Ce projet de recherche se focalise sur les infections par mycobacterium ulcerans (maladie de l’ulcère de Buruli), une maladie de la peau dévastatrice caractérisée par la formation de lésions nécrotiques progressives et l’absence d’une réponse inflammatoire. Bien que négligée, cette infection est la troisième maladie mycobactérienne la plus répandue après la tuberculose et la lèpre et des cas sont rapportés dans plus de 30 pays à travers le monde. Mycobacterium ulcerans sécrète une toxine polycétidique complexe, appelée mycolactone A/B, qui est directement responsable des effets pathogènes de la maladie. Depuis sa découverte, les propriétés biologiques inhabituelles de la mycolactone A/B ont suscité de nombreux efforts de recherche dans différents domaines. Dans ce contexte, ce projet s’intéresse à l’élucidation du mécanisme d’action des mycolactones en utilisant la synthèse totale comme outil principal. Dans cette optique, notre équipe a mis en place une voie de synthèse permettant un accès facile et robuste à différents mimes de mycolactone. L’utilisation de cette méthode a conduit à la préparation de 13 mimes de la toxine au cours de cette thèse. D’autre part notre équipe s’intéresse également à la préparation de mimes possédant un ou plusieurs atomes de fluor. Ces derniers présentent un intérêt particulier pour améliorer la compréhension des interactions ayant lieu entre la toxine et sa cible cellulaire. Les travaux réalisés autours de la synthèse de mycolactones fluorés ont conduit à la mise au point d’une méthode générale et simple pour introduire un groupe trifluorométhyle sur un alcyne terminal, permettant ainsi des modulations inédites de la structure de la toxine. / This research project focuses on mycobacterium ulcerans infection (Buruli ulcer disease), a severe skin disease characterized by the formation of progressive necrotic lesions and the lack of an acute inflammatory response. Although neglected, this infection is the third most common mycobacteriosis after Mycobacterium tuberculosis and Mycobacterium leprae, and cases are reported in more than 30 countries worldwide. Mycobacterium ulcerans secretes a complex polyketidic macrolide, called mycolactone A/B, which is directly involved in the biological effects of the disease. Since its discovery, the unusual biology triggered by this toxin has spurred research efforts. In this context, this research project aims at a better understanding of mycolactone A/B molecular interactions by using total synthesis as main tool. To this end, our research team has developed an efficient synthetic pathway allowing the preparation of different mimetics of the toxin. This synthesis has been used to prepare thirteen new mycolactone mimetics during this thesis. Moreover our team has also been interested in the synthesis of fluorinated mycolactone analogs. Such fluorinated mycolactones are of great interest to improve the interactions that occur between the toxin and its biological binding site. Work in this field led to the development of a simple and general method to introduce a trifluoromethyl group onto a terminal alkyne, allowing novel modulation of the structure of the toxin.

High quality ammonium oxofluorotitanate discoid crystal is successfully grown on glass with an aqueous solution of ammonium hexafluorotitanate and boric acid at the molar ratio of 0.6. The concentration of hydrofluoric acid is less on the glass substrate surface and enhances the ammonium oxofluorotitanate nucleation growth. The growth rate is much higher than that grown on dioctadecyldimethylammonium. From the examinations of X-ray diffraction and high-resolution transmission electron microscopy, the crystal shows high crystalline quality and uniformity. Each titanium oxide octahedral is linked with fluorine and nitrogen atoms. Therefore, ammonium oxofluorotitanate has high potential to be thermally decomposed into high crystalline fluorine and nitrogen co-doped titanium oxide. A simple process for the preparation of nanocrystalline anatase phase titanium oxide converted from ammonium oxofluorotitanate by thermal treatment was developed. The nanocrystalline anatase phase titanium oxide shows a large bandgap reduction due to the co-doping of high concentrations of fluorine and nitrogen. Due to the excellent nanocrystalline quality and the co-doping of higher concentrations of fluorine and nitrogen at the thermal treatment temperature of 800 OC, it is 1.3 times the photocatalytic activities of P-25 due to the visble region usage of Hg lamp light source. The 11.2 times the visible photocatalytic activities of P-25 using blue light-emitting diode as the light source is obtained from thermal treatment temperature of 600 OC. There is one to one correspondence between carrier lifetime and photocatalytic activity. As a result, a highly reactive and visible-light-driven photocatalysis is achieved. The heterostructure of zinc selenide/titanium oxide and zinc sulfide/titanium oxide were prepared by metal-organic chemical vapor deposition on the above-prepared titanium oxide. The energy bandgap of zinc sulfide is much larger than that of titanium oxide and can act as a window for titanium oxide. It would not hinder titanium oxide absorption and preserve the role of fluorine and nitrogen co-doping. The energy bandgap of zinc selenide is near the maximum intensity of solar spectrum and acts as a sensitizer of titanium oxide. The lifetime of electron and hole pairs of heterostructure are about 240 and 207 nsec, which are longer than 65 nsec of titanium oxide prepared at 800 oC thermal treatment. Their photocatalytic activities are further improved to 2.0 and 1.5 times higher than that of commercial P-25. The photocatalysis of titanium oxide is very sensitive to the surface states. Titanium oxide surface defects can act as trapping sites for photo-induced holes and facilitate the separation of photo-induced carriers. Zinc selenide and zinc sulfide can passivate the surface well. It may say that titanium oxide surface defects removal has a negative impact. The density, height, diameter, PL wavelength and intensity of zinc selenide self-assembled quantum dots grown on zinc sulfide/gallium arsenide with the zinc sulfide thickness from 15 to 160 nm are studied. For a fixed 30 sec zinc selenide self-assembled quantum dots growth, it cannot be formed with the zinc sulfide thickness below 15 nm due to the close lattice match between zinc sulfide and gallium arsenide. The zinc sulfide/gallium arsenide is fully lattice relaxed with the zinc sulfide thickness higher than 130 nm examined by X-ray diffraction. The higher quality and density of zinc selenide self-assembled quantum dots can be obtained on zinc sulfide/gallium arsenide with the zinc sulfide thickness far beyond its critical thickness. The maximum zinc selenide self-assembled quantum dots density of 4.9 x 109 cm-2 with the strongest photoluminescence intensity is obtained at the zinc sulfide/gallium arsenide thickness of 130 nm. Clusters are formed on the surface of zinc selenide/gallium arsenide. The selenium segregation is the main mechanism for the formation of clusters. The dislocations will enhance the selenium segregation. Higher zinc selenide cluster corresponds to higher density of dislocations. The non-spherical cluster is formed from the mergence of the two clusters. High quality zinc oxide rods and zinc hydroxide slices are successfully grown on gallium arsenide with the aqueous solution of zinc nitrate and hexamethylenetetramine. The growth can be controlled by the appropriate nitric acid concentration incorporation in the solution. After thermal annealing, the zinc oxide slices transformed from zinc hydroxide slices can contribute much higher photocatalytic activity to 1.2 times to P-25.

