Thesis (Ph. D.)--University of Oklahoma. / Bibliography: leaves 106-134.

Thesis (Ph.D. in Toxicology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 156-176). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Alabama at Birmingham, 2008. / Title from first page of PDF file (viewed on June 8, 2009). Includes bibliographical references.

No description available.

The obligate intracellular bacterium Chlamydia trachomatis is the most common bacterial STD agent in the US. This bacterium has a unique biphasic developmental cycle in which the infectious elementary body (EB) infects a host mucosal epithelial cell and differentiates into the replicative form (the reticulate body or RB) within a modified vacuole called an inclusion. The RB later divides and develops back into an EB and is released, perpetuating the infectious cycle. When developing chlamydiae are exposed to unfavorable environmental conditions, they deviate from the normal developmental cycle into a non-infectious but viable state termed persistence. Previous data from our laboratory indicate that i) during C. trachomatis/HSV co-infection, the chlamydiae become persistent and ii) HSV gD interaction with host cell surface is sufficient to induce this response. During viral entry, HSV gD interacts with one of four host co-receptors, one of which is the host adhesion molecule nectin-1. Interestingly, Western blotting demonstrated that nectin-1 is significantly decreased in C. trachomatis-infected HeLa cells. Additional studies indicated that active C. trachomatis replication is required for nectin-1 down-regulation and nectin-1 is likely down-regulated post-translationally. CPAF, a chlamydia-secreted protease, is responsible for degrading several host proteins. Both in vivo experiments using CPAF-specific chemical inhibitors and cell-free cleavage assays using recombinant CPAF indicate that nectin-1 is degraded by CPAF in C. trachomatis-infected cells. Further studies suggest that nectin-1 is the most likely candidate involved in triggering HSV-induced chlamydial persistence. Co-infection experiments using nectin-1-specific HSV-1 mutants suggest that nectin-1 is, indeed, required for persistence induction. Additional studies in single co-receptor-expressing CHO cells demonstrate that, despite the fact that HSV-1 enters both HVEM- and nectin-1-expressing cells, viral co-infection reduces chlamydial infectivity only in the CHO-nectin-1 cell line. These data confirm that HSV/nectin-1 interaction is sufficient for chlamydial persistence induction. Although nectin-1 ligation is known to activate Cdc42, pull-down assays indicate that Cdc42 is not activated in co-infected HeLa cells. Taken together, these data suggest that: i) HSV gD-nectin-1 binding activates a novel host epithelial cell pathway that restricts chlamydial development and ii) the chlamydiae may degrade nectin-1 to evade this inhibitory host response.

Le WEB2086 figure parmi les molécules ayant une activité inverse élevée pour le récepteur du PAF. Cette molécule a déjà été utilisée comme traitement pour l'asthme et représente une avenue intéressante pour une potentielle thérapie anti-cancéreuse. Nos résultats suggèrent que le WEB2086 permet la diminution de l'expression de son récepteur, le PAFR.Le même phénomène est observé avec l'agoniste PAF. Ces deux ligands n'ont cependant pas toutes les mêmes caractéristiques pharmacologiques. Nous avons donc décidé d'étudier les différentes voies de signalisation et de circulation intracellulaires activées suite à une stimulation au PAF ou au WEB2086.Le PAFR, qui peut être désensibilisé par les PKC, est phosphorylé suite à une stimulation au PAF ou au WEB2086 par des PKC d'isoformes différentes. Il semble que les PKC [bêta], [thêta] et [zêta] soit préférablement utilisées par le WEB2086, alors que les PKC [delta], [alpha], [epsilon] et [zêta] sont stimulées lorsque les récepteurs sont en mis présence de PAF. Les mécanismes régissant la signalisation et la circulation intracellulaire du PAFR ne sont pas connus. Nos travaux tentent d'élucider ces mécanismes, en comparant les voies de signalisation induites par le PAF et le WEB2086.Le mécanisme de circulation intracellulaire permettant la dégradation du PAFR utilise la voie des endosomes précoces et tardifs lors de la stimulation au PAF. Cependant, le mécanisme est différent suite à une stimulation au WEB2086. Les récepteurs sont ciblés à la dégradation indépendamment du type de ligand utilisé, soit un agoniste ou un agoniste inverse. L'utilisation d'inhibiteurs du protéasome et des lysosomes a permis d'établir que ces deux systèmes sont utilisés pour diminuer l'expression du récepteur du PAF. La compréhension des mécanismes de la régulation négative du PAFR et de la signalisation induite par les agonistes inverses donneront des outils pour la mise en place de modèles généraux de la régulation du récepteur et faciliteront le développement de thérapies ciblant efficacement la signalisation du récepteur.--Résumé abrégé par UMI.

