No description available.

This dissertation describes the synthesis of an unsymmetrically-substituted triarylborane. This term describes a three-coordinate boron atom that is bound to three different aromatic systems, namely 2,6-dimethylphenyl, mesityl, and 4-(N,N-dimethylamino)-2,6-dimethylphenyl. It is also demonstrated that the amine functionality can be converted with methyl triflate into an ammonium moiety. The investigation of photophysical and electrochemical properties of this compound in comparison with the non-aminated and di-aminated analogues of the triarylborane is described besides other investigations of e. g. singlet oxygen sensitization, rotational barriers, and fundamental DFT calculations. Based on these investigations, selectively mono-, bis- and tris-dimethylamino- and trimethylammonium-substituted bis-triarylborane bithiophene chromophores were synthesized and their photophysical, and electrochemical properties were investigated together with the water solubility and singlet oxygen sensitizing efficiency of the cationic compounds Cat1+, Cat2+, Cat(i)2+, and Cat3+. Comparing these properties with the results obtained for the mono-triarylboranes reveals a large influence of the bridging unit on the investigated properties of the bis-triarylboranes. In addition, the interaction of the cationic bis-triarylboranes with different polynucleotides were investigated in buffered solutions as well as the ability of these selectively charged compounds to enter and localize within organelles of human lung carcinoma and normal lung cells. All these investigations demonstrate that the number of charges and their distribution influences the interactions and staining properties as well as most of the other properties investigated. In addition, preliminary investigations on H2O2-cleavable boronate esters in the presence of stochiometric amounts of H2O2 are described for three different aryl boronate esters. / In dieser Doktorarbeit wird die Synthese eines unsymmetrisch subsitutierten Triarylborans beschrieben. Dieser Ausdruck beschreibt ein dreifach koordiniertes Boratom, das an drei unterschiedliche Aromaten, hier 2,6-Dimethylphenyl, Mesityl, and 4-(N,N-Dimethylamino)-2,6-dimethylphenyl, gebunden ist. Es wird gezeigt, dass die Aminofunktionalität durch Methyltriflat in eine Amminoeinheit überführt werden kann. Die Untersuchung der photophysikalischen und elektrochemischen Eigenschaften dieser Verbindung im Vergleich zu den nicht-aminierten und di-aminierten Analoga des Triarylborans ist beschrieben ebenso wie beispielsweise die Untersuchungen der Bildung von Singulet Sauerstoff, der Rotationsbarrieren und fundamentale DFT Berechnungen. Basierend auf diesen Untersuchungen wurden genau einfach, zweifach und dreifach Dimethylamino- und Trimethylammonium-substituierte Chromophore hergestellt und deren photophysikalische und elektrochemische Eigenschaften untersucht, zusammen mit der Wasserlöslichkeit und der Bildung von Singulet Sauerstoff der kationischen Verbindungen Cat1+, Cat2+, Cat(i)2+ und Cat3+. Der Vergleich dieser Eigenschaften mit den Ergebnissen der mono-Triarylborane zeigt den großen Einfluss der Brücke auf die untersuchten Eigenschaften der bis-Triarylborane. Zusätzlich wurden die Wechselwirkungen der kationischen bis-Triarlyborane mit unterschiedlichen Polynukeotiden in Pufferlösungen zusammen mit deren Fähigkeit in Lungenkrebs- und Lungenzellen zu gelangen und sich in bestimmten Organellen darin anzulagern. Diese Untersuchungen zeigen, dass die Anzahl und Verteilung der Ladungen nicht nur die Wechselwirkungen und intrazelluläre Lokalisation beeinflussen, sondern auch fast alle übrigen untersuchten Eigenschaften. Zusätzlich sind erste Untersuchungen von drei H2O2-spaltbaren Borsäureestern in der Gegenwart von stöchiometrischen Mengen Wasserstoffperoxid gezeigt.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 105-119).

Upon termination of translation, the fate of ribosomes is determined largely by the rate at which cells are growing. During periods of exponential growth, ribosomes are rapidly recycled, translation is re-initiated, and the ribosomes are extremely stable. However, when nutrient sources become limiting, and ribosomes are not actively translating, they may become substrates for degradation. While this phenomenon is well known, details of how the process is initiated and what are the signals for degradation have, until now, remained elusive. Here, I present in vitro and in vivo data showing that free ribosome subunits are the targets of degradative enzymes, whereas 70S particles that remain associated are protected from such degradation. Conditions that increase the formation of subunits both in vitro and in vivo lead to enhanced degradation. Thus, the simple presence of free 50S and 30S subunits is sufficient to serve as the mechanism that initiates ribosome degradation. In order to identify RNases involved in ribosome degradation, both in vitro and in vivo assays were developed. Together, they have provided evidence for a multi-step degradation process involving both endo- and exoribonucleases. Examination of extracts from strains deficient in known RNases revealed that the endoribonucleases, RNase E and RNase G, may be involved in the initial cleavages. The resulting fragments, some of which are small enough oligoribonucleotides that they remain part of the acid-soluble fraction are degraded to mononucleotides primarily by the 3'-5' exoribonucleases, RNase R and polynucleotide phosphorylase.