Krebserkrankungen stellen in Deutschland die zweithäufigste Todesursache dar und die Anzahl der Neuerkrankungen nimmt stetig zu. Frühzeitige Diagnosen und Therapiemöglichkeiten sind daher dringend erforderlich. Cyklinabhängige Proteinkinasen (Cdk) spielen eine entscheidende Rolle bei der Regulation des Zellzyklus. Viele Tumore zeigen eine deregulierte Cdk4‑Aktivität und/oder ‑Expression. Insgesamt zeigen ca. 80% aller Tumore eine Fehlregulation der für den Zellzyklus zentralen Cdk4/CykD1/INK4/pRb/E2F Signalkaskade. Somit besitzen Cdks ein enormes therapeutisches Potential im Kampf gegen Krebs. Die spezifische Inhibierung der Cdks verhindert die Zellproliferation und damit das Tumorwachstum. In den letzten Jahren wurden verschiedenste Strukturklassen vorgestellt, die als Cdk4-Inhibitor wirken. Im Rahmen der Promotion sollen die Möglichkeiten einer funktionellen Tumordiagnose mittels cyklinabhängiger Kinasen untersucht werden. Die Entwicklung von radioaktiv markierten Inhibitoren der Cdk4/6 als Radiotracer und ihre radiopharmakologische Charakterisierung stellt dabei einen neuen Ansatz dar. Um die Rolle der Cdk4/6 im Zellzyklus von gesunden und deregulierten (z.B. Tumor-) Zellen aufzuklären, sollten mit Iod-124 und Fluor-18 markierte Inhibitoren eingesetzt werden, die hochselektiv diese Cdks blockieren. Zunächst wurden verschiedene Inhibitoren der Cdk4/6 und deren Vorstufen für die Radiomarkierung dargestellt. Die bereits aus den Vorarbeiten von VanderWel et al., 2005 und Toogood et al., 2001 bekannten Syntheserouten mussten dazu optimiert werden und für neue Verbindungen, wie die fluorethylierten Substanzen, wurden neue Reaktionswege gefunden. Die dargestellten Referenzverbindungen CKIA-E wurden anschließend mittels Durchflusszytometrie an den Zelllinien HT-29 und FaDu auf ihre inhibitorischen Wirkung untersucht. Die Untersuchungen der Verbindungen CKIA/B/E zeigte, dass ein Zellzyklusarrest unter Einwirkung der Inhibitoren erreichbar ist. Die weiteren Untersuchungen zur Radiomarkierbarkeit sowie die radiopharmakologische Evaluation sollten daher an den Verbindungen CKIA, CKIB und CKIE stattfinden. Die Darstellung der Verbindungen [124I]CKIA und [124I]CKIB erfolgte in zwei Schritten über die elektrophile Substitution durch regioselektive Destannylierung mit anschließender Entschützung der Seitenkette. Die Darstellung der fluorethylierten Verbindung erfolgte ebenfalls über eine Zweischrittsynthese beginnend mit der Synthese der prosthetischen Gruppe [18F]BFE aus der Tosylmarkierungsvorstufe. Die zur Markierung des sekundären Amins zur Auswahl stehenden prosthetischen Gruppen [18F]Fluorethyltosylat ([18F]FETos) und [18F]Bromfluorethan ([18F]BFE) wurden auf ihre Eignung untersucht, ebenso wie die Auswahl einer geeigneten Markierungsvorstufe für die Darstellung der prosthetischen Gruppe. Die optimierten Syntheserouten ermöglichten die Isolierung von ausreichenden Mengen an Produktaktivität für die radiopharmakologischen Untersuchungen. Es fanden, neben der Bestimmung der spezifischen Aktivität und der Lipophilie der Verbindungen, Zellaufnahmeuntersuchungen und Bestimmungen zur Stabilität der Verbindungen in vitro, ex vivo und in vivo statt. Die radioiodierten Verbindungen konnten des Weiteren zur Untersuchungen der Bioverteilung in normalen männlichen Wistar-Ratten eingesetzt werden. Für alle drei Verbindungen konnte eine sehr hohe in vitro-Stabilität festgestellt werden. Die Zellaufnahmeuntersuchungen zeigten vor allem für die Verbindungen [124I]CKIA und [124I]CKIB eine beträchtliche Zellaufnahme von über 1000% ID/mg Protein nach 2 h. Die Zellaufnahme der Verbindung CKIE ist geringer, sollte allerdings für eine in vivo-Anwendung ausreichend sein. Die Untersuchung der in vivo‑Stabilität der Verbindungen [124I]CKIA, [124I]CKIB und [18F]CKIE im Blut von Wistar Ratten ergab allerdings, dass alle Verbindungen schnell metabolisiert werden. Die Untersuchung der Bioverteilung der radioiodierten Verbindungen belegen eine in vivo Radiodeiodierung sowie eine hohe hepatobliliäre Auscheidungsrate. Im Hinblick auf eine Anwendung als Radiotracer konnten im Rahmen dieser Arbeit neue Erkenntnisse gewonnen werden. Die dargestellten Inhibitoren sind in der Lage am Zellmodell den Zellzyklusarrest in der G1-Phase zu induzieren. Eine Radiomarkierung der ausgewählten Strukturen liefert das Produkt mit reproduzierbarer Ausbeute in hoher radiochemischer Reinheit und ausreichender spezifischer Aktivität, allerdings ist eine Herstellung der fluorethylierten Verbindung unter GMP-Bedingungen nur schwer realisierbar. Die radiomarkierten Verbindungen zeigen eine hohe in vitro-Stabilität und werden energieabhängig in die Zelle aufgenommen. Anhand der Stabilitätsuntersuchungen in vivo wurde gezeigt, dass alle drei Verbindungen in vivo instabil sind und sehr schnell hepatobiliär eliminiert.

