Made available in DSpace on 2014-12-17T14:55:57Z (GMT). No. of bitstreams: 1 FranciscoJTV.pdf: 1100058 bytes, checksum: 1324dc0f705af005f06ca71bee5c289c (MD5) Previous issue date: 2005-12-19 / The method of artificial lift of progressing cavity pump is very efficient in the production of oils with high viscosity and oils that carry a great amount of sand. This characteristic converted this lift method into the second most useful one in oil fields production. As it grows the number of its applications it also increases the necessity to dominate its work in a way to define it the best operational set point. To contribute to the knowledge of the operational method of artificial lift of progressing cavity pump, this work intends to develop a computational simulator for oil wells equipped with an artificial lift system. The computational simulator of the system will be able to represent its dynamic behavior when submitted to the various operational conditions. The system was divided into five subsystems: induction motor, multiphase flows into production tubing, rod string, progressing cavity pump and annular tubing-casing. The modeling and simulation of each subsystem permitted to evaluate the dynamic characteristics that defined the criteria connections. With the connections of the subsystems it was possible to obtain the dynamic characteristics of the most important arrays belonging to the system, such as: pressure discharge, pressure intake, pumping rate, rod string rotation and torque applied to polish string. The shown results added to a friendly graphical interface converted the PCP simulator in a great potential tool with a didactic characteristic in serving the technical capability for the system operators and also permitting the production engineering to achieve a more detail analysis of the dynamic operational oil wells equipped with the progressing cavity pump / O m?todo de eleva??o artificial por bombeio de cavidades progressivas tem se mostrado muito eficiente na produ??o de ?leos com alta viscosidade ou que carreiem uma grande quantidade de areia. Essa caracter?stica tem feito do mesmo o segundo m?todo de eleva??o mais utilizado nos campos de produ??o de petr?leo. ? medida em que aumenta o n?mero de suas aplica??es cresce tamb?m a necessidade de um maior dom?nio do conhecimento quanto ao seu funcionamento operacional, de maneira ? definir um ponto ?timo de opera??o. A fim de contribuir para a expans?o do conhecimento operacional do m?todo de eleva??o por bombeio de cavidades progressivas, este trabalho prop?e o desenvolvimento de um simulador computacional para po?os de petr?leo equipados com esse sistema, capaz de representar o comportamento din?mico do mesmo quando submetido ?s mais diversas condi??es operacionais. O sistema foi dividido em cinco subsistemas: motor de indu??o, escoamento multif?sico na coluna de produ??o, coluna de haste, bomba de cavidades progressivas e anular revestimento-coluna de produ??o. A modelagem e a simula??o de cada subsistema permitiram avaliar as caracter?sticas din?micas que definiram os crit?rios de acoplamento dos mesmos. Com a realiza??o do acoplamento foi poss?vel obter as caracter?sticas din?micas das principais vari?veis pertinentes ao sistema, tais como: press?o de recalque, press?o de suc??o, vaz?o da bomba, rota??o da coluna de haste e o torque aplicado ? haste polida. Os resultados apresentados, somados a uma interface gr?fica amig?vel, fazem do simulador de BCP uma ferramenta de grande potencial, tanto de car?ter did?tico, auxiliando na capacita??o t?cnica dos operadores do sistema, quanto para aux?lio na engenharia de produ??o, permitindo realizar uma an?lise mais detalhada da din?mica operacional dos po?os de petr?leo equipado com esse m?todo de eleva??o

