The first part of this thesis is concerned with the development of a general synthetic approach to-the bicyclo(3.2.1)octane ring system. This work resulted in the successful synthesis of 1,5-dimethylbicyclo (3.2.1)octan-8-one(54) , 1,5-dimethylbicyclo (3.2.1)oct-6-en-8-one(55) and 1,5-dimethyl-6-isoproxybicyclo (3.2.1)oct-6-en-8-one(56). Alkylation of 2,6-dimethylcyclohexanone with allyl bromide afforded the olefinic ketones (78) and (79). Ozonolysis of the latter gave the keto aldehydes (80) and (81), which underwent a facile intramolecular aldol condensation to afford a mixture of the bicyclic alcohols (68) and (69). The mesylates (94_) and (9_5_) were obtained from the bicyclic alcohols (68J and (69_) . The keto iodide (70) , obtained by treating a mixture of the mesylates (94) and (95) with sodium iodide in refluxing acetone, was dehydrohalogenated to afford the desired bicyclic ketone (55). Hydrogenation of the latter over palladium afforded the desired bicyclic ketone (_54) . Oxidation of a mixture of the keto alcohols (68) and (69) with Collin's reagent afforded the diketone (71) which, under appropriate conditions, underwent O-alkylation to give the desired bicyclic enol ether (56) . In the second part of this thesis, the stereochemistry of nucleophilic addition to the 8-keto group present in the bicyclic ketones (54) , (55) and (56) was briefly investigated. The bicyclic ketones (54), (55) and (56) were reduced with lithium aluminium hydride to afford the bicyclic alcohols (112), (113) and (114) . Hydrogenation of the alcohol (113) yielded the alcohol (112). Hydrolysis of the enol ether (114) gave the ketol (116) . Hydrogenolysis of the dithioketal(117) of the keto alcohol (116) with Raney-nickel afforded alcohol (112). Thus, the bicyclic alcohols (112), (113) and (114) must have the same relative stereochemistry at C-8. The bicyclic ketones (5_4_) , (5_5) and (5_6_) were also treated with one equivalent of methylithium under identical conditions to form the corresponding tertiary alcohols (128), (129) and (130). These alcohols are also formed stereo-selectively and have the same relative stereochemistry as the corresponding secondary alcohols (112) , (113) and (114) . / Science, Faculty of / Chemistry, Department of / Graduate

L’analyse vibratoire est l’une des principales méthodes pour le suivi et la maintenance des machines tournantes. Cette thèse s’inscrit dans cette thématique et en particulier dans le diagnostic des roulements en régime variable. L’objectif de la thèse est l’élaboration de capteurs logiciels. Ces derniers pourraient fournir un indicateur pertinent sur l’état de roulements défectueux à partir des signaux de vibrations de type aléatoire qu’ils génèrent dans les cas où ils sont mélangées à d’autres sources vibratoires provenant de défaut d’engrenages générant des signaux déterministes. Afin de diagnostiquer d’une façon efficace les cas d’avarie, il faut au préalable séparer le signal de vibration relatif à chaque source de vibration. En régime stationnaire, l’outil utilisé pour estimer la composante déterministe est la moyenne synchrone. En régime variable, la partie déterministe du signal de vibration évolue avec le régime même si l’enregistrement du signal de vibration est synchronisé avec la vitesse de rotation. Dès lors, cette composante ne devient plus périodique et son estimation par la moyenne synchrone classique devient inappropriée. Nous avons alors proposé une nouvelle approche qui consiste à caractériser les signaux de vibrations en régime variable en introduisant la notion de cyclo-non-stationnarité. Ceci nous a permis de développer un outil d'estimation des composantes déterministes du signal qui sont caractérisées par la cyclo-non-stationnarité d'ordre 1 et de développer un outil de caractérisation de la cyclo-non-stationnarité d'ordre 2 basé sur la synchronisation de la corrélation spectrale. Les performances de cet outil ont été testées avec succès sur des signaux synthétiques ainsi que sur des signaux réels. / Vibration analysis is one of the main methods for rotating machinery monitoring. The topic of this thesis is the diagnosis of rolling element bearings under varying rotating speed. The objective is the development of software sensors providing a relevant indicator of bearing condition monitoring from the vibration signal generated by the bearing. Often, rotating machinery is composed from gears and bearings. Gear faults generate deterministic components and bearing faults generate random components. To diagnose, the key point is to extract the vibration signal caused by bearing from the total vibration signal. In constant rotating speed condition, the used tool to estimate the deterministic component is the time synchronous averaging. In varying rotating speed, the signal becomes "cyclo-non-stationary". Therefore, the traditional synchronous averaging method becomes inappropriate and one needs to extend its definition and estimation process. In this thesis, we develop a new approach based on a decomposition of the vibration signal into a set of cyclo-non-stationary components. This allowed us to develop a tool for estimating the deterministic components of the signal which are characterized by cyclo-non-stationarity order 1 and to develop a tool for characterizing the cyclo-non-stationarity order 2 based on synchronized spectral correlation. The performance of this tool has been tested on synthetic signals and on real signals.