Die Cyclin-abhängigen Kinasen 4 und 6 (Cdk4/6) wurden als essentielle Enzyme für die Regulation des Zellzyklus mit kritischem Beitrag zur gestörten Zellproliferation während der Kanzerogenese identifiziert. Als Konsequenz davon erwiesen sich die Cdk4/6 als attraktive Zielproteine für die Entwicklung neuer therapeutischer Konzepte zur pharmakologischen Tumorbehandlung. Verbindungen aus der Substanzklasse der Pyrido[2,3-d]pyrimidine zeigten vielversprechende inhibitorische Wirkungen auf die Aktivität der Cdk4/6 bei gleichzeitiger herausragender Selektivität gegenüber anderen Cdk. Anschließende Untersuchungen in vitro und in vivo verdeutlichten das Potential einiger Pyrido[2,3 d]pyrimidine zur Inhibierung des Tumorzellwachstums. Die Weiterentwicklung und Nutzung selektiver Cdk4/6-Inhibitoren zur funktionellen Charakterisierung der Cdk4/6 in Tumoren in vivo mit Hilfe der nicht-invasiven Bildgebungstechnik Positronen-Emissions-Tomographie (PET) ist ein neuer vielversprechender Forschungsansatz und von großem Interesse für die Evaluierung neuer Strategien zur Diagnose und Therapie maligner Erkrankungen. Das Ziel der vorliegenden Arbeit ist die biochemische und radiopharmakologische Charakterisierung neuer potentieller Cdk4/6-Inhibitoren aus der Verbindungsklasse der Pyrido[2,3-d]pyrimidine und deren Bewertung hinsichtlich ihrer therapeutischen Wirksamkeit zur gezielten Cdk4/6-Inhibierung in ausgewählten Tumorzelllinien sowie ihres Potentials zur funktionellen Bildgebung der Cdk4/6 in Tumoren mittels PET am Tiermodell. Die biochemische Charakterisierung der Pyrido[2,3 d]pyrimidine CKIA, CKIB, CKIC, CKID und CKIE hinsichtlich ihrer zellulären und molekularen Wirkung erfolgte in den kontinuierlich proliferierenden humanen Tumorzelllinien HT-29, FaDu und THP 1 und in differenzierten THP-1-Makrophagen. Die Zweckmäßigkeit der untersuchten Zelllinien zur Charakterisierung potentieller Cdk4/6-Inhibitoren wurde anhand von Studien zur mRNA-Expression und Proteinbiosynthese der Kinasen Cdk4/6 nachgewiesen. Des Weiteren wurde das Vorkommen der Cdk4/6 in den humanen Xenograft-Tumoren HT-29 und FaDu, sowie in ausgewählten Organen und Geweben von nu/nu-NMRI-Mäusen charakterisiert. In vitro wurden für alle untersuchten Pyrido[2,3-d]pyrimidine signifikante, konzentrations- und zeitabhängige inhibitorische Effekte auf die Tumorzellproliferation beobachtet. Durchflusszytometrische Zellzyklusanalysen 24 Stunden nach Inkubation mit den Pyrido[2,3 d]pyrimidinen zeigten eine konzentrationsabhängige Zunahme des Anteils der HT-29-, FaDu- und THP-1-Zellen in der G1-Phase bis auf 90%. Für die nicht-proliferierenden THP 1-Makrophagen wurden bei Inkubation mit den Pyrido[2,3 d]pyrimidinen geringe Veränderungen ihrer Zellzahl und Zellzyklusphasen-verteilung detektiert. Die zellulären Studien identifizierten deutliche qualitative und quantitative Unterschiede der untersuchten Pyrido[2,3 d]pyrimidin-Derivate. Nanomolare Konzentrationen von CKIA, CKIB bzw. CKIE erzielten bereits 24 Stunden nach Inkubation deutliche Effekte, während für CKIC und CKID die 10- bis 100-fache Konzentration eingesetzt werden musste, um eine ähnliche Wirkung zu erhalten. Die molekularen Ursachen der Wachstumshemmung und des Zellzyklusarrests wurden durch Untersuchungen zur Pyrido[2,3 d]pyrimidin-abhängigen Beeinflussung des Cdk4/6-Cyclin D-Retinoblastom-E2F-Signalwegs geklärt. In allen