O veneno de C. d. terrificus tem sido descrito como sendo de pouca complexidade, tendo 4 frações caracterizadas, convulxina, giroxina. crotoxina e crotamina. O presente trabalho visou o isolamento e caracterização da Delta toxina cuja existência havia sido aventada em trabalhos anteriores. Após a realização de uma varredura de tampões em uma coluna de exclusão molecular Superdex-75 acoplada a um sistema FPLC, na presença de três diferentes tampões, chegou-se a uma condição ideal de fracionamento do veneno crotálico. Em seqüência realizou-se a segunda etapa de purificação em sistema HPLC em uma coluna C4, onde foi possível identificar o pico de interesse. O pico puro passou por análises em MALDI-ToF sendo sua massa estimada em 14.074,92 Da, Quando analisado por eletroforese em gel de poiiacrilamida, a delta toxina apresentou massa molecular de cerca de 14 kDa e uma migração anômala, Por eletroforese 2D, a proteína apresentou caráter ácido, com pl entre 4 e 5 e massa molecular de aproximadamente 42 kDa, revelando \"spots\" muito semelhantes podendo ser isoformas com características de uma proteína glicosiiada. Após digestão dos spots com tripsina, os fragmentos foram confrontados com o banco de dados do \"swissprot\", mostrando alto grau de homologia \"até 43% de cobertura\" com a troca ri na, um ativador de protrombina do veneno de Tropidechis carinatus, esses dados foram confirmados com a análise de aminoácidos. De posse desses resultados, optou-se por testar a capacidade da fração purificada de ativar fator X e II, usando substratos sintéticos. Os resultados apontaram para uma ativação direta do fator X, uma vez que não houve ativação do fator II, atividade que também não foi detectada no veneno total. A mesma se mostrou um potente ativador da agregação de forma direta, uma vez que os ensaios de agregação plaquetária foram realizados com plaquetas lavadas, logo na ausência de fatores séricos. Quando os ensaios de agregação foram realizados na presença de alguns inibidores observou-se que nem a atividade metalo proteinase, nem a serino proteinase, tampouco um domínio lectina estavam envolvidos no processo, uma vez que EDTA, benzamidina e D-galactose não inibiram a atividade da proteína. No presente trabalho isolamos a Delta toxina do veneno de C. d. terrificus. A mesma se comportou como previsto por Vital Brazil em 1980, eluindo na posição por ele aventada, sendo uma proteína ativadora de Fator X que ativa agregação plaquetária mesmo em concentrações muito baixas e de massa molecular de 40 kDa levando nos a crer se tratar de um homotrímero cujos componentes são unidos por ligações fracas. / The Crotalus durissus terrificus venom has been so far described as being of low complexity, with four major components described: convulxin, gyroxin, crotoxin and crotamine. In recent studies, other components of this venom were characterized as, for example, an analgesic factor. In 1980, Vital Brazil predicted the existence of a toxin which could be involved in platelet aggregation, and named it delta toxin. However, this toxin has never been isolated or characterized. The aim of the present work was to purify and characterize this toxin. After FPLC size exclusion chromatography followed by reverse phase HPLC, an homogeneous fraction was obtained, with a molecular weight of 14,074.92 Da. When analyzed by SOS-PAGE, this toxin presented an anomalous behavior, with a molecular weight of 14 kDa, while in 2D gels, spots around 40 kDa and with an isoelectrical point between 4 and 5 were observed suggesting isoforms with glicosilation microheterogeneity. After trypsin digestion, the fragments were submitted to the swissprot databank showing high homology (43% coverage, 15 matching peptides) with trocarin, a prothrombin activator from Tropidechis carinatus. These data were further confirmed by aminoacid analysis. The toxin was tested for its ability to activate factor II and X using synthetic substrates. Our data indicate a direct activation of factor X. The same toxin also behaved as a potent direct platelet aggregation activator on washed platelets. Assays with specific inhibitors indicate that neither metalloproteinase, nor serinoproteinase or t lectin domains are involved in the aggregating activity, since EDTA, benzamidin and D-galactose did not inhibit the toxin. In the present work, we were able to identify, purify and characterize a new toxin from the brazilian rattlesnake. It behaved as predicted by Vital-Brazil and displayed direct factor X activating properties, also inducing platelet aggregation, even at low concentrations. Our data also indicate that it is probably a homotrimer with the subunities linked by hydrophobic and/or electrostatic interactions.