Made available in DSpace on 2014-06-11T19:31:11Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-08-01Bitstream added on 2014-06-13T19:20:19Z : No. of bitstreams: 1 bersano_pro_dr_botfmvz.pdf: 1282735 bytes, checksum: 6dcfee17d1682327cfe2db3f30be0f3c (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O comportamento biológico altamente metastático e localmente agressivo é marca dos tumores ósseos malignos do cão, sua importância deve-se à dificuldades na conduta terapêutica. Para testar a hipótese, o trabalho se propôs a investigar a Cox2, proteína alvo para tratamento, com identificação e quantificação dos marcadores da proliferação: Ki-67; apoptose: caspase-3, p53, Bcl-2 e Bax; invasão: MMP-2 e MMP-9; e formação de vasos: VEGF. Cinco cães com suspeitas clínicas e radiológicas, e posterior diagnóstico citopatológico e histopatológico de osteossarcoma de ocorrência espontânea, que foram submetidos à cirurgia. Obtive-se de cada animal o isolamento e caracterização dos cultivos primários. Os diferentes cultivos foram expostos a inibidor seletivo da Cox2, o celecoxibe, no qual houve diferença (p ≤ 0,05) para os cultivos Cox2 positivos em relação aos negativos, com maior indução à necrose. Em 72h observou-se maior porcentagem de células vivas, menor de células em apoptose e maior de células em necrose em relação aos grupos de 24h e 48h. Houve tendência (p=0,08) na redução da porcentagem de células em necrose para os grupos de 100M.L-1 quando comparados aos grupos de 10M.L-1. Independente do método, os fatores que induzem crescimento vascular, proliferação, morte e invasão, inerentes as etapas da carcinogênese, são complexos Não foi possível determinar o papel individual de cada um destes fatores. A metodologia empregada se mostrou eficaz para determinar o padrão histológico das amostras. Logo, o cultivo primário se comportou como modelo ideal para estudo do OSA, visto que suas características de agressividade tumoral não se perderam quando comparados aos métodos citados / A high metastatic behavior and site aggressiveness are common features of bone malignant tumors in the dog and they are important owing to their therapeutic difficulties. The objective of this study was to validate such hypothesis by investigating the Cox2 (therapy’s target protein) via the identification and quantification of markers for proliferation (Ki-67), apoptosis (caspase-3, p53, Bcl-2 and Bax), invasion (MMP-2 and MMP-9) and vessel formation (VEGF). Five dogs diagnosed as having spontaneous osteosarcoma (clinical, radiological, cytopathological and histopathological diagnosis) underwent tumor removal surgery and it was isolated and characterized the primary culture of each animal. All cultures were exposed to a Cox2 selective inhibitor named celecoxibe. There was difference (p ≤ 0,05) between Cox2 positive cultures and Cox2 negative ones. The positive ones presented higher necrosis induction. The 72h group presented higher percentage of live cells and cells undergoing necrosis when compared to the 24h and 48h groups. Nevertheless, the 72h group presented lower percentage of cells in apoptosis. Cells undergoing necrosis tended be in a lower percentage (p=0,08) for the 100M.L-1 groups when compared to the 10M.L-1. Regardless of methodology, the factors that induce vascular growth, proliferation, death, and invasion in different phases of the carcinogenesis are complex. It was no yet possible to determine the individual role from each of these factors. The methodology applied in this experiment was efficient in determining the histological standard of these samples. Hence, the primary culture may be used as an ideal model for the OSA study as the tumor aggressive characteristics were not lost when compared to the previously mentioned methods

No description available.