Zelllinien wurde eine deutliche Inhibierung der Cdk4/6-spezifischen Phosphorylierung der Aminosäure Serin-780 des Retinoblastom-Proteins (pRb) beobachtet. Als Konsequenz dieser Inhibierung wurde für CKIA, CKIB und CKIE die signifikante konzentrationsabhängige Unterbrechung der Genexpression von E2F-1 und PCNA nachgewiesen. Die Radiomarkierung der Cdk4/6-Inhibitoren CKIA und CKIB mit 124I bzw. von CKIE mit 18F ermöglichte erstmals die Charakterisierung von in vivo-Interaktionen und des Metabolismus im Blut zirkulierender Pyrido[2,3 d]pyrimidin-Derivate. Die radiopharmako-logischen Eigenschaften von [124I]CKIA, [124I]CKIB und [18F]CKIE wurden in Untersuchungen zur zellulären Radiotracer-Aufnahme, der metabolischen Stabilität und der Bioverteilung bei Ratten sowie abschließend in Kleintier-PET-Untersuchungen bei FaDu-Tumor-tragenden nu/nu-NMRI-Mäusen analysiert. In vitro-Experimente mit [124I]CKIA, [124I]CKIB und [18F]CKIE verdeutlichten eine hohe Stabilität und schnelle Aufnahme der Radiotracer in humane Tumorzellen bei 37°C. Allerdings deutet die Zelltyp-unabhängige Anreicherung auf eine geringe Abhängigkeit der Pyrido[2,3 d]pyrimidin-Akkumulation vom Cdk4/6-Status der Zellen hin. Die signifikant geringeren Aufnahmewerte aller untersuchten Pyrido[2,3-d]pyrimidine bei 4°C und die Blockierung der Aufnahme von [18F]CKIE mit nichtradioaktivem CKIE unterstützen die Vermutung spezifischer Transportmechanismen für die Aufnahme der Pyrido[2,3 d]pyrimidine. Untersuchungen von [124I]CKIA, [124I]CKIB und [18F]CKIE in vivo identifizierten eine schnelle, innerhalb weniger Minuten stattfindende Eliminierung aus dem Blut und die primäre Aufnahme in die Leber als grundlegende Stoffwechseleigenschaft aller drei Radiotracer. Aus den PET-Untersuchungen mit [124I]CKIA und [124I]CKIB bei FaDu-Tumor-tragenden Mäusen wurden nur marginale Anreicherungen der radioaktiven Substanzen im Bereich des Tumors festgestellt. Für [18F]CKIE wurde eine Akkumulation im proliferierenden Randbereich des Tumors beobachtet. Die schnelle Metabolisierung von [18F]CKIE im Blut sowie das konstante, geringe Verhältnis der Aktivität im Tumor zur Aktivität im Skelettmuskel wiesen allerdings auf eine unspezifische Anreicherung hin. Schlussfolgernd aus den Ergebnissen der vorliegenden Arbeit wurde die Effektivität der Pyrido[2,3 d]pyrimidine CKIA, CKIB und CKIE hinsichtlich der Inhibierung der Cdk4/6-vermittelten Zellzyklusprogression gezeigt. Die antiproliferative Aktivität der Substanzen unterstützt eine weiterführende Evaluierung dieser Cdk4/6-Inhibitoren zur pharmakologischen Tumortherapie. Auf der Basis der erhaltenen radiopharmakologischen Ergebnisse werden die kurze biologische Halbwertszeit und unspezifische Tumoranreicherung von [124I]CKIA, [124I]CKIB und [18F]CKIE als limitierende Faktoren für die Eignung dieser Verbindungen als Radiotracer zur nicht-invasiven Bildgebung der Cdk4/6 im Zielgewebe mittels PET angesehen. Es bleibt zu klären, ob eine längere Verweildauer und höhere Stabilität radiomarkierter Pyrido[2,3-d]pyrimidine im Blut die Chance der Cdk4/6-spezifischen Gewebeanreicherung erhöhen oder Transport-mechanistische Effekte allein ausschlaggebend für die Anreicherung in Zellen und Geweben sind. Die Untersuchung optimierter Cdk4/6-selektiver Inhibitoren für die Charakterisierung und Therapie von Tumoren bleibt weiterhin ein interessanter Aspekt in der Tumorforschung.