Made available in DSpace on 2014-12-17T14:57:56Z (GMT). No. of bitstreams: 1 PauloASP.pdf: 2445269 bytes, checksum: 158c07b839a3cc9c6803734bb383eed0 (MD5) Previous issue date: 2009-12-15 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The use of Progressing Cavity Pumps (PCPs) in artificial lift applications in low deep wells is becoming more common in the oil industry, mainly, due to its ability to pump heavy oils, produce oil with large concentrations of sand, besides present high efficiency when compared to other artificial lift methods. Although this system has been widely used as an oil lift method, few investigations about its hydrodynamic behavior are presented, either experimental or numeric. Therefore, in order to increase the knowledge about the BCP operational behavior, this work presents a novel computational model for the 3-D transient flow in progressing cavity pumps, which includes the relative motion between rotor and stator, using an element based finite volume method. The model developed is able to accurately predict the volumetric efficiency and viscous looses as well as to provide detailed information of pressure and velocity fields inside the pump. In order to predict PCP performance for low viscosity fluids, advanced turbulence models were used to treat, accurately, the turbulent effects on the flow, which allowed for obtaining results consistent with experimental values encountered in literature. In addition to the 3D computational model, a simplified model was developed, based on mass balance within cavities and on simplification on the momentum equations for fully developed flow along the seal region between cavities. This simplified model, based on previous approaches encountered in literature, has the ability to predict flow rate for a given differential pressure, presenting exactness and low CPU requirements, becoming an engineering tool for quick calculations and providing adequate results, almost real-time time. The results presented in this work consider a rigid stator PCP and the models developed were validated against experimental results from open literature. The results for the 3-D model showed to be sensitive to the mesh size, such that a numerical mesh refinement study is also presented. Regarding to the simplified model, some improvements were introduced in the calculation of the friction factor, allowing the application fo the model for low viscosity fluids, which was unsuccessful in models using similar approaches, presented in previous works / O uso de bombas de cavidades progressivas (BCP) para eleva??o artificial de petr?leo em po?os de baixa a m?dia profundidade est? se tornando mais comum na ind?stria do petr?leo devido, dentre outras caracter?sticas, ? sua habilidade de bombear ?leos pesados, produzir ?leo com grandes concentra??es de part?culas s?lidas, e tolerar grandes fra??es de g?s livre. Al?m disso, apresenta alta efici?ncia, quando comparado a outros sistemas de eleva??o artificial. Apesar de este sistema ser bastante utilizado na eleva??o de ?leo, poucas investiga??es acerca do seu comportamento hidrodin?mico podem ser encontradas na literatura. Desta forma, devido ? necessidade de obter um maior dom?nio do comportamento operacional da BCP, este trabalho apresenta um novo modelo computacional para escoamento 3-D transiente em bombas de cavidades progressivas, utilizando o m?todo dos volumes finitos baseado em elementos, no qual se inclui o movimento relativo entre o rotor e o estator. O modelo desenvolvido ? capaz de predizer com exatid?o par?metros operacionais, como efici?ncia volum?trica e perdas viscosas, assim como fornecer informa??es detalhadas dos campos de press?o e velocidade no interior da bomba. Para tornar poss?vel o estudo com BCP s bombeando fluidos de baixa viscosidade, foram utilizados modelos de turbul?ncia avan?ados para tratar adequadamente os efeitos turbulentos no escoamento, os quais permitiram obter informa??es coerentes com a literatura do comportamento da BCP em resultados experimentais. Al?m do modelo tridimensional, este trabalho apresenta um modelo simplificado baseado em um balan?o de massa entre as cavidades e na simplifica??o das equa??es do movimento para escoamento completamente desenvolvido ao longo das linhas de selo entre as cavidades. O modelo simplificado, baseado em abordagens j? utilizadas para este tipo de bomba, tem capacidade de predizer valores de vaz?o para um dado diferencial de press?o, apresentando exatid?o e requerendo pouco trabalho de CPU, tornando-se uma ferramenta de engenharia de uso f?cil, permitindo respostas num?ricas aproximadas em tempo-real. Os resultados apresentados neste trabalho consideraram uma BCP de estator r?gido e os modelos apresentados foram validados com resultados experimentais da literatura. Os resultados para o modelo tridimensional mostraram-se sens?veis ao tamanho da malha, de maneira que um estudo de refinamento de malha ? apresentado. J? no modelo simplificado foram introduzidos alguns melhoramentos no c?lculo do fator de atrito permitindo a aplica??o para fluidos de baixa viscosidade, o que n?o era poss?vel em modelos similares apresentados em trabalhos anteriores