O veneno de C. d. terrificus tem sido descrito como sendo de pouca complexidade, tendo 4 frações caracterizadas, convulxina, giroxina. crotoxina e crotamina. O presente trabalho visou o isolamento e caracterização da Delta toxina cuja existência havia sido aventada em trabalhos anteriores. Após a realização de uma varredura de tampões em uma coluna de exclusão molecular Superdex-75 acoplada a um sistema FPLC, na presença de três diferentes tampões, chegou-se a uma condição ideal de fracionamento do veneno crotálico. Em seqüência realizou-se a segunda etapa de purificação em sistema HPLC em uma coluna C4, onde foi possível identificar o pico de interesse. O pico puro passou por análises em MALDI-ToF sendo sua massa estimada em 14.074,92 Da, Quando analisado por eletroforese em gel de poiiacrilamida, a delta toxina apresentou massa molecular de cerca de 14 kDa e uma migração anômala, Por eletroforese 2D, a proteína apresentou caráter ácido, com pl entre 4 e 5 e massa molecular de aproximadamente 42 kDa, revelando \"spots\" muito semelhantes podendo ser isoformas com características de uma proteína glicosiiada. Após digestão dos spots com tripsina, os fragmentos foram confrontados com o banco de dados do \"swissprot\", mostrando alto grau de homologia \"até 43% de cobertura\" com a troca ri na, um ativador de protrombina do veneno de Tropidechis carinatus, esses dados foram confirmados com a análise de aminoácidos. De posse desses resultados, optou-se por testar a capacidade da fração purificada de ativar fator X e II, usando substratos sintéticos. Os resultados apontaram para uma ativação direta do fator X, uma vez que não houve ativação do fator II, atividade que também não foi detectada no veneno total. A mesma se mostrou um potente ativador da agregação de forma direta, uma vez que os ensaios de agregação plaquetária foram realizados com plaquetas lavadas, logo na ausência de fatores séricos. Quando os ensaios de agregação foram realizados na presença de alguns inibidores observou-se que nem a atividade metalo proteinase, nem a serino proteinase, tampouco um domínio lectina estavam envolvidos no processo, uma vez que EDTA, benzamidina e D-galactose não inibiram a atividade da proteína. No presente trabalho isolamos a Delta toxina do veneno de C. d. terrificus. A mesma se comportou como previsto por Vital Brazil em 1980, eluindo na posição por ele aventada, sendo uma proteína ativadora de Fator X que ativa agregação plaquetária mesmo em concentrações muito baixas e de massa molecular de 40 kDa levando nos a crer se tratar de um homotrímero cujos componentes são unidos por ligações fracas. / The Crotalus durissus terrificus venom has been so far described as being of low complexity, with four major components described: convulxin, gyroxin, crotoxin and crotamine. In recent studies, other components of this venom were characterized as, for example, an analgesic factor. In 1980, Vital Brazil predicted the existence of a toxin which could be involved in platelet aggregation, and named it delta toxin. However, this toxin has never been isolated or characterized. The aim of the present work was to purify and characterize this toxin. After FPLC size exclusion chromatography followed by reverse phase HPLC, an homogeneous fraction was obtained, with a molecular weight of 14,074.92 Da. When analyzed by SOS-PAGE, this toxin presented an anomalous behavior, with a molecular weight of 14 kDa, while in 2D gels, spots around 40 kDa and with an isoelectrical point between 4 and 5 were observed suggesting isoforms with glicosilation microheterogeneity. After trypsin digestion, the fragments were submitted to the swissprot databank showing high homology (43% coverage, 15 matching peptides) with trocarin, a prothrombin activator from Tropidechis carinatus. These data were further confirmed by aminoacid analysis. The toxin was tested for its ability to activate factor II and X using synthetic substrates. Our data indicate a direct activation of factor X. The same toxin also behaved as a potent direct platelet aggregation activator on washed platelets. Assays with specific inhibitors indicate that neither metalloproteinase, nor serinoproteinase or t lectin domains are involved in the aggregating activity, since EDTA, benzamidin and D-galactose did not inhibit the toxin. In the present work, we were able to identify, purify and characterize a new toxin from the brazilian rattlesnake. It behaved as predicted by Vital-Brazil and displayed direct factor X activating properties, also inducing platelet aggregation, even at low concentrations. Our data also indicate that it is probably a homotrimer with the subunities linked by hydrophobic and/or electrostatic interactions.