The continuous microreactor-assisted solution deposition (MASD) process was used for the deposition of CdS thin films on fluorine-doped tin oxide (FTO) glass. The MASD system, including a T-junction micromixer and a microchannel heat exchanger is capable of isolating the homogeneous particle precipitation from the heterogeneous surface reaction. The results show a dense nanocrystallite CdS thin films with a preferred orientation at (111) plane. Focused-ion-beam was used for TEM specimen preparation to characterize the interfacial microstructure of CdS and FTO layers. The band gap of the microreactor-assisted deposited CdS film was determined at 2.44 eV. X-ray Photon Spectroscopy show the bindings of energies of Cd 3d₃/₂, Cd 3d₅/₂, S 2p₃/₂ and S 2p₁/₂ at 411.7 eV, 404.8 eV, 162.1 eV, and 163.4 eV, respectively. The film growth kinetics was studied by measuring the film thickness deposited from 1 minute to 15 minutes in physical (FIB-TEM) and optical (reflectance spectroscopy) approaches. A growth model that accounts for the residence time in the microchannel using empirical factor (η) obtained from previous reported experimental data. Applying this factor in the proposed modified growth model gives a surface reaction rate of 1.61*10⁶ cm⁴ mole⁻¹s⁻¹, which is considerable higher than the surface reaction rates obtained from the batch CBD process. With the feature of separating homogeneous and heterogeneous surface reaction, the MASD process provides the capability to tailor the surface film growth rate and avoid the saturation growth regime in the batch process. An in situ spectroscopy technique was used to measure the UV-Vis absorption spectra of CdS nanoparticles formed within the continuous flow microreactor. The spectra were analyzed by fitting the sum of three Gaussian functions and one exponential function in order to calculate the nanoparticle size. This deconvolution analysis shows the formation of CdS nanoparticles range from 1.13 nm to 1.26 nm using a residence time from 0.26 s to 3.96 s. Barrier controlled coalescence mechanism seems to be a reasonable model to explain the experimental UV-Vis data obtained from the continuous flow microreactor, with a rate constant k' value of 2.872 s⁻¹. Using CFD, low skewness value of the RTD curve at high flow rate (short τ) suggests good radial mixing at high flow rate is responsible for the formation of smaller CdS nanoparticles with a narrower size distribution. The combination of CdS nanoparticle solution with MASD process resulted in the hindrance of CdS thin film deposition. It is hypothesized that the pre-existing sulfide (S²⁻) ions and CdS nanoparticles changes the chemical species equilibrium of thiourea hydrolysis reaction. Consequently, the lack of thiourea slows down the heterogeneous surface reaction. To test the scalability of the MASD process, a flow cell and reel-to-reel (R2R)-MASD system were setup and demonstrated for the deposition of CdS films on the FTO glass (6" x 6") substrate. The film deposition kinetics was found to be sensitive to the flow conditions within the heat exchanger and the substrate flow cell. The growth kinetics of the CdS films deposited by R2R-MASD process was investigated by with a deposition time of 2.5 min, 6.3 min, and 9 min. In comparison with the continuous MASD process, the growth rate in R2R-MASD is higher, however more difficult to obtain a linear relationship with the deposition time. / Graduation date: 2012 / Access restricted to the OSU Community at author's request from Jan. 13, 2012 - Jan. 13, 2013

Made available in DSpace on 2014-06-11T19:33:00Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-07-28Bitstream added on 2014-06-13T19:43:56Z : No. of bitstreams: 1 casotti_ca_dr_araca.pdf: 660013 bytes, checksum: c630d7bb4fdb85d40a30ec52f8e05ca2 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A fluoretação da água de abastecimento público é reconhecida como uma das principais estratégias de saúde pública para prevenir a cárie dentária. A cidade de Baixo Guandu, localizada no Estado do Espírito Santo, Brasil, foi a primeira a implantar o método. Foi objetivo do presente estudo avaliar o impacto que a fluoretação artificial da água proporcionou à saúde bucal de indivíduos com idade de 5, 12, 15 a 19, 20 a 34, 35 a 44 e 45 a 56 anos, que sempre viveram e consumiram exclusivamente água da rede de abastecimento público dos municípios de Baixo Guandu onde a fluoretação ocorre há 52 anos e Itarana, onde o método foi implantado há 3 anos. A metodologia, os códigos e os critérios adotados foram os recomendados pela Organização Mundial da Saúde (OMS), com a inserção dos grupos etários de 20 a 34 e 45 a 56 anos. Inicialmente foram identificados os moradores permanentes das duas cidades, as quais foram examinados no domicílio, com auxílio de espelho bucal e sonda periodontal CPI. Previamente à realização do estudo, o examinador passou por uma oficina de calibração e treinamento para a padronização dos diagnósticos da cárie dentária, fluorose dentária, perda dentária e uso e necessidade de prótese, em indivíduos com grupos etários semelhantes aos do estudo. Para o cálculo da concordância kappa intra-examinador, 10% dos indivíduos foram reexaminados, sendo os resultados obtidos para a cárie dentária 0,95; fluorose dentária 0,91; perda dentária 1 e o uso e necessidade de prótese 1. No total foram examinados 1588 moradores sendo 1129 (71,1%) de Baixo Guandu e 459 (28,9%) de Itarana. Foram utilizados os testes estatísticos Qui-quadrado, Fisher, Mann Whitney e de proporções com nível de significância de 5%. As médias do ceod aos 5 anos e CPOD aos 12, 15 a 19, 20 a 34, 35 a 44 e 45 a 56 anos foram, respectivamente, em Baixo Guandu... / The water fluoridation in the public supply is recognized as one of the main public health strategies to prevent dental caries. Baixo Guandu-ES was the first city to introduce this method in Brazil. The objective of the present study was to evaluate the impact that the artificial fluoridation of the water brought to the oral health of subjects in the age of 5, 12, 15 to 19, 20 to 34,35 to 44 and 45 to 56 years, that have always lived and consumed exclusively the water from the public supply of the city of Baixo Guandu for 52 years and Itarana where the method was introduced 3 years ago. The methodology, the codes and criteria adopted were recommended by the World Health Organization (WHO), with the insertion of age groups of 20 to 34 and 45 to 56 years. Initially the identification of the permanent inhabitants of both cities, was realized and they were locally examined with the aid of plan oral mirrors number 5 and CPI periodontal probes. Previously to this study, the examiner went through a calibrator's workshop and training for the diagnosis of dental caries, dental fluorosis, tooth loss, the use and need of prosthesis with age groups similar to the ones in the study. In order to have the Kappa's intra-examiner calculated, 10% of the individuals were re-examined and the obtained result for dental caries was 0.95; for dental fluorosis 0.91; for dental loss, and for the use and need for prosthesis 1. A total of 1588 inhabitants were examined, of those 1129 (71.1%) of Baixo Guandu and 459 (29.9%) of Itarana. The Chi-square, Fisher's, Mann-Whitney's and the Proportions tests with level of significance of 5% were used to verify the significant statistic of the results. The mean dmft for 5 year old subjects and DMFT for 12, 15 to 19, 20 to 34, 35 to 44 and 45 to 56 subjects were respectively in Baixo Guandu 2.32; 1,55; 3.48; 7.69; 13,85 and 16.49 and ...(Complete abstract, click electronic address below)