Previous research has been conducted to try to explain the relationship between stress and focus for the use of different platforms for work communication. On the other hand, during a pandemic there is a lot that separates everyday life from how it was before the pandemic began and working from home was not as obvious as it is today. Applications whose number of daily users has skyrocketed can also account for a perceived stress for a user - how does this affect individuals’ ability to focus on the application? A survey was created and published for two weeks using previous research that examined the links be- tween stress and focus on social media. The study was conducted by collecting data from various forums related to relevant applications, where it is discovered that stress has a weak but significant effect on distractions outside applications and on distractions within applications. The results show that a perceived distraction outside the application can for 22% be explained by stress and within the application can for 16.6% be explained by stress. Results and the similarities between social media and platforms for work commu- nication are compared and discussed based on the presented theory and related research. / Tidigare forskning har utförts för att försöka förklara sambandet mellan stress och fokus för användandet av olika plattformar för arbetskommunikation. Däremot är det under en pandemi mycket som skiljer vardagen åt från hur det var innan pandemin började och hemmaarbete inte var en sådan självklarhet som det är i dagens läge. Applikationer som vars antal dagliga användare skjutit i höjden kan också stå för en upplevd stress för en användare- hur påverkar detta en individs förmåga att fokusera på applikationen? En enkät utformas och publicerades under två veckor med hjälp av tidigare forskning som undersökt kopplingen mellan stress och fokus för sociala medier. Undersökningen bedrivs genom att data samlas in från varierande forum tillhörande olika relevanta applikationer, där det kommer fram att stress har en svag, men signifikant påverkan på distraktioner utanför applikationen och på distraktioner inom applikationen. Resultaten visar att en upplevd distraktion utanför applikationen kan 22% förklaras av stress och inom applikationen kan 16.6% förklaras av stress. Resultaten och likheterna mellan sociala medier och plattformar för arbetskommunikation jämförs och diskuteras utifrån den presenterade teorin och relaterad forskning.

Im Rahmen dieser Diplomarbeit wurde eine Ultrahochvakuumanlage für organische Molekularstrahldeposition (OMBD) konzipiert und gefertigt, die das Aufwachsen von einzelnen organischen Schichten bis hin zu komplexen Schichtsystemen auf geeigneten Substraten erlaubt. Mit einem ebenfalls konzipierten und gefertigten Probenhalter, sind elektrische Messungen in situ möglich. Für weitere Charakterisierungsmethoden, wie der spektroskopischen Ellipsometrie und der Reflexions-Anisotropie-Spektroskopie sind ebenfalls Optionen für in-situ-Messungen an der UHV-Anlage vorgesehen. Mit dieser Anlage wurden einzelne organische Schichten von Zinkphthalocyanin (ZnPc), Fulleren C60 und Bathocuproin (BCP) hergestellt, die anschließend mit spektroskopischer Ellipsometrie ex situ untersucht wurden. Mit der Herstellung organischer Solarzellen, auf Basis der zuvor hergestellten organischen Einzelschichten, konnte gezeigt werden, dass mit der UHV-Anlage komplexe organische Schichtsysteme erzeugt werden können, an denen in-situ-elektrische Messungen durchführbar sind. / Within the scope of this diploma thesis, a ultra high vacuum chamber for organic molecular beam deposition (OMBD) was designed and built, which allows the growth of single organic layers and complex composit layer structures. With an also designed and built sample holder, it is possible to make in situ electrical measurements. Single organic layers of zinc-phthalocyanine (ZnPc), fullerene C60 and bathocuproine (BCP) were deposited inside this chamber and characterized ex situ by spectroscopic ellipsometry. The preparation of an organic photovoltaic (OPV) cell based on the before characterized single layers, demonstrates that it is possible to deposit complex layer structures and characterize them electrical in situ.

