Dissertations / Theses on the topic 'DSB substrates'

Les besoins actuels des systèmes de télécommunications et des capteurs optiques poussent à réaliser des circuits intégrés optiques présentant toujours plus de fonctions sur un même substrat. Atteindre un tel niveau d'intégration est difficile, car les matériaux et les procédés technologiques employés pour implémenter les différentes fonctions optiques ne sont pas toujours compatibles entre eux. Cette thèse s'inscrit dans la problématique de l'intégration des fonctions optiques actives (émission, amplification) et passives (multiplexage, filtrage, etc.) sur substrat passif et reporte la réalisation d'un laser DFB hybride tridimensionnel par échange d'ions sur verre passif et report de plaque d'un verre actif codopé Er3+:Yb3+. Ce laser est constitué d'un guide canal de Bragg, sélectivement enterré dans le substrat passif, et chargé par un guide plan, réalisé dans le verre actif (dopé avec une concentration massique de 2,3% en Er2O3 et 3,6% en Yb2O3). Il est caractérisé par une émission monomode de (420±15) µW à 1534 nm, pour (390±20) mW de puissance de pompe injectée. Ce dispositif ouvre ainsi la voie vers l'intégration de fonctions actives, localisées à la surface du substrat passif, avec des fonctions passives, réalisées en exploitant le volume et la surface du même substrat
The current needs of optical telecommunications and sensors require developing integrated optical circuits providing different and heterogeneous functions on the same substrate. The main issue is the incompatibility between the manufacturing processes of these optical functions. This work deals with the integration of active (emission, amplification) and passive (multiplexing, filtering, etc.) functions on a passive glass substrate. Its aim is to develop a DFB three-dimensional hybrid laser by ion-exchange in passive glass. This laser is made of a Bragg channel waveguide, selectively buried in the passive glass substrate, loaded by a plane waveguide, defined at the surface of an Er3+:Yb3+ codoped active glass wafer. It emits a (420±15) µW laser signal at 1534 nm for (390±20) mW injected pump power. Hence this device opens the way towards the integration of active functions, located at the surface of the passive glass substrate, with passive ones, spreading at its surface and in its volume

A presente dissertação de mestrado tem o propósito de relatar o desenvolvimento de um sensor planar eletroquímico com três eletrodos para o monitoramento de nitrito e seus interferentes (ácido úrico, ácido ascórbico e paracetamol). Para tal, no procedimento experimental é descrito o desenvolvimento do aparato extracorpóreo (mini-bomba compressora, válvulas solenóides e cela de análise). Esse é acoplado a um sistema de Análise por Injeção em Fluxo Automatizada (do inglês, Flow-injection Analysis) FIA-automatizada. Este trabalho também descreve a fabricação dos sensores em substrato de alumina, o processo de limpeza padrão dos eletrodos, o processo de obtenção dos eletrodos de referência em HCl (0,1 mol L-1) e ativação eletroquímica da superfície do eletrodo de trabalho com H2SO4 (0,1 mol L-1). Em seguida, é mostrada a estabilização dos eletrodos de referência Ag/AgCl em solução salina contendo cloretos, o estudo da secagem dos eletrodos em temperatura ambiente com exposição à luz e a caracterização dos mesmos por três métodos distintos, a saber: i) teste de reversibilidade (corrente redox vs. potencial); ii) variação do potencial com o tempo (coeficiente de degradação termodinâmica) e iii) variação do potencial com a concentração de cloretos. Também, apresenta-se a caracterização da área efetiva exposta do eletrodo de medição (trabalho) e os resultados da deposição de 1-2 Diaminobenzeno (DAB) como polímero seletivo. Além disso, são apresentados os resultados e discussões dos pseudo-eletrodos (recoberto ou não com Náfion® 117). Um ponto relacionado, a histerese associada à irreversibilidade dos eletrodos de referência resultou da ordem de 40 mVAg/AgCl Náfion® 117. Os potencias termodinâmicos observados foram da ordem de 100 ± 6 mVAg/AgCl Náfion® 117 para pseudo-eletrodo de referência sem e com Náfion® 117. Depois, foi realizado um estudo criterioso dos potenciais de resposta ao nitrito e aos interferentes por meio da técnica de DPV (do inglês, Differencial Pulse Voltammetry). Para evitar degradação acelerada (corrosão) do polímero sobre o eletrodo de trabalho, optou-se por realizar as medições com potencial igual a 0,50 VAg/AgCl Náfion® 117. Nessa condição, apresenta-se a monitoração de nitrito por FIA-automatizada na faixa de concentração de 50 a 250 mol L-1. Os resultados para a medição de nitrito mostraram que o sensor planar eletroquímico amperométrico desenvolvido é promissor, pois esse apresentou ótimo desempenho de reprodutividade (99,66%), sensibilidade (90 microA mol-1 L mm-2), seletividade (0,32%), repetitividade (91,28%) e estabilidade (15 ± 0,3 pA).
In this Master of Science, it has been developed an electrochemical planar sensor defined with three electrodes for nitrite measuring and its interferents (uric acid, ascorbic acid and paracetamol). In the experimental procedure, it is shown the development of an extracorporis set up (mini-pump, solenoid valves and analysis cell) which is coupled to a system of Flow-injection Analysis (FIA). This work also describes the sensor fabrication using alumina substrates, the standard cleaning of the electrodes, the process to obtain Ag/AgCl reference electrodes in HCl (0.1 mol L-1) and electrochemical activation of the working-electrode surface with H2SO4 (0.1 mol L-1). Following, it is shown the study of drying for electrodes at room temperature with exposition to room light and their characterization using three different methods: i) reversibility test (redox current vs. potential); ii) variation of the potential with the time (thermodynamics degradation coefficient) and iii) variation of the potential with the chloride concentration. Also, it is presented the characterization of the effective exposed area of the working electrode and the results of the deposited 1-2 Diaminobenzene (DAB) is presented as a selective polymer. In addition, the results and discussions of the pseudo-reference (recovered or not with Nafion® 117) are presented. Related to irreversibility is the hysteresis associated to the reference electrodes which resulted in the order of 40 mVAg/AgCl Nafion® 117. The observed thermodynamic potentials were of (100 ± 6) mVAg/AgCl Nafion® 117 for pseudo-electrodes recovered with Nafion® 117. Later on, a careful study of the response to nitrite and to its interferents was performed by means of the DPV technique (Differential Pulse Voltammetry). In order to avoid accelerated degradation (corrosion) of the polymer, measurements were performed at a potential of 0,5 VAg/AgCl Nafion® 117. In this condition, nitrite was monitored using the automatized-FIA (Flow Injection Analysis) for concentration in the range of 50 to 250 mol L-1. The results have shown that the planar amperometric sensor for nitrite measuring is usefull since it was observed excellent performance related to reproducibility (99.66%), sensitivity (90 microA mol-1 L mm-2), selectivity (0.32%), repetibility (91.28%) and stability (15.0 ± 0.3 pA).

In electronic packaging, solder joints in surface mount technology are used for not only electrical connections, but mechanical connections as well. Due to the mismatch of the coefficients of thermal expansion of different components in chip-substrate systems, solder joints under thermal cycles could develop thermal stress inside and therefore experience fatigue failure after a certain number of load cycles. In this work, the disturbed state concept (DSC) model, a unified and hierarchical approach to model a variety of materials such as soils, rocks, ceramics, metals, and alloys, was appropriately modified to characterize a 63Sn-37Pb solder. This includes a modified hardening function that eliminates some inconsistency in the HISS-delta0 model when the bonding stress is nonzero, and a different fully adjusted state that is properly assumed from test data on the 63Sn-37Pb solder. A generalized computer procedure was then developed for 3-D constitutive level back prediction with the DSC model. In addition, a modified computer procedure for parameter determination was proposed and implemented to calculate the relative intact stress-strain curve for simple shear test data automatically. The above procedures for parameter determination and back prediction were used to model simple shear tests of the 63Sn-37Pb solder at different temperatures and strain rates. Based on material properties determined from test data at different combinations of temperature and strain rate, constitutive level back predictions were performed for each test data set using (1) specific material properties and (2) temperature and rate dependent material properties. Further, a 3-D DSC FEA (finite element analysis) program was used to simulate the same stress-strain behaviors of solder joints at different temperatures and strain rates. The results from back prediction and 3-D FEA simulation show that the test data have been better characterized by the modified DSC model. Moreover, 2-D and 3-D DSC FEA programs were employed to study the fatigue failure of a 144-Pin PBGA solder ball under cyclic thermomechanical loading. An accelerated-approximate procedure was incorporated into the 3-D FEA program to reduce the computational effort for fatigue analysis. Results of 3-D FEAs show that the 3-D geometry of a solder joint has significant influences on its fatigue life. The comparison of 2-D and 3-D results with the test results for the 144-pin PBGA solder ball indicates the FEA results are consistent with the initiation of failure observed in laboratory test. In addition, failure criteria based on fractional volume were also proposed for 2-D and 3-D FEAs by calibration of the available test results.