Orientador: Nemre Adas Saliba / Banca: Edgard Michel Crosato / Banca: Beatriz Unfer / Banca: Rogério Moreira Arcieri / Banca: Renato Herman Sundfeld / Resumo: A fluoretação da água de abastecimento público é reconhecida como uma das principais estratégias de saúde pública para prevenir a cárie dentária. A cidade de Baixo Guandu, localizada no Estado do Espírito Santo, Brasil, foi a primeira a implantar o método. Foi objetivo do presente estudo avaliar o impacto que a fluoretação artificial da água proporcionou à saúde bucal de indivíduos com idade de 5, 12, 15 a 19, 20 a 34, 35 a 44 e 45 a 56 anos, que sempre viveram e consumiram exclusivamente água da rede de abastecimento público dos municípios de Baixo Guandu onde a fluoretação ocorre há 52 anos e Itarana, onde o método foi implantado há 3 anos. A metodologia, os códigos e os critérios adotados foram os recomendados pela Organização Mundial da Saúde (OMS), com a inserção dos grupos etários de 20 a 34 e 45 a 56 anos. Inicialmente foram identificados os moradores permanentes das duas cidades, as quais foram examinados no domicílio, com auxílio de espelho bucal e sonda periodontal CPI. Previamente à realização do estudo, o examinador passou por uma oficina de calibração e treinamento para a padronização dos diagnósticos da cárie dentária, fluorose dentária, perda dentária e uso e necessidade de prótese, em indivíduos com grupos etários semelhantes aos do estudo. Para o cálculo da concordância kappa intra-examinador, 10% dos indivíduos foram reexaminados, sendo os resultados obtidos para a cárie dentária 0,95; fluorose dentária 0,91; perda dentária 1 e o uso e necessidade de prótese 1. No total foram examinados 1588 moradores sendo 1129 (71,1%) de Baixo Guandu e 459 (28,9%) de Itarana. Foram utilizados os testes estatísticos Qui-quadrado, Fisher, Mann Whitney e de proporções com nível de significância de 5%. As médias do ceod aos 5 anos e CPOD aos 12, 15 a 19, 20 a 34, 35 a 44 e 45 a 56 anos foram, respectivamente, em Baixo Guandu ...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The water fluoridation in the public supply is recognized as one of the main public health strategies to prevent dental caries. Baixo Guandu-ES was the first city to introduce this method in Brazil. The objective of the present study was to evaluate the impact that the artificial fluoridation of the water brought to the oral health of subjects in the age of 5, 12, 15 to 19, 20 to 34,35 to 44 and 45 to 56 years, that have always lived and consumed exclusively the water from the public supply of the city of Baixo Guandu for 52 years and Itarana where the method was introduced 3 years ago. The methodology, the codes and criteria adopted were recommended by the World Health Organization (WHO), with the insertion of age groups of 20 to 34 and 45 to 56 years. Initially the identification of the permanent inhabitants of both cities, was realized and they were locally examined with the aid of plan oral mirrors number 5 and CPI periodontal probes. Previously to this study, the examiner went through a calibrator's workshop and training for the diagnosis of dental caries, dental fluorosis, tooth loss, the use and need of prosthesis with age groups similar to the ones in the study. In order to have the Kappa's intra-examiner calculated, 10% of the individuals were re-examined and the obtained result for dental caries was 0.95; for dental fluorosis 0.91; for dental loss, and for the use and need for prosthesis 1. A total of 1588 inhabitants were examined, of those 1129 (71.1%) of Baixo Guandu and 459 (29.9%) of Itarana. The Chi-square, Fisher's, Mann-Whitney's and the Proportions tests with level of significance of 5% were used to verify the significant statistic of the results. The mean dmft for 5 year old subjects and DMFT for 12, 15 to 19, 20 to 34, 35 to 44 and 45 to 56 subjects were respectively in Baixo Guandu 2.32; 1,55; 3.48; 7.69; 13,85 and 16.49 and ...(Complete abstract, click electronic address below) / Doutor

Made available in DSpace on 2014-10-09T12:30:48Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:00:26Z (GMT). No. of bitstreams: 1 01387.pdf: 2053886 bytes, checksum: 9c61b3e10e5af0bb9a9a9cb8707260c7 (MD5) / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP

The research work reported in this thesis is focused on the chiral analysis, quantification of enantiomeric composition, assignment of absolute configuration of molecules with chosen functional groups. The weak intra-molecular hydrogen bonding interactions are detected by exploiting several multinuclear and multi-dimensional techniques. Pulse sequences have been designed to manipulate the spin dynamics to derive specific information from the complex NMR spectra encountered in diverse situations. Broadly, the thesis can be classified in to three sections. The section I containing two chapters reports the introduction of new chiral auxiliaries and protocols developed for enantiomeric discrimination, measurement of enantiomeric contents, assignment of absolute configuration for molecules possessing specific functional groups using chiral solvating and derivatizing agents. The section II, reports NMR experimental evidence for the observation of the rare type of intramolecular hydrogen bonds involving organic fluorine in biologically important organic molecules, that are corroborated by extensive DFT based theoretical calculations. The section II also discusses the H/D exchange mechanism as a tool for quantification of HB strengths in organic building blocks. The section III reports the two different novel NMR methodologies designed for deriving information on the scalar interaction strengths in an orchestrated manner. The designed sequences are able to completely eradicate the axial peaks, prevents the evolution of unwanted couplings and also yields ultrahigh resolution in the direct dimension, permitting the accurate measurement of scalar couplings for a particular spin. The brief summary about each chapter is given below. Chapter 1 provides a general introduction