University, Diss., 2005--Würzburg. / Parallelt.: Development and application of fluorescent cAMP und cGMP biosensors.

Thesis advisor: Larry W. McLaughlin / Thesis advisor: Mary F. Roberts / Nucleic acids are the only biopolymers capable of encoding and transferring information, this property has placed them at the fundamental core of all living organisms, and made them a topic of intense research for over a century. The former studies in our laboratory on simplified nucleic acid backbones provided insight into how we might rationally alter nucleic acid structure into one that possesses properties not observed in natural DNA and RNA. Here the work began as an investigation into rigidified nucleic acid systems capable of functioning as DNA. The first rigidified nucleic acid system we designed, the cyclo-2'-deoxynucleic acids, has a linkage between the C5' of the ribose sugar and base to lock the ӽ angle into the similar angle as the native nucleoside. These rigidified bases show great impact towards the DNA structure, destabilizing double helix formation. This in fact can also be found in nature to inhibit the TATA binding protein associating with its target region. The next generation of rigidified nucleosides has an extended 7-membered ring instead of the 6-membered ring that was present in the first generation to push the base closer to the helical center. Both diastereomers of the ring-expanded-cyclo-2'-deoxyadenosine have been successfully synthesized and characterized and are ready to perform further studies. The third system is the hydroxymethyl-cyclo-nucleosides. The modified nucleosides in this project not only have the same linkage as the cyclo-nucleosides in the first system to restrict the base rotation, but also have an extra carbon (C6') to give the backbone more flexibility which might better stabilize a double helix than the first generation. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Desmosomen sind Zell-Zell-Kontakte, die eine starke interzelluläre Haftung vermitteln. Sie sind daher besonders wichtig für die Integrität von Geweben wie der Haut, die laufend einer starken mechanischen Beanspruchung ausgesetzt sind. Pemphigus vulgaris ist eine Autoimmundermatose, die zur Ausbildung schlaffer Blasen durch Spaltbildung in der Epidermis führt. Als ursächlich dafür wurden Autoantikörper gegen die desmosomalen Cadherine Dsg1 und 3 herausgestellt, die in Desmosomen vorkommen. cAMP ist ein wichtiger Botenstoff des Zellstoffwechsels und an der Regulierung und Modulation einer Vielzahl von zellulären Prozessen beteiligt, darunter auch die Stabilisierung der Endothelbarriere über Stärkung der Haftung eines klassischen Cadherins, nämlich VE-Cadherin. In Anknüpfung an die vorliegenden Daten aus Pemphigus- und Endothelforschung beschäftigt sich diese Arbeit mit der Rolle von cAMP bei Pemphigus vulgaris. Es wurde in Keratinozytenkultur sowie im neonatalen Pemphigus-Mausmodell untersucht, ob die Erhöhung der intrazellulären cAMP-Spiegel einen Einfluss auf PV-IgG-induzierte morphologische und funktionelle Veränderungen hat. Eine Erhöhung des intrazellulären cAMP-Spiegels konnte sowohl in vitro als auch in vivo als protektiv herausgestellt werden. In Keratinozytenkultur konnte gezeigt werden, dass eine Erhöhung des intrazellulären cAMP-Spiegels durch Forskolin/Rolipram oder Isoproterenol in der Lage war, die PV-IgG-induzierten morphologischen Veränderungen, die Dsg3-Depletion, sowie den Adhäsionsverlust zu blockieren. Weiterhin konnte die Blasenbildung in vivo durch cAMP-Erhöhung vollständig verhindert werden. Im Anschluss wurde untersucht, ob die Inkubation mit PV-IgGs einen Einfluss auf die intrazellulären cAMP-Spiegel in vitro