The knowledge about the reliability of power electronics is necessary for the design of converters. Especially for offshore applications it is essential to know, which fatigue processes happen and how the lifetime can be estimated. Numerical simulation is an important tool for the development of power electronic systems. This thesis analyse the thermal and thermal-mechanical behaviour of packages for power semiconductor devices with the help of simulations. One topic is the evaluation of different thermal models. The main focus is on the description of the thermal cross-coupling between the devices and the influence to the lifetime estimation. The power module is a well established package for power semiconductor devices. It will be explained how the heating period of power cycles influences the failure mode of this package type. Additionally, it will evaluated how SiC devices and DAB substrates influence the power cycling capability. The press-pack is in focus for high power applications as the package short-circuits during an electrical failure without external auxiliary systems. However, the knowledge about the power cycling behaviour is currently limited. With the help of simulations this behaviour will be analysed and possible weak points will be also derived. In the end of the work it will be discussed, how the lifetime can be estimated with help of FEM simulations
Für die Entwicklung von Umrichtern ist die Kenntnis über die Zuverlässigkeit der Leistungselektronik ein wichtiges Kernthema. Insbesondere für Offshore-Anwendungen ist das Wissen über die stattfindenden Ermüdungsprozesse und die Abschätzung der zu erwartenden Lebensdauer der Bauteile essentiell. Hierfür hat sich die Simulation als ein wichtiges Werkzeug für die Entwicklung und Lebensdauerbewertung von leistungselektronischen Anlagen etabliert. In der folgenden Arbeit wird das thermische und das thermisch-mechanische Verhalten der Leistungselektronik mittels Simulationen untersucht. Hierzu wird ein Vergleich zwischen verschiedenen thermischen Modellen für Leistungsbauelemente durchgeführt. Schwerpunkt ist die Beschreibung der thermischen Kopplung zwischen den Chips und deren Einfluss auf die Lebensdauerabschätzung. Ein weiterer Schwerpunkt ist das Leistungsmodul, welches sich als ein Standardgehäuse etabliert hat. Dazu wird erklärt, wie die Variation der Einschaltzeit im aktiven Lastwechseltest den Fehlermodus dieses Gehäusetyps beeinflusst. Weiterhin wird untersucht, wie SiC als Leistungshalbleiter und DAB als Substrat die Zuverlässigkeit beein- flusst. Der Press-Pack ist für Hochleistungsapplikationen von hohem Interesse, da dieses Gehäuse im elektrischen Fehlerfall ohne äußere Unterstützung kurzschliesst. Jedoch ist das Wissen über diese Gehäusetechnologie unter aktiven Lastwechselbedingungen sehr limitiert. Mit Hilfe von Simulationen wird dieses Verhalten untersucht und mögliche Schwachpunkte abgeleitet. Am Ende der Arbeit werden Möglichkeiten untersucht, wie Mithilfe von FEM Simulationen die Lebensdauer von Leistungsmodulen evaluiert werden kann

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 70-72). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 176-186). Also available in electronic version. Access restricted to campus users.

The knowledge about the reliability of power electronics is necessary for the design of converters. Especially for offshore applications it is essential to know, which fatigue processes happen and how the lifetime can be estimated. Numerical simulation is an important tool for the development of power electronic systems. This thesis analyse the thermal and thermal-mechanical behaviour of packages for power semiconductor devices with the help of simulations. One topic is the evaluation of different thermal models. The main focus is on the description of the thermal cross-coupling between the devices and the influence to the lifetime estimation. The power module is a well established package for power semiconductor devices. It will be explained how the heating period of power cycles influences the failure mode of this package type. Additionally, it will evaluated how SiC devices and DAB substrates influence the power cycling capability. The press-pack is in focus for high power applications as the package short-circuits during an electrical failure without external auxiliary systems. However, the knowledge about the power cycling behaviour is currently limited. With the help of simulations this behaviour will be analysed and possible weak points will be also derived. In the end of the work it will be discussed, how the lifetime can be estimated with help of FEM simulations.
Für die Entwicklung von Umrichtern ist die Kenntnis über die Zuverlässigkeit der Leistungselektronik ein wichtiges Kernthema. Insbesondere für Offshore-Anwendungen ist das Wissen über die stattfindenden Ermüdungsprozesse und die Abschätzung der zu erwartenden Lebensdauer der Bauteile essentiell. Hierfür hat sich die Simulation als ein wichtiges Werkzeug für die Entwicklung und Lebensdauerbewertung von leistungselektronischen Anlagen etabliert. In der folgenden Arbeit wird das thermische und das thermisch-mechanische Verhalten der Leistungselektronik mittels Simulationen untersucht. Hierzu wird ein Vergleich zwischen verschiedenen thermischen Modellen für Leistungsbauelemente durchgeführt. Schwerpunkt ist die Beschreibung der thermischen Kopplung zwischen den Chips und deren Einfluss auf die Lebensdauerabschätzung. Ein weiterer Schwerpunkt ist das Leistungsmodul, welches sich als ein Standardgehäuse etabliert hat. Dazu wird erklärt, wie die Variation der Einschaltzeit im aktiven Lastwechseltest den Fehlermodus dieses Gehäusetyps beeinflusst. Weiterhin wird untersucht, wie SiC als Leistungshalbleiter und DAB als Substrat die Zuverlässigkeit beein- flusst. Der Press-Pack ist für Hochleistungsapplikationen von hohem Interesse, da dieses Gehäuse im elektrischen Fehlerfall ohne äußere Unterstützung kurzschliesst. Jedoch ist das Wissen über diese Gehäusetechnologie unter aktiven Lastwechselbedingungen sehr limitiert. Mit Hilfe von Simulationen wird dieses Verhalten untersucht und mögliche Schwachpunkte abgeleitet. Am Ende der Arbeit werden Möglichkeiten untersucht, wie Mithilfe von FEM Simulationen die Lebensdauer von Leistungsmodulen evaluiert werden kann.

Cette thèse porte sur l'étude de la durée de vie de composants et modules de puissance dans des environnements thermiques sévères, lorsque les modules de puissance sont amenés à travailler à haute température ou sous des cycles de température de forte amplitude. Les domaines visés par cette étude concernent plus particulièrement des applications extrêmement contraignantes telles que les applications aéronautiques. Un module de puissance est un assemblage de plusieurs matériaux (semi-conducteurs, brasures, céramiques, conducteurs) présentant des propriétés mécaniques, notamment de coefficient de dilatation thermiques (CTE) différentes. Les pertes dans les puces actives et les variations de température ambiante (profils de mission) sont responsables de contraintes mécaniques liées aux différences de CTE entre les différents matériaux. Les modes de défaillance étant principalement d'origine mécanique, ces travaux ont été effectués entre le SATIE et le LMT (Institut Farman). L'étude mécanique a complété des travaux expérimentaux de caractérisation et de vieillissement accéléré de modules de puissance à semi-conducteur. Ces études ont nécessité la mise en place d'essais de vieillissement spécifiques, passifs (utilisation d'une étuve permettant de contraindre les assemblages de puissance sous des cycles thermiques de grande amplitude) et actifs (utilisation de régimes extrêmes de fonctionnement tels que le court-circuit pour accélérer le vieillissement de certaines parties de l'assemblage).La première partie de ce manuscrit présente les principes physiques mis en jeu. Nous présenterons succinctement les concepts de mécanique de la rupture ainsi que les couplages physiques.La deuxième partie porte sur la rupture de la céramique des substrats DCB. Cette rupture peut provenir de défauts répartis aléatoirement dans le matériau. Nous aborderons le problème par une approche statistique où nous introduirons la "Théorie du maillon faible". L'autre cause de rupture est la présence de défauts géométriques que l'on appelle singularité où il existe une concentration des contraintes nécessitant une approche déterministe. A cette occasion nous introduirons le facteur d'intensité des contraintes qui permet d'étudier les problèmes de rupture liés à des zones singulières.La dernière partie portera sur la dégradation de la métallisation du transistor. La recherche d'indicateurs de vieillissement a demandé la mise au point de différents bancs de caractérisation électrique pour la mesure fine de différentes grandeurs électriques (courants de fuite, tensions de seuil, chute de tension à l'état passant...) dans un environnement thermique contrôlé. De plus ces caractérisations électriques sont corrélées à des observations de la métallisation effectuées par un microscope électronique à balayage de manière régulière. Nous chercherons à montrer comment la modification de la morphologie de la métallisation peut modifier les caractéristiques électriques des transistors de puissance testés.

Le développement d'un procédé de détection de gaz atmosphériques à l'état de traces en temps réel, fiable, robuste, sélectif, sensible et portable, est impératif pour répondre à des enjeux sanitaires, écologiques et industriels. La spectroscopie par diodes laser accordables est une des voies envisagées pour pourvoir à ce besoin. Elle nécessite le développement de diodes lasers mono-fréquences émettant en régime continu à température ambiante entre 2 µm et 3.3 µm. Nous reportons ici les modélisations et développements technologiques nécessaires à la fabrication de lasers à contre-réaction répartie – à couplage par l'indice, réseau du 1er et 2nd ordre, sur substrat antimoniure – ainsi que les résultats obtenus. Dans la première partie de ce document, après avoir dressé le contexte de l'étude, nous introduirons la théorie des lasers à contre-réaction répartie et présenterons les modélisations qui ont permis de décrire nos structures. La seconde partie est dédiée aux développements des procédés technologiques qui ont permis de mettre en place deux filières de fabrication de composants – à savoir des lasers DFB à ailettes et lasers DFB à réseau enterré. La troisième partie expose les performances des composants fabriqués et présente les premières mesures d'analyse de gaz effectuées. Ces travaux ont conduit au développement de deux nouvelles filières de fabrication de composants : des diodes lasers mono-fréquences présentant une puissance élevée et une forte sélectivité modale. Les prototypes fabriqués seront utilisés sur des systèmes de spectroscopie
Development of a reliable, real-time, selective, sensitive and suitable technique for atmospheric trace gas spectroscopy is a critical challenge in science and engineering, for sanitary, ecological and industrial issues. Tunable single-frequency lasers in the 2µm to 3.3µm wavelength range, working in continuous regime at room temperature, can be used in absorption spectroscopy to identify and quantify several atmospheric gases. We report here on the design, the technological development and the performances of 1st and 2nd order index-coupled distributed-feedback (DFB) antimonide-lasers diodes in the 2µm to 3.3µm wavelength range. The first part of this document establishes the context of the thesis, introduces the DFB theory and our modelisation. The second part presents the technological fabrication of the two different components: the side wall corrugated DFB lasers and the buried DFB lasers. The third part shows the performances of the components and the first tests on gas measurement.This work has led to the development of two different kinds of single-frequency laser diodes with high optical power and spectral purity. The fabricated prototypes will soon be used on gas spectroscopy set-up

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2002.
Includes bibliographical references (leaves 109-110). Also available in electronic version. Access restricted to campus users.