to one and two dimensional NMR spectroscopy. The pedagogical approach has been followed to discuss the conceptual understanding of spin physics and the NMR spectral parameters. The basic introduction to chirality, existing approaches in the literature for discrimination of enantiomers and the assignment of absolute configuration of molecules with chosen functional groups and their limitations are briefly discussed. The brief introduction to hydrogen bond, experimental methods to obtain the qualitative information about the strengths of hydrogen bonds, and the theoretical approaches employed in the thesis to corroborate the NMR experimental findings have been provided. The mechanism of H/D exchange, the utilization of exchange rates to derive strengths of intra-molecular hydrogen bond in small molecules have also been discussed. This chapter builds the bridge for the rest of the chapters. Each of these topics are discussed at length in the corresponding chapters. Part I: NMR Chiral Analysis: Novel Protocols Chapter 2 discusses a simple mix and shake method for testing the enantiopurity of primary, secondary and tertiary chiral amines and their derivatives, amino alcohols. The protocol involves the in-situ formation of chiral ammonium borate salt from a mixture of C2 symmetric chiral BINOL, trialkoxyborane and chiral amines. The proposed concept has been convincingly demonstrated for the visualization of enantiomers of a large number of chiral and pro-chiral amines and amino alcohols. The protocol also permits the precise measurement of enantiomeric composition. The significant advantage of the protocol is that it can be performed directly in the NMR tube, without any physical purification. The structure of the borate complex responsible for the enantiodifferentiation of amines has also been established by employing multinuclear NMR techniques and DFT calculations. From DOSY and 11B NMR experiments it has been ascertained that there are only two possible complexes or entities which are responsible for differentiating enantiomers. From the combined utility of DFT calculations and the 11B NMR chemical shifts, the structure of the borate complex has been determined to be an amine-coordinated complex with the N atom of the amine. Chapter 3 discusses a simple chiral derivatizing protocol involving the coupling of 2-formylphenylboronic acid and an optically pure [1,1-binaphthalene]-2,2-diamine for the rapid and accurate determination of the enantiopurity of hydroxy acids and their derivatives, possessing one or two optically active centres. It is established that this protocol is not only rapid method for discrimination of enantiomers but also highly effective for assigning the absolute configuration of various chiral hydroxy acids and their derivatives. The developed protocol involves the coupling of 2-formylphenylboronic acid with (R)-[1,1-binaphthalene]-2,2-diamine, and 2-formylphenylboronic acid with (S)-[1,1-binaphthalene]-2,2-diamine as chiral derivatizing agents. The absence of aliphatic peaks from the derivatizing agent, large chemical shift separation between the discriminated peaks of diastereomers, and the systematic change in the direction of displacement of peaks for an enantiomer in a particular diastereomeric complex, permitted the unambiguous assignment of absolute configuration. Part II : Rare Type of Intramolecular Hydrogen Bonding In chapter 4 The rare occurrence of intramolecular hydrogen bonds of the type N–H˖˖˖F–C, in the derivatives of imides and hydrazides in a low polarity solvent, is convincingly established by employing multi-dimensional and multinuclear solution state NMR experiments. The observation of 1hJFH, 2hJFN, and 2hJFF of significant strengths, where the spin polarization is transmitted through space among the interacting NMR active nuclei, provided strong and conclusive evidence for the existence of intra-molecular hydrogen bonds. Solvent induced perturbations and the variable temperature NMR experiments unambiguously supported the presence of intramolecular hydrogen bond. The two dimensional HOESY and 15N–1H HSQC experiments reveals the existence of multiple conformers in some of the investigated molecules. The 1H DOSY experimental results discarded any possibility of self or cross-dimerization of the molecules. The results of DFT based calculations, viz., Quantum Theory of Atoms In Molecules (QTAIM) and Non Covalent Interaction (NCI), are in close agreement with the NMR experimental findings. In chapter 5 the rates of hydrogen/deuterium (H/D) exchange determined by 1H NMR spectra have been utilized to derive the strength of hydrogen bonds and to monitor the electronic effects in the site-specific halogen substituted Benz amides and anilines. The theoretical fitting of the time dependent variation in the integral areas of 1H NMR resonances to the first order decay function permitted the determination of H/D exchange rate constants (k) and their precise half-lives (t1/2) with high degree of reproducibility. The comparative study also permitted the determination of relative strengths of hydrogen bonds and the contribution from electronic effects on the H/D exchange rates. Part III: Novel NMR Methodologies for the Precise Measurement of 1H-1H Couplings Chapter 6 describes two novel NMR methodologies developed for the precise measurement of 1H-1H couplings. Poor chemical shift dispersion and the pairwise interaction among the entire coupled network of protons results in the severely complex and overcrowded one dimensional 1H NMR spectra, hampering both the resonance assignments and the accurate determination of nJHH. The available two-dimensional selective refocusing (SERF) based experiments suffer from the evolution of magnetization from uncoupled protons as intense uninformative axial peaks. This creates ambiguity in the identification of peaks belonging to the coupled partners of a selectively excited proton, hindering the extraction of their interaction strengths. This challenge has been circumvented by designing two novel experimental technique, cited as “Clean-G-SERF” and “PS-Clean-G-SERF”. The Clean-G-SERF technique completely eradicates the axial peaks and suppresses the evolution of unwanted couplings while retaining only the couplings to the selectively excited proton. The method permits the accurate determination of spin-spin couplings even from a complex proton NMR spectrum in an orchestrated manner. The PS-Clean-G-SERF technique has been designed for the complete elimination of axial peaks and undesired couplings, with a blend of ultra-high resolution achieved by real time broad band mononuclear decoupling has been discussed in this chapter. The spin dynamics involved in both these pulse sequences have been discussed. The diverse applications of both these novel experiments have been demonstrated.