hat. Dabei konnte gezeigt werden, dass die Zellen mit einer Erhöhung der cAMP-Spiegel reagieren, wenn auch in einem geringeren Ausmaß als durch die eingesetzten Mediatoren. Somit kann cAMP als Rettungsmechanismus der Zellen angesehen werden und es wurde daraufhin der Einfluss von cAMP auf die Regeneration von Keratinozyten nach PV-IgG-Inkubation untersucht. Dieser Prozess konnte durch eine cAMP-Erhöhung verbessert werden und erwies sich als partiell abhängig von PKA. Schlussendlich konnte nachgewiesen werden, dass cAMP in vitro wie in vivo über die Blockade der p38MAPK-Aktivierung protektiv wirkt. Zusammenfassend konnte so ein neuer Einblick in die zelluläre Antwort von Keratinozyten auf Pemphigus-Autoantikörperbindung gewonnen werden. Dieser könnte auch im Hinblick auf die Entwicklung neuer therapeutischer Strategien bei Pemphigus vulgaris wichtig sein. / Desmosomes are cell-cell-contacts which provide strong intercellular adhesion. They are most abundant and important in tissues which are continuously exposed to mechanical stress such as the epidermis of the skin. Pemphigus vulgaris (PV) is a severe autoimmune blistering disease characterized by intraepidermal cleavage leading to flaccid blisters of the skin. These phenomena are caused by autoantibodies against the desmosomal adhesion molecules desmoglein (Dsg) 1 and 3. Cyclic adenosine-monophosphat (cAMP) is an important second messenger in various signaling pathways and takes part in regulation and modulation of distinct cellular processes. For instance, cAMP is capable to stabilize the endothelial barrier by increasing adhesion of another cadherin-type adhesion molecule referred to as vascular endothelial cadherin (VE-cadherin). Therefore, based on the knowledge from pemphigus and endothelial barrier research, the scope of the present dissertation is to clarify the role of cAMP in pemphigus vulgaris pathogenesis. Investigations in keratinocyte cell cultures and in a neonatal pemphigus mouse model were carried out to analyze the effect of an increased intracellular cAMP level on desmosomal adhesion. The data demonstrated that increased cAMP-levels are protective against the pathogenic effects of autoantibodies from PV patients (PV-IgG) in vitro and in vivo. In cultured keratinocytes the increase of intracellular cAMP mediated by application of either forskolin/rolipram or isoproterenol was capable to block PV-IgG-induced morphological changes as well as Dsg3-depletion and loss of cell cohesion. Further, development of flaccid blisters and intraepidermal cleavage formation was abolished in vivo. Since cAMP signaling in vitro as well as in vivo was effective to prevent autoantibody-induced p38MAPK activation, which is a central mechanism in pemphigus pathogenesis, it can be concluded that the signaling function of desmogleins was modulated by this approach. Next, we examined whether incubation with PV-IgG affected intracellular cAMP levels in vitro. Interestingly, we found that keratinocytes responded to PV-IgG incubation with an increase in cAMP levels, albeit to a smaller extend compared to the applied mediators. These data indicate that increase of cAMP may serve as a cellular rescue mechanism to enhance intercellular cohesion. Finally, the effect of cAMP on spontaneous regeneration of cell cohesion after PV-IgG incubation was examined. Here, it was observed that regeneration was improved by increase of cAMP and was at least partially PKA-dependent. In summary, this work provides new insights into the cellular responses of keratinocytes to the binding of pemphigus autoantibodies. This may be important especially with respect to development of new therapeutic approaches in pemphigus vulgaris.