Cette thèse a pour but d'étudier le transport de charges pho- togénérées dans des matériaux hybrides composés de polymères π-conjugués et de nanocristaux de semi- conducteurs, conçus pour des applications en opto-électronique. La synthèse chimique permet d'obtenir des nanocristaux de CdSe à l'échelle du gramme ayant une faible polydispersité et des formes contrôlées (sphériques, branchées). Les ligands de surface des nanocristaux de CdSe sont échangés par de petites molécules (pyridine, éthanedithiol, phénylènediamine, butylamine, benzènedithiol) afin d'augmenter leur conductivité. L'échange de ligands modifie les niveaux énergétiques des nanocristaux, comme le montrent des études optiques et électrochimiques. Le poly(3-hexylthiophène) déposé sous forme de couches minces présente différents degrés de couplage intermoléculaire et de désordre énergétique selon la méthode de dépôt et le solvant utilisé. Dans les films hybrides, des mesures de diffraction de rayons X en incidence rasante montrent que la structuration cristalline de la matrice organique est modifiée par la présence des nanocristaux. Les mesures de Temps-de-Vol dans les couches hybrides montrent que les mobilités des trous et des électrons varient avec le contenu en nanocristaux, ainsi qu'avec leur forme et leurs ligands. De faibles fractions de nanocristaux provoquent une amélioration de la mobilité des trous, tandis que de plus grandes fractions la détériorent. Les mobilités électroniques sont soumises à une fraction-seuil, as- similable à un seuil de percolation. La fraction optimale de nanocristaux, du point de vue des mobilités des trous et des électrons, est de 36% en volume pour les nanocristaux sphériques avec les ligands de synthèse. Enfin, les simulations Monte-Carlo des courants transitoires photo-générés, dans un échantillon de poly(3-hexylthiophène) et dans un hybride, montrent d'une part que la distribution énergétique du poly(3-hexylthiophène) domine l'allure des courants simulés et d'autre part que les nanocristaux peuvent être assimilés à des sites difficilement accessibles du réseau cubique. / The aim of this work is the study of photogenerated charge transport in hybrid films composed of π-conjugated polymers and of semiconductor nanocrystals, designed for applications in optoelectronics. Chemical synthesis provides gram-scale samples of CdSe nanocrystals, of low polydispersity and con- trolled shapes (spherical, branched). In order to enhance their conductivity, the surface ligands of CdSe nanocrystals (stearic acid, oleylamine) are exchanged by smaller molecules, namely pyridine, ethanedithiol, phenylenediamine, butylamine and benzenedithiol. Optical and electrochemical studies show that the lig- and exchange modifies the nanocrystals' energy levels. Poly(3-hexylthiophene) thin films exhibit varying degrees of energetical disorder and of intermolecular coupling, depending on the processing method and on the solvent used. In hybrid films, the crystallinity of P3HT, probed by grazing incidence X-ray diffraction, is modified by the presence of nanocrystals. Time-of-Flight measurements of hybrid films show that elec- tron and hole mobilities vary with the content of nanocrystals, with their shape, and with their ligands. Small volume fractions of nanocrystals enhance the hole mobility, and large fractions degrade it. Electron mobilities are percolation-limited: they reach a stable value for a threshold fraction of nanocrystals. The optimal fraction of nanocrystals for electron and hole mobilities is 36 vol% in hybrids made of spherical nanocrystals with their synthesis synthesis ligands. Finally, Monte Carlo simulations of photogenerated current transients in pristine poly(3-hexylthiophene) and in a hybrid sample show on one hand that the energy distribution of poly(3-hexylthiophene) rules the shape of the simulated transients, and on the other hand that nanocrystals can be described as little accessible sites of the hopping lattice.

