Le taxol est un composé découvert en 1965 à partir d'un extrait d'if du Pacifique (Taxus Brevifolia Nutt.). Son mode d'action original et inconnu au moment de sa découverte a fait de cette molécule une référence en matière de traitement anti-cancéreux. La présence de nombreuses fonctionnalités oxygénées, le nombre élevé de centres asymétriques et surtout sa structure tétracyclique comportant de plus un cycle à huit chaînons ont suscité l'intérêt des chercheurs depuis sa publication. Découverte dans les années 1970, la réaction de métathèse cyclisante des oléfines est un outil synthétique extrêmement puissant permettant la préparation de cycles de diverses tailles. Cette étude envisage l'utilisation de cette réaction pour préparer le cycle à huit chaînons du taxol. La première rétrosynthèse étudiée se fonde sur la fermeture du cycle B à huit chaînons par une réaction de métathèse cyclisante entre les carbones C9 et C10. La première étape de ce travail est consacrée à la préparation des précurseurs de métathèse. Un travail d'optimisation des conditions ne nous a cependant pas permis d'obtenir les diènes souhaités, par réaction de Shapiro ou de Nozaki-Hiyama-Kishi. Une nouvelle stratégie est ensuite étudiée: la fermeture du cycle B s'envisage cette fois-ci entre les carbones C10 et C11. Le diène souhaité serait obtenu par une réaction d'addition nucléophile entre le précurseur linéaire du cycle A et le cycle C. Ces deux précurseurs sont tout d'abord préparés par des méthodes rapides, simples et efficaces, donnant accès à des composés énantiomériquement purs. Les réactions d'additions nucléophiles sont ensuite étudiées. Une optimisation des paramètres permet d'obtenir le composé souhaité sous la forme d'un mélange 1:1 de deux diastéréoisomères, qui sont séparés à l'étape suivante. L'étude des figures de diffraction aux rayons X de ces deux composés permet d'attribuer la configuration relative de chaque centre asymétrique. Les composés obtenus sont ensuite transformés en quelques étapes en précurseurs de métathèse. Le diastéréoisomère comportant la mauvaise stéréochimie pour la sythèse du taxol se dégrade en présence du catalyseur de métathèse. En revanche, le diastéréoisomère comportant la bonne stéréochimie pour la sythèse du taxol cyclise en quelques heures en présence de catalyseur de Grubbs II pour donner le cyclooctène cis. De plus, en présence de catalyseur de Grubbs I, l'obtention d'un mélange des cyclooctènes cis et trans est mise en évidence. Enfin, une étude menée en annexe démontre la possibilité d'introduire la fonctionnalité oxygénée manquante sur le cycle C en utilisant comme produit de départ la cétone de Wieland-Miescher.

<p>Ribose 5-phosphate isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms <i>Mycobacterium tuberculosis</i> (<i>Mt</i>) and <i>Escherichia coli</i> (<i>Ec</i>) from a structural and functional point of view.</p><p>Since the <i>E. coli</i> genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that <i>Ec</i>RpiB has a different role as an allose-6-phosphate isomerase. Activity measurements presented here show that <i>Ec</i>RpiB does have this second activity. </p><p>In the <i>M. tuberculosis</i> genome there is only a gene for RpiB. The crystal structure of <i>Mt</i>RpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for <i>Mt</i>RpiB and for other RpiBs in general. Activity measurements of <i>Mt</i>RpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between <i>Ec</i>RpiB and <i>Mt</i>RpiB explain these kinetic results. </p><p>Activity measurements and a structure of an <i>Ec</i>RpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. </p><p>Inhibition studies have uncovered a compound that selectively inhibits <i>Mt</i>RpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.</p>

Nanoparticles were synthesized and modified for target drug delivery. The research involved the aqueous synthesis of near infrared (NIR) sensitive Au-Au₂S nanoparticles. An anti-cancer drug (<i>cis-platin</i>) was subsequently adsorbed onto the Au-Au₂S nanoparticle surface via the 11-mercaptoundecanoic acid layers. The results showed that the degree of adsorption of cis-platin onto Au-Au₂S nanoparticles was controlled by the pH value of solution, and the rate of drug release was sensitive to NIR irradiation. The results of the synthesis, drug-release properties and nanoparticle-cell interactions will be discussed. / Singapore-MIT Alliance (SMA)

The research presented in this thesis examines a lithic assemblage from the multi-component habitation site Gorelyi Les in the Belaia river valley, Cis-Baikal region, Siberia. The distinctive traits of this collection are the relatively small size of the lithic assemblage and the large proportion of debitage. The chosen methodological framework for this research concentrates on obtaining maximum information from the available materials through application of typological, technological, use-wear, and spatial analyses. The results suggest that there were differences in the organization of the technological process of lithic tool manufacture during the Early Neolithic and Late Neolithic. During the Early Neolithic, lithic tool manufacture and use were rather intensive and diversified, while during the Late Neolithic, tool manufacture and use were limited to a narrower range of technological operations and functions.