DNA lesions arise constantly in cells and are repaired by a variety of DNA repair pathways. Polynucleotide kinase 3’-phosphatase (PNKP) aids repair by phosphorylating 5’-hydroxyl DNA termini and dephosphorylating 3’-phosphate DNA termini for the completion of repair by DNA ligases. This activity is critical in vivo because DNA breaks do not usually possess ligatable termini. PNKP knockdown sensitizes cells to several DNA damaging agents, including the topoisomerase I (TOP1) inhibitor camptothecin - analogs of which are being developed into chemotherapeutic drugs - because the resolution of stalled TOP1-DNA complexes requires processing by PNKP. We hypothesize that small molecule inhibitors of PNKP could bolster the effects of radio- and chemotherapies on cancer cells. I have identified eight compounds that effectively inhibit human PNKP and, with reduced potency, T4 PNK in vitro. These compounds act by reversibly inhibiting the substrate-enzyme interaction but they do not appear to sensitize U2OS cells to camptothecin.

DNA lesions arise constantly in cells and are repaired by a variety of DNA repair pathways. Polynucleotide kinase 3’-phosphatase (PNKP) aids repair by phosphorylating 5’-hydroxyl DNA termini and dephosphorylating 3’-phosphate DNA termini for the completion of repair by DNA ligases. This activity is critical in vivo because DNA breaks do not usually possess ligatable termini. PNKP knockdown sensitizes cells to several DNA damaging agents, including the topoisomerase I (TOP1) inhibitor camptothecin - analogs of which are being developed into chemotherapeutic drugs - because the resolution of stalled TOP1-DNA complexes requires processing by PNKP. We hypothesize that small molecule inhibitors of PNKP could bolster the effects of radio- and chemotherapies on cancer cells. I have identified eight compounds that effectively inhibit human PNKP and, with reduced potency, T4 PNK in vitro. These compounds act by reversibly inhibiting the substrate-enzyme interaction but they do not appear to sensitize U2OS cells to camptothecin.

No description available.

No description available.