Les poly (ADP-ribose) polymérases dépendantes de l’ADN (PARPs) PARP1, PARP2 et PARP3 agissent comme des détecteurs de cassures d'ADN signalant des dommages à l'ADN. Lors de la détection des dommages à l'ADN, ces PARPs utilisent le nicotinamide adénine dinucléotide comme substrat pour synthétiser un monomère ou un polymère d'ADP-ribose (MAR ou PAR, respectivement) attaché de manière covalente au résidu accepteur des protéines cibles. Récemment, il a été démontré que les protéines PARP1–3 peuvent directement ADP-ribosyler les cassures d'ADN en attachant les oligomères MAR et PAR aux phosphates terminaux.Néanmoins, peu de choses sont connues sur les mécanismes régissant la reconnaissance et la spécificité du substrat de PARP1, qui représente la majeure partie de l'activité de PARylation cellulaire, ainsi que sur les protéines responsables de la détection et de l'élimination des adduits d'ADN ADP-ribosylés et son rôle dans une multitude de processus cellulaires. Dans cette étude, nous avons caractérisé de manière détaillée la spécificité du substrat (ADN) de PARP1 et des mécanismes de la PARylation de l'ADN. Nous avons montré que le résidu phosphate 3'-terminal aux extrémités des cassures de l'ADN double brin servait de site accepteur majeur pour la PARylation catalysée par PARP1 en fonction de l'orientation et de la distance entre les cassures du brin d'ADN dans une seule molécule d'ADN. De plus, une préférence pour l’ADP-ribosylation des molécules d'ADN contenant du phosphate 3'-terminal a été observée par rapport à l'auto-ADP-ribosylation de PARP1, et un modèle de modification de l'ADN par PARP1 a été proposé. Des résultats similaires ont été observés avec l’enzyme PARP1 recombinante purifiée et des extraits provenant des cellules HeLa. Ainsi, les effets biologiques de l’ADP-ribosylation médiée par PARP peuvent dépendre fortement de la configuration des cassures complexes des brins d'ADN. De plus, nous avons élaboré une nouvelle approche permettant d’identifier et valider les protéines responsables de la détection («readers») ou de l'élimination («erasers») des adduits ADN- ADP-ribose. Nos données protéomiques ont révélé que les adduits de l'ADN MARylé modulaient sélectivement la reconnaissance de l'ADN par un grand nombre de protéines impliquées dans différentes voies de signalisation cellulaire. Environ 90 protéines, y compris des complexes protéiques, ont été sélectionnées comme lecteurs («readers») potentiels d'adduits ADN-MARylé. Le rôle de l'ADP-ribosylation de l’ADN dans la jonction d'extrémités non homologues (NHEJ) a été partiellement caractérisé dans une étude in vitro. Nous avons démontré que l'ADP-ribosylation de l’extrémité de la cassure double brin («DSB») peut conduire à l'inhibition de la réparation de la DSB bout franc par la voie NHEJ canonique si elle n'est pas éliminée par la glycohydrolase PARG. Au contraire, la présence d'une coupure («nick») proximale avec un site apurinique / apyrimidinique stabilisé conduit à une efficacité NHEJ accrue, apparemment de manière indépendante de l'ADP-ribosylation. Enfin, nous avons recherché de nouveaux inhibiteurs de PARP1, PARP2 et PARP3 parmi les dérivés de 1,4-dihydropyridine, ayant une capacité de liaison à l'ADN. Nos résultats ont révélé que certains analogues de NAD + pourraient être utilisés par les PARPs pour la modification de l'ADN conduisant à la stabilisation des adduits MARylés et PARylés correspondants, en raison de leur résistance à l'activité d'hydrolyse des PARG. Ensemble, ces données mettent en évidence la pertinence physiologique et les résultats biologiques possibles de l’ADP-ribosylation de l’ADN catalysée par les protéines PARPs, tels que la fourniture d'une référence stable de l'emplacement d'une cassure du brin d'ADN sur une carte de chromatine, le recrutement de protéines de réparation de l'ADN et l'inhibition du mécanisme NHEJ toxique
DNA-dependent poly(ADP-ribose) polymerases (PARPs) PARP1, PARP2 and PARP3 act as DNA break sensors signaling DNA damage. Upon detecting DNA damage, these PARPs use nicotine adenine dinucleotide as a substrate to synthesize a monomer or polymer of ADP-ribose (MAR or PAR, respectively) covalently attached to the acceptor residue of target proteins. Recently, it was demonstrated that PARP1–3 proteins can directly ADP-ribosylate DNA breaks by attaching MAR and PAR moieties to terminal phosphates. Nevertheless, little is still known about the mechanisms governing substrate recognition and specificity of PARP1, which accounts for most of cellular PARylation activity, as well, about proteins responsible for detection and removal of ADP-ribosylated DNA adducts and its role in multitude of cellular processes.In this study we provide a detailed characterization of PARP1 DNA substrate specificity and mechanisms of DNA PARylation. We showed that the 3′-terminal phosphate residue at double-strand DNA break ends served as a major acceptor site for PARP1-catalysed PARylation depending on the orientation and distance between DNA strand breaks in a single DNA molecule. Moreover, a preference for ADP-ribosylation of DNA molecules containing 3′-terminal phosphate over PARP1 auto-ADP-ribosylation was observed, and a model of DNA modification by PARP1 was proposed. Similar results were obtained with purified recombinant PARP1 and HeLa cell-free extracts. Thus, the biological effects of PARP-mediated ADP-ribosylation may strongly depend on the configuration of complex DNA strand breaks. Furthermore, we elaborated a new research technique to identify and validate proteins responsible for ADP-ribose-DNA adducts detection (“readers”) or removal (“erasers”). Our proteomic data revealed that MARylated DNA adducts selectively modulated DNA recognition of a large number of proteins involved in different cellular pathways. About 90 proteins including protein complexes were selected as potential MAR-DNA adduct readers. The role of DNA ADP-ribosylation in non-homologous end-joining (NHEJ) was partially characterized in an in vitro study. We demonstrated that ADP-ribosylation of DSB terminus can lead to inhibition of blunt DSB repair by canonical NHEJ if not removed by PARG glycohydrolase. Contrary, presence of a proximal nick with a stabilized apurinic/apyrimidinic site leads to increased NHEJ efficiency, apparently in ADP-ribosylation-independent manner. Finally we searched for novel PARP1, PARP2 and PARP3 inhibitors among derivatives of 1,4-dihydropyridine with DNA binding capacity. Our results revealed that some of NAD+ analogues analogs could be used by PARPs for DNA modification leading to stabilization of corresponding MARylated and PARylated adducts due to their PARG hydrolysis activity resistance. Taking together, these data highlight the physiological relevance and possible biological outcomes of PARP-catalyzed DNA-ADP-ribosylation such as providing a stable benchmark of the location of a DNA strand break on a chromatin map, recruitement of DNA repair proteins and inhibition of the toxic NHEJ

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 67-69). Also available in electronic version. Access restricted to campus users.

Vor dem Hintergrund des heutigen Verständnisses der biologischen Psychologie zur Entstehung von Farbempfindung als selbstregulierende Energiewandlung und -wirkung wird der Beitrag gewürdigt, den Wilhelm Ostwald vor über 100 Jahren mit seiner ‚biologischen Energetik’ einbrachte. Energetische Intentionen und Interpretationen bestimmten auch dessen ,Psychologische Farbenlehre’ als das erklärte Kernstück einer modernen Farbenlehre. Obwohl im Sinne einer kritischen Rezeption auch Grenzen jener Farbenlehre aufgezeigt werden, offenbart sich aber Ostwalds energetische Konzeption durchaus als visionäre Vorleistung, die sich in vielen Aspekten mit heutigen einschlägigen Wissensbeständen deckt. Aus der Perspektive enger Beziehungen zwischen Wissenschaft und Kunst wird zudem auf befruchtende Wechselwirkungen, insbesondere die Ausstrahlung der Ostwald’schen ‚Energetik’ auf die visuellen Künste im 20. Jahrhundert hingewiesen
With today's understanding of colour perception as a self-regulating process of energy transformation and effects, biological psychology honours the contribution made by Wilhelm Ostwald over 100 years ago with his concept of ’biological energetics’. Energy considerations and interpretations determined also his ’psychological theory of colour’, the declared core of modern colour science. Even if critical reception has revealed also the limitations of this colour theory, Ostwald's energetic concept can certainly be seen as a pioneering vision, retaining its validity in many aspects in today's scientific knowledge base. In the context of close relationships between science and art, furthermore, attention must be drawn to fertile interactions, in particular in the influences of Ostwald’s ’energetics’ on the visual arts in the 20th century