Die NO-sensitive Guanylyl-Cyclase (NO-GC) ist ein zentrales Enzym der NO/cGMP-Signalkaskade, das über die Aktivierung von NO zur Bildung des second messangers cGMP führt. Die NO-GC setzt sich aus zwei Untereinheiten zusammen, sodass zwei Isoformen des Enzyms gebildet werden können (α1β1 und α2β1). Da die genaue Verteilung der beiden Isoformen im Colon nicht bekannt ist, wurde diese im ersten Teil dieser Arbeit charakterisiert. Immunhistochemie und In-situ-Hybridisierung zeigten die Expression beider Isoformen sowohl in der glatten Muskelschicht als auch in der Submukosa und Lamina propria. Dabei war die α1β1-Isoform ubiquitär, die α2β1-Isoform dagegen hauptsächlich im Bereich des myenterischen Plexus vorzufinden. In der glatten Muskelschicht des Colons ist die NO-GC in glatten Muskelzellen (SMC), interstitiellen Zellen von Cajal (ICC) sowie Fibroblasten-ähnliche Zellen (FLC) exprimiert und hauptsächlich in die Modulation der gastrointestinalen Motilität involviert. Zur spezifischen Charakterisierung der Funktion der NO-GC in den einzelnen Zelltypen wurden Knockout-Mäuse generiert, denen die NO-GC global (GCKO) oder spezifisch in SMC (SMC-GCKO), ICC (ICC-GCKO) oder beiden Zelltypen (SMC/ICC-GCKO) fehlt. Anhand dieser Mausmodelle sollten im zweiten Teil dieser Arbeit die modulatorischen Effekte der NO-GC auf die spontanen Kontraktionen des Colons bestimmt werden. Zur Charakterisierung der spontanen Kontraktionen der zirkulären Muskelschicht wurden Myographiestudien mit 2,5 mm langen Colonringen durchgeführt. Hierbei konnten drei verschiedene Kontraktionen gemessen werden: Kleine, hochfrequente Ripples, mittlere Kontraktionen und große Kontraktionen. Die detaillierte Analyse der einzelnen Kontraktionen zeigte einerseits eine NO-unabhängige Regulation der Ripples, andererseits eine NO-abhängige Modulation der mittleren und großen Kontraktionen über die NO-GC in SMC und ICC. Die NO-GC in SMC beeinflusst die Kontraktionen vermutlich vor allem über die Regulation des Muskeltonus der zirkulären Muskelschicht. Die NO-GC in ICC dagegen modifiziert die spontanen Kontraktionen möglicherweise über eine Veränderung der Schrittmacheraktivität. Allerdings führt erst ein Funktionsverlust des NO/cGMP-Signalweges in beiden Zelltypen zu einem sichtbar veränderten Kontraktionsmuster, das dem von globalen Knockout-Tieren glich. Dies weist auf eine kompensatorische Wirkung der NO-GC im jeweils anderen Zelltyp hin. Zur Analyse der propulsiven Kontraktionen entlang des gesamten Colons wurden Videoaufnahmen der Darmbewegungen in Kontraktionsmusterkarten transformiert. Zudem wurde der Darm durchspült und die Ausflusstropfen aufgezeichnet, um die Effektivität der Kontraktionen beurteilen zu können. Hierbei zeigte sich, dass eine Beeinträchtigung des NO/cGMP-Signalweges eine verminderte Effektivität der Kontraktionen zur Folge hat und vermutlich durch eine beeinträchtige Synchronisation der Kontraktionen erklärt werden kann. In diesem Regulationsmechanismus konnte vor allem der NO-GC in SMC eine übergeordnete Rolle zugewiesen werden. Der dritte Teil der Arbeit thematisierte den Befund, dass SMC-GCKO-Tiere ca. 5 Monate nach Tamoxifen-Behandlung Entartungen der Mukosa entwickelten. Diese Entartung war lediglich in Tamoxifen-induzierten Knockout-Tieren vorzufinden. Histologische Analysen identifizierten die Entartungen als tubulovillöses Adenom. Die Genexpressionsanalyse von Mukosafalten von SMC-GCKO- und heterozygoten Kontrolltieren zeigte eine Vielzahl von Genen, welche spezifisch bei colorectalem Karzinom differenziell exprimiert sind. Einer dieser Faktoren war der BMP-Antagonist Gremlin1. Dieser Faktor erschien von besonderem Interesse, da er in Zellen der Lamina muscularis mucosae und kryptennahen Myofibroblasten exprimiert wird. Immunhistochemische Analysen ließen vermuten, dass diese Zellen sowohl