Cette thèse présente une étude de la sensibilité à la physique au delà du modèle standard et en particulier aux couplages anormaux entre les photons et les bosons W. Ceci est réalisé en détectant dans ATLAS les protons intacts après interaction et en mesurant leur temps de vol avec une précision de quelques pico-secondes de part et d’autre du détecteur central.Je décrirai également les photo-détecteurs de grande superficie avec une précision de quelques pico-secondes et les algorithmes de reconstruction de temps basés sur l’échantillonnage rapide du signal. Le circuit intégré spécifique SamPic pour une mesure de temps de très haute précision sera enfin présenté ainsi que les premiers résultats de mesure avec ce circuit. Ils montrent en particulier une précision exceptionnelle, meilleure que 5 ps, sur la mesure de temps entre deux impulsions. / In this thesis, we present a study of the sensitivity to Beyond Standard Model physics brought by the design and installation of picosecond time-of-flight detectors in the forward region of the ATLAS experiment at the LHC. The first part of the thesis present a study of the sensitivity to the quartic gauge anomalous coupling between the photon and the W boson, using exclusive WW pair production in ATLAS. The event selection is built considering the semi-leptonic decay of WW pair and the presence of the AFP detector in ATLAS. The second part gives a description of large area picosecond photo-detectors design and time reconstruction algorithms with a special care given to signal sampling and processing for precision timing.The third part presents the design of SamPic: a custom picosecond readout integrated circuit. At the end, its first results are reported, and in particular a world-class 5ps timing precision in measuring the delay between two fast pulses.

Lanthanum-mediated bond activation reactions of small hydrocarbon molecules, including alkenes, alkynes, and alkadienes, were carried out in a laser vaporization metal cluster beam source. Time-of-flight mass spectrometry and mass-analyzed threshold ionization (MATI) spectroscopy, in combination with quantum chemical and multi-dimensional Franck-Condon factor calculations, were utilized to identify the reaction products and investigate their geometries, electronic structures, and formation mechanisms. La-hydrocarbon association was only observed in the reaction of La with isoprene. C-H bond activation was observed in all reactions, hydrogen elimination was observed as the prominent reaction for the alkenes (2-butene, isobutene, 1-pentene, and 2-pentene), alkynes (1-butyne, 2-butyne, and 1-pentyne), and 1,4-pentadiene, and C-C bond activation was observed for the five-membered hydrocarbons (1-pentene, 2-pentene, 1-pentyne, isoprene, and 1,4-pentadiene). The La-hydrocarbon radicals formed in these reactions had lanthanacyclic structures in various sizes, and each of the La-hydrocarbon complexes had a doublet ground state with a 6s1 La-based electron configuration. Ionization removed the 6s electron, and the resultant ion was in a singlet state. Formations of dehydrogenated products were either through a concerted hydrogen elimination process or the dehydrogenation after ligand isomerization. The C-C bond activation proceeded through La-assisted hydrogen migration, followed by C-C bond cleavage, or vice versa.