The wide band-gap semiconductor silicon carbide has tremendous potential for use in high power, high temperature, and high frequency electronic devices. One of the more important design factors for these devices is the epitaxial layer. It is desirable that this thin film have uniform polytype, thickness, and impurity concentration, as well as be defect free. One method used for SiC to ensure epitaxial layers with homogenous polytype is to cut wafers from a boule that has been tilted towards a specific crystallographic face at a fixed angle (known as ?off cut?). The purpose of this thesis was to investigate the growth mechanisms of alternative boule tilting directions with 6H-SiC. Four alternative crystallographic tilting faces were chosen: <1230>, <1340>, <2130>, and <3140>. A lightly doped 1um-thick layer was grown on samples representing the four alternative off-cut directions and, as references, commercially available substrates off cut towards the traditional direction <1120>. The physical and electrical properties of the layers were characterized by means of optical microscopy, Fourier Transform Infrared Reflectance Spectroscopy, Atomic Force Microscopy, capacitance vs. voltage, and current vs. voltage. Three facts were observed: 1) the alternative off-cut directions affected the growth mechanisms and surface morphology, 2) the quality of the substrate affects the morphology of the epitaxy layer, and 3) the relative differences between the surface roughness attributed to the different off-cut directions affected the observed electrical characteristics of Schottky barrier diodes fabricated on the epi layers. The samples cut towards the <31-40> and <13-40> directions showed to the most promising alternative off-axis tilting direction.

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 153-177). Also available in electronic version. Access restricted to campus users.

Orientador: Sidney Luiz Stürmer.
Co-orientador: Luis Hamilton Pospissil Garbossa.
Dissertação (Mestrado em Engenharia Ambiental) - Programa de Pós-Graduação em Engenharia Ambiental, Centro de Ciências Tecnológicas, Universidade Regional de Blumenau, Blumenau.

L’objectif de ces travaux a été de développer des vernis acrylate photo-polymérisables à 100% d’extrait sec, destinés à protéger des pièces thermoplastiques en polycarbonate contre les endommagements mécaniques, en particulier contre la rayure. Les relations entre la composition, la structure et les propriétés de ces revêtements ont été explorées. Pour ce faire ont été étudiées la morphologie, les propriétés thermomécaniques ainsi que la résistance à la rayure des matériaux. Cette dernière a été évaluée par des tests de micro-scratch. La cinétique de formation des réseaux polymères a elle aussi été étudiée, par photo-DSC. Tous les matériaux étudiés présentent, en analyse thermomécanique dynamique, un module élevé à l’état caoutchoutique ainsi qu’une large relaxation mécanique. Un vernis pétro-sourcé à 100% d’extrait sec, qualifié de standard, a servi de point de départ à ces travaux. Il a tout d’abord été comparé à un vernis commercial solvanté photo-polymérisable, spécialement conçu pour la protection de pièces thermoplastiques. Celui-ci s’est avéré être plus efficace en termes de résistance à la rayure. Dans un deuxième temps, a été étudiée l’influence sur les propriétés du vernis standard pétro-sourcé d’un monomère multicyclique entrant dans sa composition. La modification de son pourcentage n’a permis d’apporter aucun bénéfice en termes de résistance à la rayure. Des nanoparticules de silice, d’alumine ou de zircone, disponibles sous forme de dispersion dans un monomère acrylate, ont ensuite été incorporées dans le vernis standard pétro-sourcé. Une organisation particulière de la nano-silice et de la nano-alumine au sein des matériaux étudiés a pu être observée par microscopie électronique en transmission. Il a été constaté que le taux de charge doit être élevé pour observer une augmentation du module élastique et une amélioration de la résistance à la rayure du vernis (≥15% massique dans le cas de la nano-silice). Par ailleurs, l’ajout de 5% massique de nano-silice dans le vernis n’a conduit à aucune modification de sa cinétique de photo-polymérisation. Enfin, une partie des composés acrylate pétro-sourcés du vernis standard a été substituée par des acrylates bio-sourcés disponibles industriellement. La cinétique de photo-polymérisation des deux types de vernis est similaire. Les conclusions de la comparaison entre les vernis bio-sourcés et le vernis standard pétro-sourcé en termes de résistance à la rayure dépendent de l’épaisseur des revêtements étudiés. L’ajout d’un composé monoacrylate bio-sourcé à la formulation des vernis tend à améliorer la recouvrance élastique des revêtements de faible épaisseur. L’acrylate d’isobornyle est en particulier intéressant, car il a aussi tendance à retarder l’apparition des craquelures au cours de la rayure
The aim of this work was to develop 100% solids photo-polymerizable acrylate coatings, intended to protect thermoplastic pieces made of polycarbonate against mechanical damage, in particular scratches. The relationships between the composition, the structure and the properties of these coatings were examined. For this purpose the morphology, the thermomechanical properties and the scratch resistance of the materials, assessed by micro-scratch tests, were studied. The kinetics of the polymer network formation was also studied by photo-DSC experiments. All the materials feature a high elastic modulus and a broad mechanical relaxation in dynamic thermomechanical analysis. A 100% solids petro-based coating (standard) constituted the starting point of this work. First it was compared to a commercial photo-polymerizable coating containing solvents, specially designed to protect thermoplastic pieces. This commercial coating turned out to be more efficient against scratches. In a second time was studied the influence of the percentage of a multicyclic monomer, taking part in the composition of the standard petro-based coating, on the properties of the latter. The modification of its proportion does not bring any advantage concerning the scratch resistance. Silica, alumina and zirconia nanoparticles, dispersed in an acrylate monomer, were then incorporated in the standard petro-based coating. A particular organization of the silica or alumina nanoparticles in the materials could be observed by transmission electron microscopy. A high filler content is required to observe an increase in the elastic modulus and an enhancement of the scratch resistance of the coating (≥15% by weight for the nano-silica). Moreover, no change of the photo-polymerization kinetics was noticed through the addition of 5% by weight of nano-silica in the coating. Finally, some of the petro-based acrylate compounds of the standard coating were substituted by commercially available bio-based acrylate monomers. Both types of coatings feature similar polymerization kinetics. The conclusions concerning the comparison of the scratch resistance of the bio-based and standard petro-based coatings depend on their thickness. The incorporation of a bio-based monoacrylate compound in low thickness coatings tends to improve the elastic recovery. Isobornyl acrylate is particularly interesting since it also tends to delay the apparition of cracks along the scratch

Die vorliegende Arbeit untersucht Struktur und Mn-Wertigkeit von La0,7Sr0,3MnO3 (LSMO)/SrTiO3 (STO)-Dünnschichten, die mit außeraxialer gepulster Laserdeposition auf STO(0 0 1)-Substrat abgeschieden wurden. Aufgrund der hohen Spinpolarisation der Ladungsträger und der hohen Curie-Temperatur von LSMO sowie der geringen Gitterfehlpassung zwischen LSMO und STO erscheinen solche Dünnschichtsysteme interessant für magnetoresistive elektronische Bauelemente wie LSMO/STO/LSMO-Tunnelkontakte. Da das mit steigender Temperatur rasch abnehmende Magnetwiderstandsverhältnis derartiger Tunnelkontakte vor allem auf die atomistische Struktur der LSMO/STO-Grenz?fläche und den damit gekoppelten Ladungszustand der Mn-Ionen zurückgeht, stellt diese Arbeit eine hoch ortsaufgelöste Charakterisierung von Kristallstruktur und Mn-Wertigkeit mit im TEM aufgezeichneten Abbildungen und Elektronen-Energieverlust-Ionisationskanten vor. Der hinsichtlich des Einfl?usses auf die Qualität der Messdaten durchgeführte Vergleich verschiedener TEM-Präparationsverfahren zeigt, dass die konventionell ionengedünnten Querschnitte geringere Verbiegungen aufweisen als die mit fokussiertem Ionenstrahl präparierten und daher besser für die hochau?flösende TEM geeignet sind. Zur Quantifizierung der Mn-Wertigkeit werden die O-K- und Mn-L2,3-Nahkantenfeinstrukturen herangezogen, die sich in charakteristischer Weise mit dem Sr-Gehalt x und damit der Mn-Valenz in La1-xSrxMnO3 ändern. Hierbei reagieren die Kantenenergieabstandsmaße am empfindlichsten auf die Mn-Wertigkeit und ermöglichen eine Valenzbestimmung mit einer Genauigkeit von bis zu ?0,08. Die für das Innere der untersuchten LSMO-Schichten ermittelte Mn-Wertigkeit stimmt mit dem nominalen Wert von 3,3 überein, wohingegen an manchen LSMO/STO-Grenz?flächen zwischen Substrat und Schicht sowie zwischen den Schichten von Multilagen eine Reduktion um 0,1...0,2 zu beobachten ist. Dies kann auf eine La0,7Sr0,3O/TiO2-terminierte Grenz?fläche zurückgeführt werden und deutet darauf hin, dass sich die abschließenden Atomlagen messbar auf die grenzfl?ächennahe Mn-Wertigkeit auswirken. Weiterhin äußert sich der Ein?fluss der Grenz?fläche in dem Auftreten einer Schulter an der Flanke geringeren Energieverlusts der Mn-L3-Kante. Hierfür werden mögliche Ursachen wie Gitterdeformationen und Sauerstoffleerstellen diskutiert. Die geometrische Phasenanalyse von HRTEM-Aufnahmen und TEM-Hellfeldaufnahmen belegen eine tetragonale Verzerrung des LSMO-Schichtkristalls, der aus nadelförmigen Zwillingsdomänen besteht. Aus hochaufgelösten Raster-TEM-Abbildungen mit den unter großem Winkel gestreuten Elektronen geht hervor, dass die LSMO/STO-Grenz?flächen eine kohärente, gelegentlich mit Elementarzellenstufen versehene Gitterstruktur aufweisen. Insbesondere vermitteln die Stufen (Einfach- und Mehrfachstufen) die wellige Struktur der LSMO/STO-Multilagen. Schließlich wird gezeigt, dass die mit konvergenter Beugung bestimmte Fehlorientierung zwischen LSMO-Schicht und STO-Substrat bei geringer durchstrahlter Dicke der TEM-Lamelle die erwartete Netzebenenneigung der Zwillinge übersteigt. Dafür wird eine nachträgliche Relaxation der LSMO-Schicht während der Ionendünnung verantwortlich gemacht.