Yes / Recently, the demand for border crossing has increased massively, with the aim to increase the processing and clearance speed at border crossing points (BCP). The attempt to improve travel convenience, Border Cross Point (BCP) output, and national security result in automated border control (ABC) with biometric technology having a major effect on the efficiency, and safety of the control processes. The border processing of BCP can be increased by automating biometric recognition and facilitated by clearance procedures. This paper discussed the two structures of an e-gate (ABC) and a prospective benefit of biometrics to the EU border in terms of accuracy, integrity, robustness, and efficiency. Challenges posed by biometrics in border control systems were identified and recommendations such as multimodal systems and smart systems with AI and machine learning were suggested to assist travelers to cross border points faster. / e European Union’s Horizon-MSCA-RISE-2019-2023, Marie Skłodowska-Curie, Research, and Innovation Staff Exchange (RISE), titled: Secure and Wireless Multimodal Biometric Scanning Device for Passenger Verification Targeting Land and Sea Border Control

A bactéria fitopatogênica Xylella fastidiosa é o agente etiológico da Clorose Variegada dos Citros (CVC), que causa perdas anuais estimadas em US$ 100 milhões no Brasil. Durante o processo infeccioso, a geração extracelular de espécies ativas de oxigênio é um dos principais mecanismos de defesa da planta contra o patógeno. Em contrapartida, para se defender do estresse oxidativo imposto pelo hospedeiro, os fitopatógenos possuem mecanismos de defesa que incluem enzimas antioxidantes, como as peroxirredoxinas, alquil hidroperóxido redutase subunidade C (AhpC) e proteína comigratória com bacterioferritina (Bcp). As peroxirredoxinas são proteínas que utilizam suas cisteínas ativas para catalisar a redução de hidroperóxidos. Por análise proteômica, os produtos dos genes ahpc e bcp foram identificados no extrato celular protéico de X. fastidiosa (Smolka e col., 2003). Com o intuito de caracterizar funcional e estruturalmente as proteínas AhpC e Bcp de X. fastidiosa, clonamos e expressamos seus respectivos genes em Escherichia coli e purificamos as proteínas por cromatografia de afinidade a níquel. As proteínas recombinantes apresentaram atividade dependente de tiól de redução de peróxido de hidrogênio e hidroperóxidos orgânicos. A atividade peroxidase da AhpC e Bcp são dependentes, respectivamente, de alquil hidroperóxido redutase subunidade F (AhpF) e do sistema tiorredoxina. Paradoxalmente, a flavoproteína AhpF possui atividade NAD(P)H oxidase, que resulta na produção de peróxido de hidrogênio. As constantes de segunda ordem da reação das proteínas com peróxido de hidrogênio (da ordem de 107 M-1.s-1), determinadas pelo ensaio de cinética competitiva com peroxidase de raiz forte, indicam que ambas possuem atividades peroxidase equivalentes às apresentadas por glutationa peroxidases dependentes de selênio e catalases, ao contrário do descrito na literatura. Por SDS-PAGE não-redutor e pela quantificação de cisteínas livres por DTNB, verificamos que as proteínas possuem mecanismos catalíticos distintos: AhpC é uma 2-Cys Prx típica (com formação de ponte dissulfeto intermolecular), enquanto Bcp é uma 2-Cys Prx atípica (com formação de ponte dissulfeto intramolecular). Para AhpC, a atividade catalítica envolve as cisteínas conservadas (Cys-47 e Cys-165), em contraste, apenas através de estudos de mutação sítio-dirigida e espectrometria de massas conseguimos identificar os resíduos de cisteínas envolvidos na atividade catalítica da Bcp (Cys-47 e Cys-83). A caracterização estrutural de AhpC por cromatografia de exclusão molecular e espalhamento dinâmico de luz mostram que a proteína nativa é um decâmero estável, independentemente do estado de oxidação de suas cisteínas. A caracterização da estrutura cristalográfica de Bcp C47S, inédita para 2-Cys Prx atípicas que possuem as cisteínas ativas separadas por 35 aminoácidos, indica que a proteína possui o enovelamento característico das peroxirredoxinas e que as cisteínas ativas estão localizadas a uma distância média de 12,4 Å. Baseado em dicroísmo circular, apresentamos dados que indicam que a aproximação das cisteínas deve envolver um significativo rearranjo estrutural, que provavelmente se inicia com a formação do intermediário ácido sulfênico na cisteína peroxidásica (Cys-47). Assim, conseguimos elucidar o papel catalítico dessas proteínas, bem como identificar seus sistemas redutores, obtendo informações que podem ser relevantes para o entendimento do mecanismo da patogenicidade da X. fastidiosa. Os resultados apresentados neste trabalho podem contribuir para o desenvolvimento de novas técnicas de controle de praga para a doença CVC em citrus e outras que envolvam a bactéria X. fastidiosa. / The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of Citrus Variegated Chlorosis (CVC) that causes losses of about 100 millions dollars per year in Brazil. During infection, reactive oxygen species play a central role in plant pathogen defense. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including the peroxiredoxins alkyl hydroperoxide reductase subunit C (AhpC) and bacterioferritin comigratory protein (Bcp). Peroxiredoxins are peroxidases, which rely on an activated cysteine residue to catalyze the reduction of hydroperoxides. By proteome analysis, Smolka et al. (2003) identified the products of ahpc and bcp genes present in whole cell extract of X. fastidiosa. To characterize the function and structure of AhpC and Bcp protein, their genes were cloned in Escherichia coli and the corresponding proteins purified by nickel affinity chromatography. Recombinant proteins presented thiol-dependent peroxidase activity against hydrogen peroxide and organic hydroperoxides. AhpC and Bcp peroxidase activities are dependent on alkyl hydroperoxide reductase subunit F (AhpF), and on thioredoxin system, respectively. Paradoxically, AhpF flavoenzyme possesses hydrogen peroxide-forming oxidase activity. Contrary to classical assumptions, competitive kinetics employing horseradish peroxidase assays showed that the second-order rate constants of AhpC and Bcp reaction with hydrogen peroxide are in the order of 107 M-1.s-1, as fast as the activity of selenium-dependent glutathione peroxidases and catalases. Non-reducing SDS-PAGE and cysteine quantification using DTNB indicated different peroxidasic mechanisms: AhpC is a typical 2-Cys peroxiredoxin (with intermolecular disulfide bond formation), while Bcp is an atypical 2-Cys peroxiredoxin (with intramolecular disulfide bond formation). In contrast to the well-conserved AhpC cysteines responsible for the peroxidase activity (Cys-47 and Cys-165), only through site-specific mutagenesis and mass spectrometry we could identified the cysteine residues involved in the Bcp peroxidase activity (Cys-47 and Cys-83). Structural characterization by size exclusion chromatography and dynamic light scattering revealed that AhpC native protein forms stable and redox state independent decamers. The crystal structure of Bcp C47S, the first 2-Cys Prx with a 35-residue between the active cysteines ever characterized, shows that protein contains the common fold of peroxiredoxins and that active cysteines lies ~12.4 Å away one from the other. Based on circular dichroism, we presented data indicating that disulfide bond formation may require significant conformational changes, which probably is triggered by the peroxidatic cysteine oxidation to sulfenic acid. In conclusion, we elucidated the catalytic mechanisms and reduction systems of AhpC and Bcp proteins that may help to understand the pathogenicity mechanism of X. fastidiosa. These results can contribute to the development of plague control methods against X. fastidiosa.

Ce travail a été réalisé dans le cadre d'une convention CIFRE entre le LAAS-CNRS et la société ACTIA qui développe des outils d'aide au diagnostic de pannes automobiles. Le but était l'utilisation des réseaux de neurones artificiels pour la conception d'une nouvelle méthode de diagnostic de pannes automobiles sans modèle, ni information symbolique mais avec seulement des observations du système en bon et en mauvais fonctionnement. C'est donc une approche du diagnostic de système complexe par reconnaissance de formes. Après avoir mis au point le système d'acquisition, nous avons conçu une première maquette qui a démontré la faisabilité d'un tel système et l'intérêt des réseaux de neurones, mais qui a soulevé de nombreux problèmes. L'utilisation particulière des réseaux de neurones dans cette application a nécessité l'usage d'algorithmes de construction. Après une étude théorique des structures de l'hypercube qui n'a pas abouti à un algorithme de construction mais qui a fourni plusieurs résultats, nous avons développé une famille d'algorithmes pour la construction des réseaux de neurones binaires. La base de ces outils est une nouvelle méthode d'apprentissage d'unités à seuil très performante dénommée Barycentric Correction Procedure (BCP). L'aboutissement est un algorithme novateur car très général (entrées quelconques et sorties multiples), rapide et avec un bon pouvoir de généralisation. Nous avons finalement mis au point une nouvelle méthodologie de diagnostic, dans laquelle l'utilisation de méthodes statistiques et d'analyse de données en collaboration avec les réseaux neuronaux paru nécessaire. Cette méthodologie utilise donc des techniques très diverses : analyse en composantes principales, estimation de densité de probabilité, classification automatique, calcul d'enveloppes convexes, génération géométrique de bases d'apprentissage, construction de réseaux de neurones binaires, réseaux de neurones gaussiens et méthodes de diagnostic simples. Cette méthodologie a été appliquée avec succès au problème de la détection de pannes automobiles et a aussi montré des potentialités pour le diagnostic préventif. Elle est de plus assez générique pour avoir de nombreuses applications potentielles.