The objective of this dissertation is to provide a new design approach to a fully automated book reader for individuals with visual impairment and blindness that is portable and cost effective. This approach relies on the geometry of the design setup and provides the mathematical foundation for integrating, in a unique way, a 3-D space surface map from a low-resolution time of flight (ToF) device with a high-resolution image as means to enhance the reading accuracy of warped images due to the page curvature of bound books and other magazines. The merits of this low cost, but effective automated book reader design include: (1) a seamless registration process of the two imaging modalities so that the low resolution (160 x 120 pixels) height map, acquired by an Argos3D-P100 camera, accurately covers the entire book spread as captured by the high resolution image (3072 x 2304 pixels) of a Canon G6 Camera; (2) a mathematical framework for overcoming the difficulties associated with the curvature of open bound books, a process referred to as the dewarping of the book spread images, and (3) image correction performance comparison between uniform and full height map to determine which map provides the highest Optical Character Recognition (OCR) reading accuracy possible. The design concept could also be applied to address the challenging process of book digitization. This method is dependent on the geometry of the book reader setup for acquiring a 3-D map that yields high reading accuracy once appropriately fused with the high-resolution image. The experiments were performed on a dataset consisting of 200 pages with their corresponding computed and co-registered height maps, which are made available to the research community (cate-book3dmaps.fiu.edu). Improvements to the characters reading accuracy, due to the correction steps, were quantified and measured by introducing the corrected images to an OCR engine and tabulating the number of miss-recognized characters. Furthermore, the resilience of the book reader was tested by introducing a rotational misalignment to the book spreads and comparing the OCR accuracy to those obtained with the standard alignment. The standard alignment yielded an average reading accuracy of 95.55% with the uniform height map (i.e., the height values of the central row of the 3-D map are replicated to approximate all other rows), and 96.11% with the full height maps (i.e., each row has its own height values as obtained from the 3D camera). When the rotational misalignments were taken into account, the results obtained produced average accuracies of 90.63% and 94.75% for the same respective height maps, proving added resilience of the full height map method to potential misalignments.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 132-140).

Metabolism is the sum of all chemical processes with the purpose to maintain life, as well as enable reproduction, in a living organism. Through the study of metabolism, increased understanding of pharmacological mechanisms and diseases can be achieved. This thesis describes several ways of doing so, including targeted analysis of selected metabolites and investigations of systematic metabolic differences between selected groups through pattern recognition. A method for exploring metabolic patterns in urine samples after intake of coffee or tea was developed. The methodology was later used with the aim to find biomarkers for prostate cancer and urinary bladder cancer. Furthermore, a fully automated quantitative method was developed for concentration measurements of the double prodrug ximelagatran and its metabolites in pig liver. The method was then used to study the roll of active transporters in pig liver cells. Moreover, a fundamental study was conducted to investigate how monitoring of small, doubly charged analytes can improve the limit of detection and precision in a quantitative method. The techniques used for the experiments were liquid separation coupled to electrospray mass spectrometry. Extra efforts were made to make the separation and the ionization as compatible as possible to each other for increased quality of the collected data.

Diamond is a semiconductor with many superior material properties such as high breakdown field, high saturation velocity, high carrier mobilities and the highest thermal conductivity of all materials. These extreme properties, as compared to other (wide bandgap) semiconductors, make it desirable to develop single-crystalline epitaxial diamond films for electronic device and detector applications. Future diamond devices, such as power diodes, photoconductive switches and high-frequency field effect transistors, could in principle deliver outstanding performance due to diamond's excellent intrinsic properties. However, such electronic applications put severe demands on the crystalline quality of the material. Many fundamental electronic properties of diamond are still poorly understood, which severely holds back diamond-based electronic device and detector development. This problem is largely due to incomplete knowledge of the defects in the material and due to a lack of understanding of how these defects influence transport properties. Since diamond lacks a shallow dopant that is fully thermally activated at room temperature, the conventional silicon semiconductor technology cannot be transferred to diamond devices; instead, new concepts have to be developed. Some of the more promising device concepts contain thin delta-doped layers with a very high dopant concentration, which are fully activated in conjunction with undoped (intrinsic) layers where charges are transported. Thus, it is crucial to better understand transport in high-quality undoped layers with high carrier mobilities. The focus of this doctoral thesis is therefore the study of charge transport and related electronic properties of single-crystalline plasma-deposited (SC-CVD) diamond samples, in order to improve knowledge on charge creation and transport mechanisms. Fundamental characteristics such as drift mobilities, compensation ratios and average pair-creation energy were measured. Comparing them with theoretical predictions from simulations allows for verification of these models and improvement of the diamond deposition process.

