus mechanistische Hinweise auf die genotoxische Wirkung der Testsubstanz liefert.

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

Species identification of archived fish bones collected from the Mediterranean Sea 100 years ago using molecular techniques

With the discovery that DNA can be successfully recovered from museum collections, a new source of genetic information has been provided to extend our comprehension of the evolutionary history of species. However, historical specimens are often mislabeled or report incorrect information of origin, thus accurate identification of specimens is essential. Due to the highly damaged nature of ancient DNA many pitfalls exist and particular precautions need to be considered in order to perform genetic analysis. In this study we analyze 208 historical remains of pelagic fishes collected in the beginning of the 20th century. Through the adaptation of existing protocols, usually applied to human remains, we manage to successfully retrieve valuable genetic material from almost all of the examined samples using a guanidine and silica column-based approach. The combined use of two mitochondrial markers cytochrome-oxidase-1(mtDNA COI) and Control Region (mtDNA CR), and the nuclear marker first internal transcriber space (ITS1) allowed us to identify the majority of the examined specimens using traditional PCR and Sanger sequencing techniques. The creation of primers capable of amplifying heavily degraded DNA have great potential for future uses, both in ancient and in modern investigation. The methodologies developed in this study can in fact be applied for other ancient fish specimens as well as cooked or canned samples.

Favourable long-term outcome after immediate treatment of neonatal hyperammonemia due to N-acetylglutamate synthase deficiency

INTRODUCTION: N-Acetylglutamate synthase (NAGS) deficiency is a rare urea cycle disorder, which may present in the neonatal period with severe hyperammonemia and marked neurological impairment. CASE REPORT: We report on a Turkish family with a patient who died due to hyperammonemia in the neonatal period. Reduced activity of NAGS and carbamyl phosphate synthetase were found at autopsy. A second child who developed hyperammonemia on the second day of life was immediately treated with arginine hydrochloride, sodium benzoate and protein restriction. After NAGS deficiency was suspected by enzyme analysis, sodium benzoate was replaced by N-carbamylglutamate (NCG). A third child who developed slight hyperammonemia on the third day of life was treated with NCG before enzyme analysis confirmed reduced NAGS activity. Neither of the patients developed hyperammonemia in the following years. After the human NAGS gene was identified, mutation analysis revealed that the older sibling on NCG therapy was homozygous for a 971G>A (W324X) mutation. The parents and the younger sibling were heterozygous. Therapy was continued in the older sibling until now without any adverse effects and favourable neurodevelopment outcome. In the younger sibling, therapy was stopped without any deterioration of urea cycle function. CONCLUSION: NAGS deficiency can be successfully treated with NCG and arginine hydrochloride with favourable outcome. Molecular diagnostic rather than enzyme analysis should be used in patients with suspected NAGS deficiency.

Enzymatic Activity of Soybean Lipoxygenase-1 on Linoleoyl-Derivatized Agarose

Soybean lipoxygenase-1 (SBLO-1) catalyzes the oxygenation of linoleic acid to form 13(S) and 9(R) hydroperoxides. The manner in which substrates bind to the lipoxygenase family of enzymes is not known. It is believed fatty acid substrates may bind either with the aliphatic end first or with the carboxylate group facing the interior of the protein. This thesis tested a potential methyl-end first substrate binding mechanism by studying the activity of SBLO-1 to oxygenate immobilized linoleoyl residues attached to an insoluble polymer. Linoleic acid was attached to aminohexyl agarose in the presence of N-(3- dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride (EDC) and Nhydroxysuccinimide (NHS). The concentration of the covalently attached residues was facilitated by enriching linoleic acid with a small amount of the radioactive 14C-isotope. Functionalization yields of 3% available primary amines on the resin were obtained. Enzymatic oxygenation of the linoleoyl-residues was verified using the ferrous oxidation in xylenol orange (FOX) assay. Approximately 30% of the attached linoleoyl moieties were converted to hydroperoxides in the presence of SBLO-1. A disulfide-containing cleavable linker, cystamine, was used as part of an improved method to isolate the product in a facile manner. Cystamine was attached to NHS-activated agarose with approximately 5% overall functionalization yield of available functional groups. 14C-linoleic acid was successfully covalently linked to the cystamine moieties in the presence of EDC and NHS. The FOX assay verified the enzymatic oxygenation of the linoleoyl residues attached to cystamine-derivatized agarose. The isolation of the peroxide product was attempted in a series of extractions in organic solvents. The product was analyzed using GC/MS which did not show a new peak indicative of product. Further work is needed to successfully analyze the stereoand regiochemistry of the oxygenated product. The presence of the peroxides in this study indicated the linoleoyl residues behave as substrates of SBLO-1. It is unknown how bulky substrates bind to the active site; however, it is difficult to rationalize a carboxylate group-first binding mode. Discovery of the 13(S)-hydroperoxide product on the linoleoyl-agarose would support the claim of a potential methyl-end first binding mechanism.

Clinical efficacy of pimobendan versus benazepril for the treatment of acquired atrioventricular valvular disease in dogs

Seventy-six dogs with clinical acquired atrioventricular valvular disease were evaluated to determine the efficacy of pimobendan (n=41) versus benazepril hydrochloride (n=35) in a randomized, positive-controlled, multicenter study. The study was divided into 56-day and long-term evaluation periods. In a subgroup of dogs with concurrent furosemide treatment (pimobendan [n=31], benazepril [n=25]), the Heart Insufficiency Score improved in favor of pimobendan (P=0.0011), equating to a superior overall efficacy rating (P<0.0001) at day 56. Long-term median survival (i.e., death or treatment failure) for dogs receiving pimobendan was 415 days versus 128 days for dogs not on pimobendan (P=0.0022).