Ranking a set of candidates or items from pair-wise comparisons is a fundamental problem that arises in many settings such as elections, recommendation systems, sports team rankings, document rankings and so on. Indeed it is well known in the psychology literature that when a large number of items are to be ranked, it is easier for humans to give pair-wise comparisons as opposed to complete rankings. The problem of ranking from pair-wise comparisons has been studied in multiple communities such as machine learning, operations research, linear algebra, statistics etc., and several algorithms (both classic and recent) have been proposed. However, it is not well under-stood under what conditions these different algorithms perform well. In this thesis, we aim to fill this fundamental gap, by elucidating precise conditions under which different algorithms perform well, as well as giving new algorithms that provably perform well under broader conditions. In particular, we consider a natural statistical model wherein for every pair of items (i; j), there is a probability Pij such that each time items i and j are compared, item j beats item i with probability Pij . Such models, which we summarize through a matrix containing all these pair-wise probabilities, have been used explicitly or implicitly in much previous work in the area; we refer to the resulting matrix as the pair-wise preference matrix, and elucidate clearly the crucial role it plays in determining the performance of various algorithms. In the first part of the thesis, we consider a natural generative model where all pairs of items can be sampled and where the underlying preferences are assumed to be acyclic. Under this setting, we elucidate the conditions on the pair-wise preference matrix under which popular algorithms such as matrix Borda, spectral ranking, least squares and maximum likelihood under a Bradley-Terry-Luce (BTL) model produce optimal rankings that minimize the pair-wise disagreement error. Specifically, we derive explicit sample complexity bounds for each of these algorithms to output an optimal ranking under interesting subclasses of the class of all acyclic pair-wise preference matrices. We show that none of these popular algorithms is guaranteed to produce optimal rankings for all acyclic preference matrices. We then pro-pose a novel support vector machine based rank aggregation algorithm that provably does so. In the second part of the thesis, we consider the setting where preferences may contain cycles. Here, finding a ranking that minimizes the pairwise disagreement error is in general NP-hard. However, even in the presence of cycles, one may wish to rank 'good' items ahead of the rest. We develop a framework for this setting using notions of winners based on tournament solution concepts from social choice theory. We first show that none of the existing algorithms are guaranteed to rank winners ahead of the rest for popular tournament solution based winners such as top cycle, Copeland set, Markov set etc. We propose three algorithms - matrix Copeland, unweighted Markov and parametric Markov - which provably rank winners at the top for these popular tournament solutions. In addition to ranking winners at the top, we show that the rankings output by the matrix Copeland and the parametric Markov algorithms also minimize the pair-wise disagreement error for certain classes of acyclic preference matrices. Finally, in the third part of the thesis, we consider the setting where the number of items to be ranked is large and it is impractical to obtain comparisons among all pairs. Here, one samples a small set of pairs uniformly at random and compares each pair a fixed number of times; in particular, the goal is to come up with good algorithms that sample comparisons among only O(nlog(n)) item pairs (where n is the number of items). Unlike existing results for such settings, where one either assumes a noisy permutation model (under which there is a true underlying ranking and the outcome of every comparison differs from the true ranking with some fixed probability) or assumes a BTL or Thurstone model, we develop a general algorithmic framework based on ideas from matrix completion, termed low-rank pair-wise ranking, which provably produces an good ranking by comparing only O(nlog(n)) pairs, O(log(n)) times each, not only for popular classes of models such as BTL and Thurstone, but also for much more general classes of models wherein a suitable transform of the pair-wise probabilities leads to a low-rank matrix; this subsumes the guarantees of many previous algorithms in this setting. Overall, our results help to understand at a fundamental level the statistical properties of various algorithms for the problem of ranking from pair-wise comparisons, and under various natural settings, lead to novel algorithms with improved statistical guarantees compared to existing algorithms for this problem.