This work refers to the area of bio-nanotechnology and concerns the selective immobilization of DNA or other bio-template on microstructured gold contacts and which then permit a coordinated cooperation of several of these nanotemplate, e.g., within a microreactor. The immobilization of such nano-objects should be realized through functional thin polymer films which provide binding groups. Thus, the main aim of this work was the development of polymeric materials for thin functional films which permit to deposit on different substrates a wide variation of functional elements or metal structures and to achieve a pattern formation using optical grid methods. In order to realize this concept it was necessary to design and develop a polymer system based on suitable photolabile units and in addition having anchoring groups which attach on specific substrates like gold. In this terpolymer concept was aimed for which consists of three components with particular functions in suitable molar ratios, which allow the tune the properties of the materials, and provide: amino photolabile protected groups for the photolithographic creation of patterned areas with free amino groups, which are available for further modifications like attachment of colloids, metallization or attachment of DNA strands; disulfide derivative anchor groups providing anchoring capacity for gold surface and spacer groups for adjusting the film quality. These multifunctional terpolymers should be synthesised by free radical polymerisation of suitable monomers. Although these techniques are successful, they are limited by their complexity, rigorous synthetic demands, as well as incompatibility with many functional termolabile and highly reactive functionalities. To overcome these difficulties a polymerisation technique based on “living” free radical polymerisation has been used in this work. A highly efficient polymer-analogous modification allows to introduce the functionalities after the polymer construction reaction. The production of suitable prepolymers [poly(styrene-r-4-propargyl-oxystyrene)] was carried out with the help of a controlled synthesis methodology “nitroxide mediate radical polymerization&quot; followed by polymer analogous reaction using one of the most efficient click reactions, the Cu(I) catalyzed Huisgen 1,3-dipolar cycloaddition between terminal acetylenes and azides to attach further functionalities through the formation of a stable 1,4-disubstituted 1,2,3-triazol ring . The combination of nitroxide mediated radical polymerization (NMRP) and click chemistry was used to produce well-defined random copolymer. It could already be shown that also block copolymers can be prepared which give the chance to combine nanostructure formation in block copolymers with special functionality. Thus, the special properties of these functional polymers like the capability for photopatterning and anchoring onto gold substrates make them very interesting for nanotechnology applications
Diese Arbeit bezieht sich auf das Gebiet der Bionanotechnologie und betrifft ein neuartiges Verfahren zur selektiven Immobilisierung der DNA oder anderer Biomoleküle auf mikrostrukturierten Goldkontakten, welche dann ein koordiniertes Zusammenwirken von einzelnen Nanomolekülen ermöglichen, z.B. in einem Mikroreaktor. Die Immobilisierung solcher Nanoobjekte soll durch dünne Funktionsschichten realisiert werden, die die Anbindungsgruppen liefern. Folglich war das Hauptziel dieser Arbeit die Entwicklung von Polymermaterialien für dünne Funktionsschichten, die die Aufbringung einer großen Vielzahl von Funktionselementen oder metallischen Strukturen auf verschiedenen Substraten gestatten und die Strukturierung durch den Einsatz von lithographischen Methoden ermöglichen. Um dieses Konzept zu realisieren, war es notwendig, ein Polymersystem zu gestalten und zu entwickeln, welches auf geeignete photolabile Einheiten basiert und zusätzlich Ankergruppen hat, die mit spezifischen Substraten wie Gold verbunden ist. Dieses Terpolymerkonzept wurde gezielt aus drei Komponenten mit speziellen Funktionen in entsprechenden molaren Verhältnissen gebildet, die eine Abstimmung der Materialeigenschaften ermöglicht und folgendes bereitstellt: photolabile geschützte Aminogruppen für die photolitographische Strukturerzeugung mit freien Aminogruppen, welche für weitere Modifikationen verfügbar sind wie das Anhängen von Kolloiden, die Metallisierung oder Anfügung von DNA-Strängen; disulfide Derivate für die kovalente Anbindung auf der Goldoberfläche und Spacer-Gruppe für Verbesserung der Schichtenbildung. Diese multifunktionalen Terpolymere sollen durch eine freie radikalische Polymerisation von entsprechenden Monomeren synthetisiert werden. Obwohl diese Techniken erfolgreich sind, sind sie eingeschränkt durch ihre Komplexität, den strengen synthetischen Anforderungen, sowie der Inkompatibilität mit vielen funktionalen thermolabilen und hochreaktiven Funktionalitäten. Um diese Schwierigkeiten zu überwinden wurde eine Polymerisationstechnik für diese Arbeit genutzt, die auf der „lebenden“ freien radikalischen Polymerisation basiert. Eine hoch effiziente polymeranaloge Modifizierung erlaubt die Einführung von Funktionalitäten nach der Polymeraufbaureaktion. Die Herstellung von entsprechenden Präpolymeren Poly(Styrol-r-4-Propargyl-oxystyrol) wurde mittels einer kontrollierten Synthesemethodik „Nitroxid-mediated controled radical polymerisation“ (NMRP) durchgeführt, gefolgt von der Polymeranalogreaktion, die eine der effizientesten Click-Reaktion - die Cu(I) katalysierte 1,3-dipolar Cycloaddition von terminalen Alkinen an Aziden nach Huisgen nutzt, um weiter Funktionalitäten durch die Bildung eines stabilen 1,4-disubstituierten-[1,2,3]-Triazolringes anzufügen. Die Kombination von NMRP und Click-Chemie wurde zur Herstellung eines exakt definierten Random Copolymers genutzt. Es konnte bereits gezeigt werden, dass auch Blockcopolymere geschaffen werden können, die eine Möglichkeit zur Kombination von Nanostrukturformationen in Blockcopolymeren mit speziellen Funktionaltäten bieten. Folglich sind die speziellen Eigenschaften dieser Funktionalpolymere wie die Fähigkeit zur Photostrukturierung und Verankerung auf Goldsubstraten für nanotechnologische Anwendungen sehr interessant

Le substrat SOI (Silicon-On-Insulator) constitue aujourd'hui le support de choix pour la fabrication de fonctions optiques compactes. Cette plateforme commune avec la micro-électronique favorise l'intégration de circuits photoniques avec des circuits CMOS. Néanmoins, si le silicium peut être utilisé de manière très avantageuse pour la fabrication de composants optiques passifs, il présente l'inconvénient d'être un très mauvais émetteur de lumière. Ceci constitue un obstacle majeur au développement de sources d'émission laser, briques de constructions indispensables à la fabrication d'un circuit photonique. La solution exploitée dans le cadre de cette thèse consiste à reporter sur SOI des épitaxies laser III-V par collage direct SiO2-SiO2. L'objectif est de réaliser sur SOI des sources lasers à cavité horizontale permettant d'injecter au moins 1mW de puissance dans un guide d'onde silicium inclus dans le SOI. Notre démarche est de transférer un maximum des fonctions du laser vers le silicium, dont les procédés sont familiers au monde de la micro-électronique. Dans l'idéal, le III-V ne devrait être utilisé que comme matériau à gain ; la cavité laser pouvant être fabriquée dans le silicium. Mais cette ligne de conduite n'est pas forcément aisée à mettre en œuvre. En effet, les photons sont produits dans le III-V mais doivent être injectés dans un guide silicium placé sous l'épitaxie. La difficulté est que les deux matériaux sont séparés par plus d'une centaine de nanomètres d'oxyde de collage faisant obstacle au transfert de photons. Le développement de lasers III-V couplés à un guide d'onde SOI demande alors de nouvelles conceptions du système laser dans son ensemble. Notre travail a donc consisté à concevoir un laser hybride III-IV / silicium se pliant aux contraintes technologiques du collage. En s'appuyant sur la théorie des modes couplés et les concepts des cristaux photoniques, nous avons imaginé, réalisé, puis caractérisé un laser à contre-réaction distribuée hybride (en anglais : " distributed feedback laser ", laser DFB). Son fonctionnement optique original, permet à la fois un maximum de gain et d'efficacité de couplage grâce à une circulation en boucle des photons du guide III-V au guide SOI. Sur ces dispositifs, nous montrons une émission laser monomode (SMSR de 35 dB) à température ambiante en pompage optique et électrique pulsé. Comme attendu, la longueur d'onde d'émission est dépendante du pas de réseau DFB. Les lasers fonctionnent avec une épaisseur de collage de silice de 200 nm, ce qui offre une grande souplesse quant au procédé d'intégration. Tous les lasers fonctionnent jusqu'à des longueurs de 150 μm (la plus petite longueur prévue sur le masque). Malgré les faibles niveaux de puissances récoltés dans la fibre lors des caractérisations, la prise en compte des pertes optiques induites pas les coupleurs fibres nous indique que la puissance réellement injectée dans le guide silicium dépasse le milliwatt. Notre objectif de ce point de vue est donc rempli. Malheureusement le fonctionnement des lasers en injection électrique continue n'a pas pu être obtenu dans les délais impartis. Cependant, les faibles densités de courant de seuil mesurées en injection pulsée (300A / cm2 à température ambiante sur les lasers de 550 μm de long) laissent présager un fonctionnement prochain en courant continu.