The thesis entitled “Organic fluorine in crystal engineering: Consequences on molecular and supramolecular organization” consists of six chapters. The main theme of the thesis is to address the role of substituted fluorine atoms in altering the geometrical and electronic features in organic molecules and its subsequent consequences on crystal packing. The thesis is divided into three parts. Part I deals with compounds that are liquids under ambient conditions, crystal structures of which have been determined by the technique of in situ cryocrystallography. Part II demonstrates the utilization of in situ cryocrystallography to study kinetically trapped metastable crystalline phases that provide information about crystallization pathways. In part III, crystal structures of a series of conformationally flexible molecules are studied to evaluate the consequences of fluorine substitution on the overall molecular conformation. The genesis and stabilization of a particular molecular conformation has been rationalized in terms of variability in intermolecular interactions in the crystalline state. Part I. In situ cryocrystallography: Probing the solid state structures of ambient condition liquids. Chapter 1 discusses the crystal structures of benzoyl chloride and its fluorinated analogs. These compounds have been analysed for the propensity of adoption of Cl···O halogen bonded dimers and catemers. The influence of conformational and electronic effects of sequential fluorination on the periphery of the phenyl ring has been quantified in terms of the most positive electrostatic potential, VS,max (corresponding to σ-hole) on the Cl-atom. It is shown that fluorine also exhibits “amphoteric” nature like other heavier halogens, particularly in presence of electron withdrawing groups. Although almost all the derivatives pack through C–H···O, C–H···F, C–H···Cl, Cl···F, C–H···π and π···π interactions, the compound 2,3,5,6-tetrafluorobenzoyl chloride exhibited a not so commonly observed Cl···O halogen bonded catemer. On the other hand, the proposed Cl···O mediated dimer is not observed in any of the structures due to geometrical constraints in the crystal lattice. Chapter 2 presents the preferences of fluorine to form hydrogen bond (C–H···F) and halogen bonds (X···F; X= Cl, Br, I). Crystal structures of all three isomers of chloro-, bromo-and iodo-fluorobenzene have been probed in order to gain insights into packing interactions preferred by fluorine and other heavier halogens. It has been observed that homo halogen…halogen (Cl···Cl, Br···Br and I···I) contacts prevail in most of the structures with fluorine being associated with the hydrogen atom forming C–H···F hydrogen bond. The competition between homo and hetero halogen bonds (I···I vs I···F) is evident from the packing polymorphism exhibited by 4-iodo fluorobenzene observed under different cooling protocols. The crystal structures of pentafluoro halo (Cl, Br, I) benzenes were also determined in order to explore the propensity of formation of homo halogen bonds over hetero halogen bonds. Different dimeric and catemeric motifs based on X···F and F···F interactions were observed in these structures. Chapter 3 focuses on the effect of different cooling protocols in generating newer polymorphs of a given liquid. The third polymorph (C2/c, Z'=6) of phenylacetylene was obtained by sudden quenching of the liquid filled in capillary from a hot water bath (363 K) to the nitrogen bath (< 77 K). Also, different polymorphs were obtained for both 2¬fluoro phenylacetylene (Pna21, Z'=1) and 3-fluoro phenylacetylene (P21/c, Z'=3) when crystallized by sudden quenching in contrast to the generally followed method of slow cooling which results in isostructural forms (P21, Z'=1). The rationale for these kinetically stable “arrested” crystalline configurations is provided in part II of the thesis. Part II. Tracing crystallization pathways via kinetically captured metastable forms. Chapter 4 explains the utilization of the new approach of sudden quenching of liquids (detailed in chapter 3) to obtain kinetically stable (metastable) crystalline phases that appear to be closer to the unstructured liquids. Six different examples namely, phenylacetylene, 2-fluorophenylacetylene, 3-fluorophenylacetylene, 4-fluorobenzoyl chloride, 3-chloro fluorobenzene and ethyl chloroformate are discussed in this context. In each case, different polymorphs were obtained when the liquid was cooled slowly (100 K/h) and when quenched sharply in liquid nitrogen. The relationship between these metastable forms and the stable forms (obtained by slow cooling) combined with the mechanistic details of growth of stable forms from metastable forms provides clues about the crystallization pathways. Part III. Conformational analysis in the solid state: Counterbalance of intermolecular interactions with molecular and crystallographic symmetries. Chapter 5 describes the crystal structures of a series of conformationally flexible molecules namely, acetylene and diacetylene spaced aryl biscarbonates and biscarbamates. While most of the molecules adopt commonly anticipated anti (transoid) conformation, some adopt unusual cisoid and gauche conformations. It is shown that the unusually twisted conformation of one of the compounds [but-2-yne-bis(2,3,4,5,6¬pentafluorocarbonate)] is stabilized mainly by the extraordinarily short C–H···F intermolecular hydrogen bond. The strength of this rather short C–H···F hydrogen bond has been authenticated by combined single crystal neutron diffraction and X-ray charge density analysis. It has also been shown that the equi-volume relationship of H-and F-atoms (H/F isosterism) can be explored to access various possible conformers of a diacetylene spaced aryl biscarbonate. While biscarbonates show variety of molecular conformations due to absence of robust intermolecular interactions, all the biscarbamates adopt anti conformation where the molecules are linked with antiparallel chains formed with N–H···O=C hydrogen bonds. Chapter 6 presents a unique example where the commonly encountered crystallographic terms namely, high Z' structure, polymorphism, phase transformation, disorder, isosterism and isostructuralism are witnessed in a single molecular species (parent compound benzoylcarvacryl thiourea and its fluorine substituted analogs). The origin of all these phenomenon has been attributed to the propensity of formation of a planar molecular dimeric chain mediated via N–H···O [R2 (12)] and N–H···S [R2 (8)] dimers.