Ribose 5-phosphate isomerase (Rpi) is one of the major enzymes of the pentose phosphate pathway, where it catalyses the inter-conversion of ribose 5-phosphate (R5P) and ribulose 5-phosphate. Two forms of this isomerase with no significant amino acid sequence similarity exist, RpiA and RpiB. This thesis describes RpiB from the organisms Mycobacterium tuberculosis (Mt) and Escherichia coli (Ec) from a structural and functional point of view. Since the E. coli genome encodes both an RpiA and an RpiB, which generally is not expressed, it has been proposed that EcRpiB has a different role as an allose-6-phosphate isomerase. Activity measurements presented here show that EcRpiB does have this second activity. In the M. tuberculosis genome there is only a gene for RpiB. The crystal structure of MtRpiB was solved in complex with several different inhibitors designed to mimic the reaction intermediate as well as with the substrate, R5P. The organisation of the active site in these structures could be used to derive the reaction mechanism for MtRpiB and for other RpiBs in general. Activity measurements of MtRpiB showed that it can catalyse the R5P isomerisation, but not the allose 6-phosphate reaction. Differences observed in the active site between EcRpiB and MtRpiB explain these kinetic results. Activity measurements and a structure of an EcRpiB mutant, where histidine99 was changed to asparagine, implies that RpiB catalyses the first step of the reaction in which the sugar ring must be opened, and gives a possible explanation for how this could occur. Inhibition studies have uncovered a compound that selectively inhibits MtRpiB over RpiA from spinach, which is homologous to the human RpiA. Differences in the inhibition patterns and active site residues of these two species’ Rpi may provide information for future virtual screening approaches, with the aim of discovering new anti-tuberculosis agents.

Mycobacterium tuberculosis, the causative pathogen of tuberculosis, currently infects one-third of the world’s population, resulting in two million deaths annually. This clearly shows that tuberculosis is one of the most serious diseases of our times. The often unpleasant side effects from the current drugs, combined with the difficulty of ensuring patient compliance, and the emergence of drug-resistant and multidrug-resistant strains, makes the need for new and better drugs urgent. In this thesis, all the steps, from cloning, purification, crystallization, to activity determination, and structure determination are presented for two different M. tuberculosis enzymes. The structures, which were modeled from X-ray crystallographic data, provide the framework for structure-based drug design. Here, new potential inhibitors can be tailor-made based on the specific interactions in the enzyme’s active site. The bacteria have two different peptidyl-prolyl cis-trans isomerases that catalyze the isomerization of peptide bonds preceding proline residues, a process of high importance for correct folding. Here we present the structure of peptidyl-prolyl cis-trans isomerase A, an enzyme present inside the bacteria, and distinguish it from the B form of the enzyme, which is membrane bound, placing its active site outside the bacteria. The enzyme 1-deoxy-D-xylulose-5-phosphate reductoisomerase catalyzes the second step within the non-mevalonate pathway, which leads to the production of isopentenyl diphosphate. This compound is the precursor of various isoprenoids, vital to all living organisms. In humans, isopentenyl diphosphate is produced via a different pathway, indicating that all the enzymes within the non-mevalonate pathway may be suitable drug targets in M. tuberculosis. Several structures of both wild type and mutant 1-deoxy-D-xylulose-5-phosphate reductoisomerase in complex with different substrates, and also with the known inhibitor fosmidomycin, provide valuable information not only to the field of drug design, but also, in this case, into the catalysis.