L'objectif de ces travaux a été de développer des vernis acrylate photo-polymérisables à 100% d'extrait sec, destinés à protéger des pièces thermoplastiques en polycarbonate contre les endommagements mécaniques, en particulier contre la rayure. Les relations entre la composition, la structure et les propriétés de ces revêtements ont été explorées. Pour ce faire ont été étudiées la morphologie, les propriétés thermomécaniques ainsi que la résistance à la rayure des matériaux. Cette dernière a été évaluée par des tests de micro-scratch. La cinétique de formation des réseaux polymères a elle aussi été étudiée, par photo-DSC. Tous les matériaux étudiés présentent, en analyse thermomécanique dynamique, un module élevé à l'état caoutchoutique ainsi qu'une large relaxation mécanique. Un vernis pétro-sourcé à 100% d'extrait sec, qualifié de standard, a servi de point de départ à ces travaux. Il a tout d'abord été comparé à un vernis commercial solvanté photo-polymérisable, spécialement conçu pour la protection de pièces thermoplastiques. Celui-ci s'est avéré être plus efficace en termes de résistance à la rayure. Dans un deuxième temps, a été étudiée l'influence sur les propriétés du vernis standard pétro-sourcé d'un monomère multicyclique entrant dans sa composition. La modification de son pourcentage n'a permis d'apporter aucun bénéfice en termes de résistance à la rayure. Des nanoparticules de silice, d'alumine ou de zircone, disponibles sous forme de dispersion dans un monomère acrylate, ont ensuite été incorporées dans le vernis standard pétro-sourcé. Une organisation particulière de la nano-silice et de la nano-alumine au sein des matériaux étudiés a pu être observée par microscopie électronique en transmission. Il a été constaté que le taux de charge doit être élevé pour observer une augmentation du module élastique et une amélioration de la résistance à la rayure du vernis (≥15% massique dans le cas de la nano-silice). Par ailleurs, l'ajout de 5% massique de nano-silice dans le vernis n'a conduit à aucune modification de sa cinétique de photo-polymérisation. Enfin, une partie des composés acrylate pétro-sourcés du vernis standard a été substituée par des acrylates bio-sourcés disponibles industriellement. La cinétique de photo-polymérisation des deux types de vernis est similaire. Les conclusions de la comparaison entre les vernis bio-sourcés et le vernis standard pétro-sourcé en termes de résistance à la rayure dépendent de l'épaisseur des revêtements étudiés. L'ajout d'un composé monoacrylate bio-sourcé à la formulation des vernis tend à améliorer la recouvrance élastique des revêtements de faible épaisseur. L'acrylate d'isobornyle est en particulier intéressant, car il a aussi tendance à retarder l'apparition des craquelures au cours de la rayure.

The increasing knowledge about the properties of nanostructured materials is a source of ideas for the improvement of existing and the development of new electronic or opto-electronic devices. One approach is the implementation of nanoparticles as building blocks. Among other synthesis routes, different kinds of gas phase synthesis have been developed which open the possibility to produce nanoparticles with defined properties like chemical composition, crystal structure, shape and size distribution. In order to make use of these properties, the particles have to be transferred from the three dimensional distribution in the gas phase to a two dimensional arrangement on a carrier material, for instance a substrate surface. The first goal of the investiagtion is to describe the microscopic aspects of the deposition process of nanoparticles in the size range between 5 nm and 100 nm at ambient pressure and temperature, under the condition that the particle density (which is the number of particles per unit area)on the substrate surface is below one monolayer. For this purpose, a computer simulation program has been developed, which calculates the particle trajectory that results from the balance of forces on the particle. It takes into account the interaction of incoming individual particles with the substrate surface as well as the interactions with already deposited particles. A parameter study has been carried out in order to point out how the particle arrangement on the substrate depends on the deposition parameters. The results obtained by the computer simulations are compared with experimental results. The parameters varied were the particle diameter, the particle charge, the number of particles per unit area, the substrate properties and the electric field strength. The second goal of the investigation is the structured deposition of nanoparticles from the gas on oxidized silicon substrates by direct deposition from the gas phase of an aerosol. The particles are attracted onto charge patterns created on the plane surface by contact charging. The basic principle of which is that charges cross the interface between an insulator and a metal brought into contact. After the metal is removed, charges retain on the insulator. The charge patterns can either be transferred by a sharp metal tip which is allowed to slide over the substrate surface or by a metal stamp which is pressed on the substrate surface. In the second case even complex structures may be transferred. The resolution of the patterns obtained with these methods is in the order of below 100 nm.

Die Arbeit beschäftigt sich mit der Erweiterung der für dicke Proben schon mit Erfolg eingesetzten Werkstoffanalytischen Methode Elektronenstrahl-Mikroanalyse (ESMA) mittels Peak/Untergrund-Verhältnissen auf die Analyse von dünnen Schichten (unter 1 μm) zur qualitative und quantitativen Elementanalyse sowie zur Ermittlung von Schichtdicken. Weiterhin wird auf der Basis von einer ESMA-Methode für zwei dünne Schichten auf einem Substrat wird ein Modell zur Ermittlung des Phasenwachstumskoeffizienten für eine intermetallische Phase die sich bei der Diffusion zwischen einer dünnen Schicht und einem Substrat bildet, mittels ESMA-Messungen bei gleichzeitiger Erwärmung der Probe dargestellt
The paper deals with the application of the materials analysis method EPMA by peak-to-background ratios, which is currently being used for the analysis of thick samples successfully, to thin layers (less than 1 μm) for the quantitative element analysis as well as for thickness prediction. In addition a model has been established on the Basis of an EPMA method for two films on a substrate for deriving the phase growth coefficient of an inter-metallic phase which grows during the diffusion between a thin layer and a substrate from EPMA measurements while simultaneously heating the sample