When and how life first emerged on the Earth is an area of intense research. Signs of the first life on Earth, including morphological fossils, are scarce and hard to interpret. An alternative approach is to study organic biomarkers, which are molecular fossils commonly considered as bona fide biosignatures. The main objective of the project is to develop an approach for analysis of single oil-bearing fluid inclusions and most importantly the detection of organic biomarkers in these inclusions. Analysis of oil-bearing fluid inclusions is advantageous since the inclusions may provide an uncontaminated sample source of Precambrian hopanes and steranes, which are key biomarkers for tracing the early evolution of life on Earth. Due to the presence of several inclusion generations, single inclusion analysis is desired in order to constrain biomarkers to specific inclusions. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) could be an excellent tool for analysis of these types of samples. The development of the approach for analysis of single oil-bearing inclusions was done in a two-step process; i) a number of crude oils were analysed with ToF-SIMS and gas chromatography mass-spectrometry (GC-MS) to facilitate interpretation of ToF-SIMS spectra of these types of samples and, ii) a procedure that combines micrographs with ion etching and ToF-SIMS analysis was developed for analysis of inclusions. The feasibility of the technique was demonstrated for oil inclusions from the Siljan impact crater in which hopanes and steranes where detected. Single oil-bearing fluid inclusions trapped in mid-Proterozoic sandstones from Northern Australia were subsequently analyzed, and steranes and hopanes were detected in these inclusions. If applied on older inclusions this approach may help answer some of the questions regarding the emergence and evolution of life on Earth, and if applied on extraterrestrial samples, also the possibility of life on other planets and moons. / Livets uppkomst och tidiga utveckling på jorden är ett hett forskningsfält. Hur och när livet och dess olika domäner (arkéer, bakterier och eukaryoter) uppstod på jorden är fortfarande oklart vilket beror på att de första tecknen på liv, vilka inkluderar morfologiska fossil, spårfossil och isotoper, är få och svåra att tolka. Ett alternativt sätt att studera det tidiga livet är att studera organiska biomarkörer som är organiska molekyler som anses unika för liv. Huvudmålet med projektet är att utveckla en metod som kan detektera organiska biomarkörer i enskilda oljebärande vätskeinneslutningar. Vätskeinneslutningar, som är små mängder vätska (picoliter) infångad in en sten, är intressanta då de är en potentiell provkälla för prekambriska (äldre än 500 miljoner år) biomarkörer, som hopaner och steraner, vilka används för att utforska livets tidiga utveckling på jorden. Analys av enskilda inneslutningar är emellertid oftast nödvändigt för att kunna tidsavgränsa biomarkörer. På grund av att de flesta inneslutningar är små (10 µm i diameter) är det inte möjligt att analysera en enskild vätskeinneslutning med standardtekniken gaskromatografi-masspektrometri (GC-MS). Time-of-flight secondary ion mass spektrometri (ToF-SIMS) med sin höga känslighet, höga massupplösning och kapacitet för 2D-representation av analysdata och djupprofilering av prover är en utmärkt teknik för analys av enskilda inneslutningar. Metoden för analys av enskilda inneslutningar utvecklades i två steg. Först analyserades ett antal råoljor med ToF-SIMS och GC-MS för att underlätta förståelsen av ToF-SIMS-spektra från dessa typer av prover. Därefter utvecklades en metod som bestod av mikroskopering för att lokalisera inneslutningen, jonetsning för att öppna inneslutningen och ToF-SIMS analys av det exponerade innehållet. Metoden testades framgångsrikt på enskilda inneslutningar i hydrotermala vener av flusspat och kalcit i ordovicisk (488-443 miljoner år sedan) kalksten. Därefter användes den utvecklade metoden för att analysera enskilda vätskeinneslutningar i 1,43 miljarder år gammal sandsten från norra Australien, i vilka hopaner och steraner detekterades. De detekterade steranerna visar att trots att havet under denna tid var syrefritt existerade det lokala syrerika miljöer där eukaryoter kunde överleva. Om den utvecklade metoden används på ännu äldre inneslutningar, vilka har daterats till 3,2 miljarder år, kan den komma att svara på några de mest fundamentala frågorna kring livets uppkomst och tidiga utveckling. Om metoden används på utomjordiska prover kan den svara på frågan om det finns liv på andra planeter eller månar. / At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Submitted.