<p>This thesis is based on 9 papers dealing with the synthesis, physicochemical and biochemical properties of two types of chemically modified oligonucleotides which have the potential to down-regulate gene expression: (i) The first set is comprised of antisense oligonucleotides (AONs) conjugated with different chromophores of varying size, charge and π-electron density. Conjugation of the chromophores at the 3'- or 5'-end enhanced the target RNA binding affinity and RNase H recruitment capabilities compared to the native counterpart without changing the global helical conformation of their AON/RNA hybrid duplexes. The 3'-dipyridophenazine (DPPZ) has emerged as the most promising non-toxic chromophore in this series. (ii) The second set encompasses a new class of AONs containing <i>North</i>-<i>East</i> conformationally constrained 1',2'-oxetane-nucleosides. The introduction of oxetane-<b>T</b> and -<b>C</b> units imparts lowering of the T_m by ~ 6º and ~ 3 ºC/modification, respectively, of the AON/RNA hybrids, whereas the incorporation of the corresponding oxetane-<b>A</b> and-<b>G</b> units into AONs did not alter the thermostability in comparison with that of the native hybrid duplex. The oxetane-modified AONs have been found to possess enhanced serum stability compared to that of the native, whereas oxetane-<b>T</b> and -<b>C</b> containing AONs were more endonuclease-resistant than oxetane-<b>A</b> and-<b>G</b> modified AONs. All oxetane-modified mixmer AON/ RNA hybrid duplexes were, however, found to be excellent substrates for RNase H cleavage, which has been analyzed by Michaelis-Menten kinetics. The oxetane-modified mixmer AONs have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells, which was analyzed by QRT-PCR and Western Blot. Based on the amount of AON uptake after delivery, determined by slot blot, it was apparent that the oxetane-modified AONs are 5-6 times more effective antisense agents than the corresponding isosequential phosphorothioate analogues. The electrochemical assay based on sensitive nucleic acid mediated charge transport (CT) has revealed that the presence of oxetane-<b>T</b> unit causes more stacking perturbations in a DNA/DNA duplex than in a DNA/RNA duplex. </p>

This thesis is based on 9 papers dealing with the synthesis, physicochemical and biochemical properties of two types of chemically modified oligonucleotides which have the potential to down-regulate gene expression: (i) The first set is comprised of antisense oligonucleotides (AONs) conjugated with different chromophores of varying size, charge and π-electron density. Conjugation of the chromophores at the 3'- or 5'-end enhanced the target RNA binding affinity and RNase H recruitment capabilities compared to the native counterpart without changing the global helical conformation of their AON/RNA hybrid duplexes. The 3'-dipyridophenazine (DPPZ) has emerged as the most promising non-toxic chromophore in this series. (ii) The second set encompasses a new class of AONs containing North-East conformationally constrained 1',2'-oxetane-nucleosides. The introduction of oxetane-<b>T</b> and -<b>C</b> units imparts lowering of the Tm by ~ 6º and ~ 3 ºC/modification, respectively, of the AON/RNA hybrids, whereas the incorporation of the corresponding oxetane-<b>A</b> and-<b>G</b> units into AONs did not alter the thermostability in comparison with that of the native hybrid duplex. The oxetane-modified AONs have been found to possess enhanced serum stability compared to that of the native, whereas oxetane-<b>T</b> and -<b>C</b> containing AONs were more endonuclease-resistant than oxetane-<b>A</b> and-<b>G</b> modified AONs. All oxetane-modified mixmer AON/ RNA hybrid duplexes were, however, found to be excellent substrates for RNase H cleavage, which has been analyzed by Michaelis-Menten kinetics. The oxetane-modified mixmer AONs have shown effective down-regulation of the proto-oncogene c-myb mRNA in the K562 human leukemia cells, which was analyzed by QRT-PCR and Western Blot. Based on the amount of AON uptake after delivery, determined by slot blot, it was apparent that the oxetane-modified AONs are 5-6 times more effective antisense agents than the corresponding isosequential phosphorothioate analogues. The electrochemical assay based on sensitive nucleic acid mediated charge transport (CT) has revealed that the presence of oxetane-<b>T</b> unit causes more stacking perturbations in a DNA/DNA duplex than in a DNA/RNA duplex.

Wood is a complex material and the mechanical properties are influencedby a number of structural parameters. The objective of this study has been toinvestigate the relationship between the structure and the mechanical propertiesof hardwood. Two levels were of special interest, viz. the cellular structureand morphology of the wood, and the ultra-structure of the cell wall. In thenext step, it was of interest to examine how the mechanical properties ofhardwood change with spontaneous/induced changes in morphology and/orchemical composition beyond the natural variation found in nature. Together, this constituted the framework and basis for two larger projects,one on European aspen (Populus tremula) and hybrid aspen (Populus tremulax Populus tremuloides), and one on European oak (Quercus robur). Amethodology was developed where the concept of relative density and compositemechanics rules served as two useful tools to assess the properties ofthe cell wall. Tensile testing in the longitudinal direction was combined withchemical examination of the material. This approach made it possible to revealthe mechanical role of the lignin in the cell wall of transgenic aspen trees,and investigate the consequences of holocellulose degradation in archaeologicaloak from the Vasa ship. The study on transgenic aspen showed that a major reduction in lignin inPopulus leads to a small but significant reduction in the longitudinal stiffness.The longitudinal tensile strength was not reduced. The results are explainableby the fact that the load-bearing cellulose in the transgenic aspen retained itscrystallinity, aggregate size, microfibril angle, and absolute content per unitvolume. The results can contribute to the ongoing task of investigating andpinpointing the precise function of lignin in the cell wall of trees. The mechanical property study on Vasa oak showed that the longitudinaltensile strength is severely reduced in several regions of the ship, andthat the reduction correlates with reduced average molecular weight of theholocellulose. This could not have been foreseen without a thorough mechanicaland chemical investigation, since the Vasa wood (with exception fromthe bacterially degraded surface regions) is morphologically intact and witha micro-structure comparable to that of recent oak. The results can be usedin the ongoing task of mapping the condition of the Vasa wood. / QC 20110420