The bacterium Helicobacter pylori is recognized as one of the most successful bacterial pathogens. It colonizes the stomach of more than half of the world’s population and it presents a very high infection rate in developing countries. Most infected people are asymptomatic, however an important minority of them (15–20%) develops during their life severe gastroduodenal pathologies, including stomach and duodenal ulcers, adenocarcinomas and stomach lymphomas. Until now nine H. pylori strains have been completely sequenced and this pathogen presents high genetic variability, not only in the gene sequences but also in genes content. Many of the genes functions are annotated based on sequence similarity and there are around 45% genes with unknown function. The most remarkable differences in H. pylori virulent strains with that of non virulent is the presence or absence of the so-called cag-PAI (a 40-kb DNA sequence named cag Pathogenicity Island), that encodes a Type IV Secretion System, responsible for the translocation of the CagA toxin into host epithelial cells. Although H. pylori can be successfully eradicated by antibiotics in many patients, rising antibiotic resistance in the bacterium left overs a serious problem. New therapies are required to eradicate H. pylori infection and looking for new targets could be useful for the development of new treatment strategies. Recently, new factors important for colonization and establishment of infection have been proposed, using multiple approaches. A pull of these H. pylori proteins have been cloned, expressed in E. coli and purified for structural and functional studies. The aim of this thesis was to determine the three dimensional structural and characterize the function of H. pylori proteins important for stomach colonization and pathogenesis. In particular, we concentrate our efforts on cell wall modification enzymes (peptidoglycan deacetylase), LPS biosynthesis (ADP-L-glycero-D-manno-heptose-6-epimerase, rfaD), periplasmic substrate binding protein of ABC transporter (ceuE), key enzymes in nitrogen assimilation pathway (Glutamine synthetase), secreted immunogenic disulfide isomerase (DsbG). The strategy employed are bioinformatic analyses, molecular cloning of the gene starting from PCR amplification, construction of vectors for cloning, expression of the protein in E. coli. After expression analysis and optimization of conditions, the solubility of the recombinant proteins was checked. The soluble recombinant protein was then purified using different chromatography techniques, and eventually characterized by analytical gel filtration, mass spectrometry, UV spectroscopy. Techniques to make the protein samples more suitable for crystallization, as DLS (Dynamic light scattering), and to investigate the secondary structure, as CD (Circular dichroism), were used. The proteins were concentrated for crystallization trials. X-ray diffraction data of the crystals were measured at the ESRF synchrotron (Grenoble, France). Functional characterizations of the proteins were accomplished using fluorescence spectroscopy, CD, ITC, UV spectroscopy and ELISA. After a general introduction about the bacterium (Chapter 1), in the chapter 2 the three dimensional structure and the enzymatic activity of a putative peptidoglycan deacetylase is described. Peptidoglycan deacetlyase (HP0310, HpPgdA) from H. pylori has been indicated as the enzyme responsible for a modification of peptidoglycan that counteracts the host immune response. The enzyme, which belongs to the polysaccharide deacetylases protein family, is a homo-tetramer. The four polypeptide chains, each folded into a single domain characterized by a non-canonical TIM-barrel fold, are arranged around a four-fold symmetry axis. The active site, one per monomer, contains a heavy ion coordinated in a way similar to other deacetylases. However, the enzyme showed no in vitro activity on the typical polysaccharide substrates of peptidoglycan deacetylases. In striking contrast with the known peptidoglycan deacetylases, HpPgdA does not exhibit a solvent-accessible polysaccharide binding groove, suggesting that the enzyme binds a small substrate at the active site. The crystal structure of the last enzyme of the biosynthesis pathway of core oligosaccharides (L, D-heptose) of the LPS is discussed in details in the chapter 3. H. pylori owes much of the integrity of its outer membrane on lipopolysaccharides (LPSs). Together with their essential structural role, LPSs contribute to the bacterial adherence properties, as well as they are well characterized for the capability to modulate the immune response. In H. pylori the core oligosaccharide, one of the three main domains of LPSs, shows a peculiar structure in the branching organization of the repeating units, which displayed further variability when different strains have been compared. We present here the crystal structure of ADP-L-glycero-D-manno-heptose-6-epimerase (HP0859, rfaD), the last enzyme in the pathway that produces L-glycero-D-manno-heptose starting from sedoheptulose-7-phosphate, a crucial compound in the synthesis of the core oligosaccharide. In a recent study, a HP0859 knockout mutant has been characterized, demonstrating a severe loss of lipopolysaccharide structure and a significant reduction of adhesion levels in an infection model to AGS cells, if compared with the wild type strain, in good agreement with its enzymatic role. The crystal structure reveals that the enzyme is a homo-pentamer, and NAD is bound as a cofactor in a highly conserved pocket. The substrate-binding site of the enzyme is very similar to that of its orthologue in E. coli, suggesting also a similar catalytic mechanism. The other enzymes of the pathway are also discussed in terms of their three-dimensional structure. The crystal structure of the periplasmic ABC transporter substrate-binding protein is discussed in chapter 4. Host-derived iron sources, including heme compounds released from damaged tissues, utilized by H. pylori have been identified. H. pylori can use heme as a sole iron source. Heme transport across the periplasmic space and into the cytoplasm is affected by an active transport system comprising a soluble periplasmic binding protein, a cytoplasmic permease, and an ATPase (ABC transporter). Periplasmic heme binding protein have been identified and characterized in several different pathogenic bacteria, but until now no protein was recognized as heme binding in H. pylori. HP15161 (ceuE) was annotated as ABC transporter periplasmic iron-binding protein but the homologous gene from H. mustalae was reported to be involved in nickel transport. To elucidate the structural features of this putative periplasmic ABC transporter binding protein the protein was cloned, expressed and purified in good yield in E. coli, crystallized, its structure determined in apo form and in complex with nickel. The structure was solved by means of single anomalous dispersion experiments on crystals of a selenomethionine variant of the protein. The overall structure is folded in two main domain connected by a long alpha helix It shares the common features of other bacterial periplasmic ABC transporter heme or vitamin B12 binding protein. The substrate binding site is generated in between these two domain. The crystal structure revels that nickel is not the right substrate for this protein and that the substrate-binding cavity is more similar to a heme-binding groove. In vitro binding activity with heme was performed with different techniques (Fluorescence and ITC), which confirms that ceuE in H. pylori is a periplasmic heme binding protein responsible for heme uptake in conjunction with other members of the ABC transporters family. The crystal structure of the key enzyme in the unique nitrogen assimilation pathway glutamine synthetase is discussed in chapter 5. Glutamine synthetase catalyzes the synthesis of glutamine, a central intermediate in nitrogen metabolism, from ATP, glutamate, and ammonia in a divalent metal ion dependent reaction. Ammonia, which is also a preferred nitrogen source for H. pylori, is available in plentiful quantity owing to urease activity. It is assimilated into proteins and other nitrogenous compounds through one single nitrogen incorporation pathway, mediated by GS, an enzyme encoded by gene Hp0512. The absence of an allosteric regulation site (adenylation site) and other key enzymes in the single nitrogen assimilation pathways makes HpGS a potential target for structural studies. In order to elucidate the structural features of the synthetase and to establish a possible regulatory mechanism of the enzyme, glutamine synthetase (HpGS) from H. pylori was cloned, expressed and purified in good yield in E. coli, crystallized and its structure determined. The enzyme is dodecamer, held together mainly by hydrophobic and hydrogen bonding interactions between the two hexameric rings. The N-terminal helix assembles above the hexameric ring and is exposed to solvent. The C-terminal helix, called the `helical thong,' is inserted into a hydrophobic hole in the eclipsed subunit on the opposite hexameric ring. In addition, the central channel of the dodecamer is lined by six four-stranded L-sheets, each built from an antiparallel loop contributed by subunits in opposite rings. The structure of one monomer consists of a smaller N-terminal domain and a larger C-terminal domain. The dodecameric enzyme contains 12 active sites, which can be described as a 'bifunnel' in which ATP and glutamate bind at opposite ends. The ATP binding site is located at the top of the bifunnel, because it opens to the external 6-fold surface of GS. At the junction of the bifunnel there are two divalent cation binding sites. The adenylation site that in all the most similar homologs of HpGS contains the consensus sequence NLYDLP, is replaced in H. pylori by NLFKLT (residues 405 to 410). Since the Tyr407 residue is the well-conserved target of adenylation and H. pylori glutamine synthetase apparently lacks that residue, no adenylation occurs for this enzyme within this motif and no significant structural changes are observed in the loop of adenylation comparing with other structure of GS. In the chapter 6 the cloning, expression and characterization of secreted immunogenic protein DsbG is discussed. Proteins were identified that are involved in disulfide isomerization. They are located in the membranes or in the periplasm and are called Dsb’s for disulfide bond formation. These proteins catalyze the introduction of disulfide bridges, isomerization (shuffling) of incorrectly introduced disulfide bonds and reduction (removal) of inappropriate disulfide bonds. Secreting proteins is a way to interact with hosts by many pathogenic bacteria. In H. pylori, type IV secretion pathways have been identified. Among the secreted proteins, many of them, either residing in or transiting through the periplasmic space, form disulfide bonds after translocation. HP0231 has a sequence similarity with E. coli DsbG and contains the CXXC motif. HP0231 have been already identified as an immunogenic protein recognized by patient sera. The recombinant protein, expressed in E. coli, did not bind strongly to the affinity IMAC-Ni2+ resin, probably because of the degradation of the N-terminal His-tag. To solve the purification problems, two approaches were adopted: the cloning of a new HP0231 construct with a C-terminal His-tag and the purification and the refolding of the HP0421 protein from the inclusion bodies. Finally, refolding procedure was optimized and the protein was purified to high homogeneity. HP0231 was characterized employing immunological techniques and blot with patient blood sera. HP0231 was also further characterized using bioinformatics tools.