Chlorbenzole sind aufgrund ihrer weiten Verbreitung in Industrie und Landwirtschaft und ihrer geringen chemischen Reaktivität sowie guten Fettlöslichkeit ubiquitäre Umweltkontaminanten, die sich in der Nahrungskette anreichern. Eine natürliche, mikrobielle Dechlorierung dieser Verbindungen ist von besonderem Interesse, da die Toxizität chlorierter Benzole mit der Anzahl der Chlorsubstituenten steigt. Im Gegensatz zu Mono- und Dichlorbenzol, die durch aerobe Laborreinkulturen dechlorierbar sind, werden höher chlorierte Benzole bevorzugt unter anaeroben Bedingungen in Mischkulturen mit unbekannter Spezieszusammensetzung dechloriert. Bioreaktoren, in denen solche undefinierten Mischkulturen kontinuierlich kultiviert werden, sind eine vielversprechende Technik bei der Behandlung Chlorbenzol-kontaminierter Abwässer. Aufgrund der unbekannten Zusammensetzung der Population muß die mikrobielle Aktivität jedoch als "black box" betrachtet werden, was eine direkte Steuerung und Optimierung solcher Bioreaktoren erschwert. Zur Bestimmung der mikrobiellen Diversität einer in ihrer Zusammensetzung unbekannten, Trichlorbenzol(TCB)-dechlorierenden Bioreaktorkultur wurde aufgrund der bekannten Limitierungen kulturabhängiger Methoden die kulturunabhängige, vergleichende Sequenzanalyse direkt amplifizierter und klonierter 16S-rDNA gewählt. Die durch eine neuentwickelte Hybridisierungsmethode wesentlich vereinfachte Analyse der 16S-rDNA-Genbibliotheken erlaubte eine phylogenetische Klassifizierung der in der TCB-dechlorierenden Mischkultur abundanten Mikroorganismen (Bakterien und Archaea) und zeigte das Auftreten von 51 bakteriellen 16S-rDNA-Klonfamilien, mit einem weiten phylogenetischen Spektrum und teilweise enge Verwandtschaften zu anaerob dechlorierenden Dehalobacter spp. oder zu unkultivierten Bakterien eines vergleichbaren Biotops. Dieser Diversität stand eine dominierende Methanosaeta-ähnliche Klonfamilie innerhalb der Archaea gegenüber. Die aus der phylogenetischen Klassifizierung abgeleiteten metabolischen Eigenschaften einiger Bakterien und Archaea der TCB-dechlorierenden Mischkultur konnten durch gezielte Anreicherung und in-vitro Kultivierung bzw. die kulturunabhängige Sequenzanalyse funktioneller Gene bestätigt werden. Die dargestellten Ergebnisse lassen eine spezifische Zusammensetzung der TCB-dechlorierenden Mischkultur vermuten und geben Hinweise auf Indikatororganismen, die ein Monitoring und eine damit verbundene Effizienzsteigerung der anaeroben TCB-Dechlorierung im Bioreaktor ermöglichen könnten. / Due to their widespread application in industry and agriculture and their chemical stability chlorobenzenes (CB) are ubiquitous pollutants in soil, sediments, and aquifers. Since toxicity of CBs increases with the number of chlorine substituents, microbial dechlorination of CBs is of major interest. In contrast to di- and monochlorobenzenes (DCB and MCB) higher chlorinated benzenes are more resistant to aerobic dechlorination. However, for these compounds reductive dechlorination by different anaerobic microbial communities is well known. Bioreactors inoculated with complex dechlorinating anaerobic microbiota seem to be promising technologies for bioremediation of CB-contaminated aquifers. Several studies showed the efficiency of such bioreactors in treating chloroaromatic contaminated wastewaters. However, due to the unknown species diversity microbial activity had to be treated as a "black box" and direct optimization was hampered. To determine the microbial diversity of an anaerobic consortium in a fluidized bed reactor used for dechlorination of trichlorobenzene (TCB) I employed comparative sequence analysis of 16S rRNA genes after direct PCR-amplification and cloning from community DNA since culture-based methods have shown to be strongly biased. The application of a new hybridization based screening approach for bacterial 16S rDNA clone libraries drastically simplified the analysis and allowed the phylogenetic classification of the abundant bacteria and archaea. A total of 51 bacterial 16S rDNA clone families were found, which showed a wide distribution among the main bacterial phyla. Several bacterial 16S rDNA clone families were closely related to anaerobic, dechlorinating Dehalobacter spp. and to yet-uncultured bacteria of a similar habitat. In contrast to the high bacterial diversity archaeal 16S rDNA clone libraries were clearly dominated by a Methanosaeta concilii-like clone family. Some yet-uncultured bacteria and archaea of the TCB-dechlorinating consortium were sufficiently closely related to studied organsims that reasonable physiological hypotheses could be formulated. These hypotheses were confirmed by either cultivation of the respective organism or by culture-independent sequence analysis of specific functional genes. The results suggest a specific community structure of the TCB-dechlorinating consortium and give evidence for indiator organisms. Moleculargenetic monitoring of these indicator organisms might allow the optimization of the continous TCB-dechlorination in the fluidized bed reactor.

Systematic studies of basal nonsense suppression, orthogonality of tRNAPyl variants, and cross recognition between codons and tRNA anticodons are reported. E. coli displays detectable basal amber and opal suppression but shows a negligible ochre suppression. Although detectable, basal amber suppression is fully inhibited when a pyrrolysyl-tRNA synthetase (PylRS)-tRNAPyl_CUA pair is genetically encoded. trnaPyl_CUA is aminoacylated by an E. coli aminoacyl-tRNA synthetase at a low level, however, this misaminoacylation is fully inhibited when both PylRS and its substrate are present. Besides that it is fully orthogonal in E. coli and can be coupled with PylRS to genetically incorporate a NAA at an ochre codon, tRNAPyl_UUA is not able to recognize an UAG codon to induce amber suppression. This observation is in direct conflict with the wobble base pair hypothesis and enables using an evolved M. jannaschii tyrosyl-tRNA synthetase-tRNAPyl_UUA pair and the wild type or evolved PylRS-tRNAPyl_UUA pair to genetically incorporate two different NAAs at amber and ochre codons. tRNAPyl_UCA is charged by E. coli tryptophanyl-tRNA synthetase, thus not orthogonal in E. coli. Mutagenic studies of trnaPyl_UCA led to the discovery of its G73U form which shows a higher orthogonality. Mutating trnaPyl_CUA to trnaPyl_UCCU not only leads to the loss of the relative orthogonality of tRNAPyl in E. coli but also abolishes its aminoacylation by PylRS.