La caractérisation des modules cis-régulateurs (CRM) ainsi que de leur activité sont essentiels pour comprendre la régulation des gènes au cours du développement des métazoaires. La technique de l'immunoprécipitation de la chromatine suivie du séquenage à haut débit de l'ADN (ChIP-seq) constitue une approche puissante pour localiser les CRM. Afin de localiser des facteurs génériques au sein de tissus spécifiques, nous avons développé une approche ChIP-seq sur des noyaux triés par cytométrie de flux et localisons des modifications post-traductionelles de l'histone H3, ainsi que l'ARN polymérase II (PolII) dans le mésoderme de la Drosophile. Nous montrons que les CRM actifs sont caractérisés par la présence d'H3 modifiés (K27Ac et K79me3) et de PolII. De plus, la présence et la forme des signaux correspondants à ces marques corrèlent dynamiquement avec l'activité des CRM. Enfin, nous prédisons la présence de CRM actifs et confirmons leur activité in vivo à 89%. Paralllement, nous étudions comment cinq facteurs essentiels au développement cardiaque se coordonnent en cis au sein du mésoderme dorsal, précurseur des mésodermes cardiaque (MC) et viscéral (MV). Nous démontrons que ces facteurs sont recrutés en tant que collectif au niveau des CRM cardiaques via un nombre limité de sites de fixation et en l'absence de contraintes architecturales. En outre, nous découvrons que ces facteurs cardiaques sont recrutés au niveau de CRM actifs dans le MV voisin et activement réprimés dans le MC, reflétant ainsi l'origine tissulaire commune de ces deux populations cellulaires. Nous concluons que les CRM impliqués dans le développement peuvent présenter une empreinte développementale.

This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.

This thesis performs de novo predictions for functionally significant sequence motifs in the Arabidopsis genome under two separate contexts. Each study applies the use of genomic positional information, statistical over-representation and several biologically contextual filters to maximize the visibility of biological signal in prediction results. Numerous literature supported motifs are prevalent in the results of both studies and a number of novel motif patterns possess a strong potential for in planta significance. The first study examines the statistical over-representation of C-terminal tripeptides as a means for identifying eukaryotic conserved protein targetting signatures. Comparative genomics is applied to the analysis of tripeptide frequencies in the C-terminus of 7 eukaryotic proteomes. While biological signal is maximized through the filtering of both simple sequences and homologous sequences present across protein families. The second study introduces a methodology for the effective prediction of transcription factor binding sites in Arabidopsis. A collection of motif prediction algorithms and a novel enumerative strategy are applied to the prediction of cis-acting regulatory elements within the promoters of genes found coexpressed within distinct tissues and under specific abiotic stress treatments. Overall, the analysis identifies 4 known motifs in expected contexts, 5 known motifs in novel contexts and 7 novel motifs with a high potential for biological function.

Includes vita. / Title from PDF of title page. / Document formatted into pages; contains 204 pages. / Thesis (Ph.D.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: The goal of this research project was to contribute to the understanding of CuGaSe2/CdS photovoltaic devices, and to improve the performance of these devices. The initial part of the research dealt with the optimization of a Sequential Deposition process for CuIn(Ga)Se2 absorber formation. As an extension of this, a recipe (Type I Process) for CuGaSe2 absorber layer fabrication was developed, and the deposition parameters were optimized. Electrical characterization of the thin films and completed devices was carried out using techniques such as Two-Probe and Three-Probe Current-Voltage, Capacitance-Frequency, Capacitance-Voltage, and Spectral Response measurements. Structural/chemical characterization was done using XRD and EDS analysis. Current densities of up to 15.2 mA/cm2, and Fill Factors of up to 58% were obtained using the Type I CuGaSe2 Process. VOC's, however, were limited to less than 700 mV. / ABSTRACT: Several process variations, such as changes in the rate/order/temperature of depositions and changes in the thickness of layers, resulted in little improvement. With the aim of breaking through this VOC performance ceiling, a new absorber recipe (Type II Process) was developed. VOC's of up to 735 mV without annealing, and those of up to 775 mV after annealing, were observed. Fill Factors were comparable to those obtained with Type I Process, whereas the Current Densities were found to be reduced (typically, 10-12 mA/cm2, with the best value of 12.6 mA/cm2). This performance of Type II devices was correlated to a better intermixing of the elements during the absorber formation. To gain an understanding of the performance limitations, two simulation techniques, viz. SCAPS and AMPS, were used to model our devices. / ABSTRACT: Several processing experiments and SCAPS modeling indicate that a defective interface between CuGaSe2 and CdS, and perhaps a defective absorber layer, are the cause of the VOC limitation. AMPS simulation studies, on the other hand, suggest that the back contact is limiting the performance. Attempts to change the physical back contact, by changes in the absorber processing, were unsuccessful. Processing experiments and simulations also suggest that the CuGaSe2/CdS solar cell involves a true heterojunction between these two layers. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.