Il batterio Helicobacter pylori è riconosciuto essere uno dei più diffusi patogeni umani: esso colonizza circa la metà della popolazione umana, con una elevata velocità di propagazione nei paesi in via di sviluppo. Benché molti soggetti infetti siano asintomatici, una significativa minoranza di questi (15-20%) sviluppano durante la loro vita patologie duodenali gravi, che includono ulcera gastrica e duodenale, adeno-carcinoma e linfoma dello stomaco. Fino ad oggi sono stati completamente sequenziati nove diversi ceppi di H. pylori, in quanto esso presenta una alta variabilità genetica, non solo nelle sequenze geniche, ma anche nel contenuto di geni. Molti dei geni del batterio sono annotati solo sulla base dell’omologia di sequenza e per circa il 45% di essi la funzione è incerta o addirittura ignota. La differenza più significativa tra i ceppi virulenti del batterio rispetto ai ceppi non virulenti è la presenza o assenza della cosiddetta cag-PAI (una sequenza del DNA di 40-kb definita “isola di patogenicità cag”), un inserto genico che codifica per un sistema di secrezione di tipo IV, responsabile della trasloscazione della tossina CagA nelle cellule epiteliali. Benché H. pylori in molti pazienti possa essere sradicato mediante antibiotici, l’aumento della resistenza in alcuni ceppi rappresenta un problema emergente. Nuove terapie sono richieste per combattere il batterio e l’individuazione di nuovi bersagli farmacologici può essere utile per sviluppare nuove strategie di trattamento. Recentemente, nuovi fattori importanti per la colonizzazione e per lo stabilirsi dell’infezione sono stati identificati. In questo lavoro di tesi, un gruppo di queste proteine sono state clonate, espresse in E. coli e purificate allo scopo di effettuare studi strutturali. Obiettivo della tesi era quello di determinare la struttura tridimensionale e caratterizzare la funzione di proteine importanti per la colonizzazione dello stomaco e per la patogenesi. In particolare, gli sforzi sono stati concentrati sugli enzimi coinvolti nella modifica della parete cellulare (peptidoglicano deacetilasi), nella biosintesi di LPS (ADP-L-glycero-D-manno-eptoso-6-epimerasi, rfaD), su una periplasmic-substrate binding protein di un trasportatore ABC (ceuE), su enzimi chiave nel ciclo di assimilazione dell’azoto (glutamina sintasi) e sulla disolfuro ìsomerasi (DsbG), una proteina immunogenica secreta. La strategia applicata è consistita in una analisi bioinformatica preliminare, nell’ottenimento del gene a partire da amplificazione mediante PCR, nella costruzione di un vettore per la clonazione e nell’espressione della proteina in E. coli. Dopo analisi dell’espressione e ottimizzazione delle condizioni, è stata analizzata la solubilità della proteina ricombinante. Quest’ultima è stata quindi purificata mediante diverse tecniche cromatografiche, ed eventualmente caratterizzata per gel-filtrazione analitica, spettrometria di massa, spettroscopia UV. Sono state usate tecniche per rendere i campioni di proteina più adatti per la cristallizzazione, quali DLS (dynamic light scattering) e per investigarne la struttura secondaria, quali CD (dicroismo circolare). La proteina è stata quindi concentrata prima di essere sottoposta ai test di cristallizzazione. I dati di diffrazione sono stati misurati ai sincrotroni ESRF (Grenoble, Francia). La caratterizzazione funzionale delle proteine è stata eseguita usando spettroscopia di fluorescenza, CD, ITC, UV ed ELISA. Dopo una introduzione generale sul batterio (Capitolo 1), nel capitolo 2 viene descritta la struttura tridimensionale e l’attività enzimatica di una putativa peptidoglicano deacetilasi. HP0310 (HpPdgA) da H. pylori è stato indicato come l’enzima responsabile della modifica del peptidoglicano che serve a minimizzare la risposta immunitaria da parte dell’ospite. L’enzima, che appartiene alla famiglia delle polisaccaride deacetilasi, è un omo-tetramero. Le quattro catene polipeptidiche, ciascuna avvolta in un dominio singolo caratterizzato da un TIM-barrel non canonico, sono arrangiate attorno ad un asse di rotazione quaternario. Il sito attivo, uno per monomero, contiene uno ione coordinato in modo simile ad altre deacetilasi. L’enzima non presenta però in vitro attività sui tipici substrati delle peptidoglicano deaetilasi. In netto contrasto con altre peptidoglicano deacetilasi conosciute, HpPdgA non ha un sito di legame accessibile ad una molecola ingombrante quale un polisaccaride, suggerendo che l’enzima leghi nel proprio sito attivo un substrato di piccole dimensioni. Nel capitolo 3 viene discussa in dettaglio la struttura cristallina dell’ultimo degli enzimi del ciclo della biosintesi dell’oligosaccaride (L,D-eptoso) del core di LPS. H. pylori deve molta dell’integrità della sua membrana esterna ai lipopolisaccaridi (LPS). Insieme al loro esenziale ruolo strutturale, gli LPS contribuiscono alle proprietà di aderenze del batterio, come anche alla modulazione della risposta immunitaria. L’oligosaccaride del core del batterio, uno dei tre principali domini dell’LPS, presenta una struttura peculiare nell’organizzazione della ramificazione delle unità che si ripetono. Queste mostrano ulteriore variabilità quando si confrontano ceppi diversi. In questo capitolo viene presentata la struttura cristallina della ADP-L-glicero-D-manno-eptoso-6-epimerasi (HP0859, rfaD), l’ultimo enzima del ciclo che produce L-glicero-D-manno-eptoso partendo da sedoeptuloso-7-fosfato, un composto cruciale nella sintesi dell’oligosaccaride del core. In uno studio recente è stato caratterizzato un mutante knok-out di HP0859 che mostra, in un modello di infezione in cellule AGS, una seria perdita di struttura del lipopolisaccaride e una significativa riduzione dei livelli di adesione, se paragonato ai ceppi wild-type. La struttura cristallina rivela che l’enzima è un omo-pentamero e che NAD è legato come cofattore in una cavità altamente conservata. Il sito di legame del substrato è molto simile a quello del suo ortologo in E. coli, suggerendo anche un simile meccanismo catalitico. Altri enzimi del ciclo sono discussi nei termini della loro struttura tridimensionale. Nel capitolo 4 viene descritta la struttura tridimensionale di una binding-protein al trasportatore periplasmico ABC. E’ noto dalla letteratura che il batterio può utilizzare l’eme come sola sorgente di ferro, e sono state identificate sorgenti di ferro, derivanti dall’ospite, utilizzate da H. pylori, inclusi composti provenienti da gruppi eme da tessuti danneggiati. Il trasporto di eme entro il citoplasma è effettuato da un sistema di trasporto attivo che comprende una proteina periplasmica solubile, una permease citoplasmatica e una ATPase (ABC transporter). La proteina periplasmica che lega l’eme è stata identificata e caratterizzata in vari batteri patogeni, ma fino ad ora non era ancora stata identificata una heme-binding protein in H. pylori. Hp1561 (ceuE) era annotata come una “ABC transporter periplasmic binding protein”, ma un gene omologo from H. mustalae era stato riportato essere coinvolto nel trasporto del nichel. Per chiarire le caratteristiche strutturali di questa putativa proteina di trasporto, essa è stata clonata, espressa e purificata con buona resa in E. coli, cristallizzata e la sua struttura determinata nella forma apo- and in complesso con il Ni(II). La struttura è stata risolta per mezzo di esperimenti di dispersione anomala singola (SAD) su cristalli di Se-metionina. Il modello molecolare è costituito da due domini, collegati da una lunga α-elica e presenta le caratteristiche generali di altre “heme-binding periplasmic ABC transporters” o di “B12 binding-proteins”. Il sito di legame del substrato è localizzato tra i due domini. La struttura cristallina suggerisce che il Ni(II) non è il legante naturale della proteina e che la cavità di legame assomiglia di più a quella delle heme-binding proteins. Sono stati effettuati anche studi di legame in vitro con tecniche diverse (fluorescenza e ITC), che hanno confermato che ceuE in H. pylori è una “periplasmic heme-binding protein”, responsabile per l’assunzione dell’eme. La struttura cristallina di un enzima chiave nell’unico ciclo di assimilazione dell’azoto in H. pylori è discusso nel capitolo 5. La glutamina sintetasi (GS) catalizza la sintesi di glutamina, un intermedio centrale nel metabolismo dell’azoto, da ATP, glutammato e ammoniaca, in una reazione dipendente da un catione bivalente. L’ammoniaca, che è anche una sorgente preferita di azoto per H. pylori, è disponibile in grande quantità, grazie all’attività ureasica del batterio. E’ assimilato in proteine e altri composti contenenti azoto attraverso un singolo ciclo di incorporazione dell’azoto, mediato da GS, un enzima codificato dal gene hp0512. L’assenza di un sito si regolazione allosterico (sito di adenilazione) e di altri enzimi chiave nel ciclo di assimilazione dell’azoto rende HpGS un interessante soggetto per gli studi strutturali, per chiarirne le caratteristiche strutturali e il meccanismo regolatorio. La glutamina sintetasi (HpGS) di H. pylori è stata clonata, espressa, purificata e cristallizzata e la sua struttura determinata. L’enzima è un dodecamero, i cui monomeri sono tenuti assieme soprattutto da interazioni idrofobiche e legami ad idrogeno tra i due anelli esamerici. L’elica N-terminale, chiamata “elica stringa”, è inserita in una cavità idrofobia nella subunità eclissata sull’anello esamerico opposto. In aggiunta, il canale centrale del dodecamero è formato da sei fogli L a quattro fogli beta, ciascuno costituito da un loop antiparallelo cui contribuiscono subunità in anelli opposti. La struttura di un monomero consiste di un piccolo dominio N-terminale e di un dominio C-terminale più grande. L’enzima dodecamerico contiene 12 siti attivi, ciascuno dei quali può essere descritto come un “bifunnel”, in cui ATP e glutammato legano da lati opposti. Il sito di legame dell’ATP è localizzato nella parte alta del bifunnel, alla giunzione del quale ci sono due siti per il legame di cationi bivalenti. Il sito di adenilazione che in tutti gli enzimi omologhi contiene la sequenza consenso NLYDLP è sostituita in H. pylori da NLFKLT (residui da 405 a 410). Poiché la tirosina 407 è il bersaglio conservato dell’adenilazione e H. pylori apparentemente manca di questo residuo, non c’è adenilazione nell’enzima e non si osservano modifiche strutturali significative in questo loop a confronto con altre strutture di GS. Nel capitolo 6 vengono discussi la clonazione, l’espressione e la caratterizzazione della proteina immunogenica secreta DsbG. Le proteine coinvolte nell’isomerizzazione dei ponti disolfuro sono ben note. Esse sono localizzate nelle membrane o nel periplasma e sono chiamate Dsb (Disulfide bond formation). Questi enzimi catalizzano l’introduzione di ponti disolfuro, la loro isomerizzazione o rimozione. Secernere alcune proteine è un modo attraverso il quale un batterio può interagire con l’ospite e in H. pylori questo avviene attraverso uno dei tre sistemi di secrezione di tipo IV presenti. Molte tra le proteine secrete, alcune delle quali risiedono nello spazio periplasmico, formano ponti a disolfuro dopo la traslocazione. HP0231 ha una sequenza simile a Dsbg di E. coli e contiene un motivo CXXC. Essa è stata già identificata come una proteina immunogenica riconosciuta da sieri di pazienti. La proteina ricombinante, espressa in E. coli, non lega fortemente alla resina di affinità IMAC-Ni2+, probabilmente a causa di degradazione dell’His-tag presente all’N-terminale. Per risolvere il problema della purificazione sono perciò stati adottati due approcci: è stato preparato un nuovo costrutto di HP0231 con His-tag al C-terminale, e in parallelo la proteina è stata purificata dai corpi di inclusione e poi rifoldata. La proteina è infine stata purificata ad alta omogeneità. HP0231 è stata caratterizzata usando tecniche immunologiche e blot con sieri di pazienti.