die NO-GC als auch die Cre-Rekombinase unter dem SMMHC-Promotor exprimieren. Diese Arbeit liefert demnach Hinweise darauf, dass die NO-GC einen wichtigen Regulator innerhalb der Stammzellnische bildet. Die Deletion der NO-GC führt vermutlich zu einer verstärkten Bildung bzw. Sekretion von Gremlin1, was die Homöostase der mukosalen Erneuerung stört und somit zur Entwicklung von Adenomen führt. / The NO/cGMP signalling cascade is involved in many physiological processes. NO sensitive Guanylyl-Cyclase (NO-GC) is a key enzyme of this cascade, which is activated by NO and leads to the generation of the second messenger cGMP. As NO GC is comprised of two subunits two different isoforms exist (α1β1 und α2β1). However, the detailed distribution of the two isoforms in the colon is not known. Therefore, in the first part of this thesis, immunohistochemical staining and in situ hybridization was performed. The results showed NO-GC expression in the smooth muscle layer as well as in the submucosa and the lamina propria. Whereas the α1β1 isoform is expressed ubiquitously, expression of the α2β1 isoform is concentrated on the myenteric plexus. In the smooth muscle layer, NO-GC expression has been shown specifically in smooth muscle cells (SMC), interstitial cells of Cajal (ICC) and fibroblast-like cells (FLC). The function has been mainly associated with modulation of gastrointestinal motility. To specify the role of NO-GC on colonic motility in each cell type, mice lacking NO-GC globally (GCKO) or specifically in SMC (SMC-GCKO), ICC (ICC-GCKO) or in both (SMC/ICC-GCKO) were used. First, organ bath studies were performed using small proximal colonrings of 2,5 mm size. These measurements showed three different contractions: small, high frequency ripples, intermediate contractions and large contractions. For each contraction type, NO dependence was analyzed: Ripples are regulated in a NO-independent manner. In contrast, intermediate and large contractions are modulated by NO via a mechanism involving both ICC and SMC: NO GC in SMC most likely modulates contractions by setting muscle tone, whereas NO GC in ICC interacts with the pacemaker activity. Moreover, contractions along the whole colon were analyzed using spatiotemporal maps. These measurements revealed NO-GC to be related to the efficiency of propulsive long distance contractions (LDC). Impaired NO-GC signalling (e.g. in SMC-GCKO, GCKO mice or in the presence of NOS inhibitor L-NAME) resulted in altered appearance of LDC. Some of these LDC did not produce outflow. Thus, our results indicate a prominent role of NO-GC in SMC to synchronize contractions along the colon to result in effective propulsion. The third part of the study focused on the potential involvement of NO-GC in adenoma development. This hypothesis was set up based on the observation that colon of SMC GCKO mice showed mucosal swellings five month after tamoxifen treatment. Histological analysis assumed the mucosal swellings as tubulovillous adenoma. Subsequent gene expression analysis of mucosal folds of SMC-GCKO and heterozygous control mice identified a variety of differentially expressed genes, among them the BMP antagonist Gremlin1. This factor is potentially influenced by NO-GC as it is expressed by cells of the lamina muscularis mucosae and myofibroblasts closely located to the colonic crypts. Immunohistochemical analysis of tdTomato reporter mice assumed the same cells to express NO-GC under control conditions and active Cre recombinase in SMC-GCKO colon. Thus, this work hypothesizes NO-GC to be involved in the regulation of mucosal renewal. Deletion of NO-GC likely increases Gremlin1 generation and/or secretion, leading to altered mucosal renewal and finally resulting in adenoma development.

Univ., Diss., 2010--Würzburg.

Techn. Universiẗat, Diss., 2006--München.