Toxoplasma gondii ist ein obligat intrazellulär lebender einzelliger Parasit, der sich durch ein breites Wirtsspektrum sowie lebenslang persistierende Infektionen bei Menschen und Tieren auszeichnet. T. gondii hat für ein langfristiges Überleben unterschiedliche Mechanismen entwickelt, die ein Gleichgewicht zwischen der Pathogenität des Erregers und der intakten Immunabwehr des Wirtes gewährleisten. In diesem Kontext stellt die Modulation von Signalwegen der Wirtszelle eine wichtige Überlebensstrategie von Toxoplasmen dar. Frühere Arbeiten mit murinen Monozyten haben gezeigt, dass T. gondii die Expression von MHC-Klasse-II-Molekülen auf der Oberfläche infizierter Wirtszellen hemmt und dadurch eine effektive Antigenpräsentation an T-Helfer-Lymphozyten verhindert. Das Ziel der vorliegenden Dissertation war es herauszufinden, inwieweit Toxoplasma gondii auch die Interferon-γ-induzierte MHC-Expression von Monozyten des Menschen vermindert. Analysen mittels Immunfluoreszenzfärbung und Durchflusszytometrie zeigten, dass sowohl primäre, aus PBMC des Menschen isolierte Monozyten als auch permanente humane Monozyten (THP-1) durch eine Infektion mit T. gondii in der Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ signifikant gehemmt werden. Das Ausmaß der Inhibition ist dabei von der Infektionsdosis des Parasiten abhängig und betrifft sowohl die HLA-Expression auf der Zelloberfläche als auch den intrazellulären HLA-Pool. Interessanterweise kann dieser Effekt auch durch hohe Konzentrationen des stimulierenden Zytokins Interferon-γ nicht aufgehoben werden. Es zeigt sich außerdem eine signifikant reduzierte Expression von HLA-DR, -DP, -DQ bei Parasit-negativen Zellen einer T. gondii-infizierten Kultur, was möglicherweise durch sezernierte Proteine des Parasiten oder durch die Produktion hemmender Zytokine durch infizierte Wirtszellen begründet sein kann. Die HLA-Expression in in vitro-infizierten und nicht-infizierten primären Monozyten wurde darüberhinaus zwischen T. gondii-negativen Individuen und Spendern mit chronischer Toxoplasmose verglichen. Chronisch mit T. gondii infizierte Blutspender wurden serologisch anhand von spezifischen IgG-Antikörpern identifiziert. Durchflusszytometrische Analysen zeigten, dass Monozyten aus chronisch mit T. gondii infizierten Blut-Spendern signifikant mehr HLA-A, -B, -C und HLA-DR, -DP, -DQ exprimieren als Monozyten aus Toxoplasma-negativen Spendern. Eine Erklärung für diese gesteigerte MHC-Expression könnte eine Dominanz bestimmter Monozyten-Subpopulationen in Abhängigkeit vom Infektionsstatus ihres Spenders sein. Die Expression von HLA-A, -B, -C und HLA-DR, -DP, -DQ wird jedoch sowohl bei Monozyten von T. gondii-positiven als auch nicht-infizierten Individuen durch eine Infektion mit Toxoplasmen in vitro signifikant inhibiert. Analysen mit Hilfe von RT-qPCR zeigten deutlich, dass T. gondii mit der HLA-DR-, -DP-, -DQ-Expressions-Kaskade interferiert und die Synthese der Transkripte von IRF-1 und CIITA dosisabhängig inhibiert. Außerdem sind die Transskripte für HLA-A und HLA-DRα in infizierten Monozyten deutlich reduziert. Dies legt die Annahme nahe, dass T. gondii die Aktivierung von STAT1-abhängigen Promotoren effektiv inhibiert und so die Synthese der sich anschließenden HLA-Expressionskaskade supprimiert. Die Ergebnisse dieser Arbeit eröffnen interessante Ansätze für weitere Untersuchungen, insbesondere eine genauere Charakterisierung von Monozyten-Subpopulationen bei T. gondii-positiven Individuen sowie die Erforschung einer möglicherweise gesteigerten Immunreaktivität gegen andere Infektionserreger im Rahmen einer chronischen Toxoplasmose.