A bactéria fitopatogênica Xylella fastidiosa é o agente etiológico da Clorose Variegada dos Citros (CVC), que causa perdas anuais estimadas em US$ 100 milhões no Brasil. Durante o processo infeccioso, a geração extracelular de espécies ativas de oxigênio é um dos principais mecanismos de defesa da planta contra o patógeno. Em contrapartida, para se defender do estresse oxidativo imposto pelo hospedeiro, os fitopatógenos possuem mecanismos de defesa que incluem enzimas antioxidantes, como as peroxirredoxinas, alquil hidroperóxido redutase subunidade C (AhpC) e proteína comigratória com bacterioferritina (Bcp). As peroxirredoxinas são proteínas que utilizam suas cisteínas ativas para catalisar a redução de hidroperóxidos. Por análise proteômica, os produtos dos genes ahpc e bcp foram identificados no extrato celular protéico de X. fastidiosa (Smolka e col., 2003). Com o intuito de caracterizar funcional e estruturalmente as proteínas AhpC e Bcp de X. fastidiosa, clonamos e expressamos seus respectivos genes em Escherichia coli e purificamos as proteínas por cromatografia de afinidade a níquel. As proteínas recombinantes apresentaram atividade dependente de tiól de redução de peróxido de hidrogênio e hidroperóxidos orgânicos. A atividade peroxidase da AhpC e Bcp são dependentes, respectivamente, de alquil hidroperóxido redutase subunidade F (AhpF) e do sistema tiorredoxina. Paradoxalmente, a flavoproteína AhpF possui atividade NAD(P)H oxidase, que resulta na produção de peróxido de hidrogênio. As constantes de segunda ordem da reação das proteínas com peróxido de hidrogênio (da ordem de 107 M-1.s-1), determinadas pelo ensaio de cinética competitiva com peroxidase de raiz forte, indicam que ambas possuem atividades peroxidase equivalentes às apresentadas por glutationa peroxidases dependentes de selênio e catalases, ao contrário do descrito na literatura. Por SDS-PAGE não-redutor e pela quantificação de cisteínas livres por DTNB, verificamos que as proteínas possuem mecanismos catalíticos distintos: AhpC é uma 2-Cys Prx típica (com formação de ponte dissulfeto intermolecular), enquanto Bcp é uma 2-Cys Prx atípica (com formação de ponte dissulfeto intramolecular). Para AhpC, a atividade catalítica envolve as cisteínas conservadas (Cys-47 e Cys-165), em contraste, apenas através de estudos de mutação sítio-dirigida e espectrometria de massas conseguimos identificar os resíduos de cisteínas envolvidos na atividade catalítica da Bcp (Cys-47 e Cys-83). A caracterização estrutural de AhpC por cromatografia de exclusão molecular e espalhamento dinâmico de luz mostram que a proteína nativa é um decâmero estável, independentemente do estado de oxidação de suas cisteínas. A caracterização da estrutura cristalográfica de Bcp C47S, inédita para 2-Cys Prx atípicas que possuem as cisteínas ativas separadas por 35 aminoácidos, indica que a proteína possui o enovelamento característico das peroxirredoxinas e que as cisteínas ativas estão localizadas a uma distância média de 12,4 Å. Baseado em dicroísmo circular, apresentamos dados que indicam que a aproximação das cisteínas deve envolver um significativo rearranjo estrutural, que provavelmente se inicia com a formação do intermediário ácido sulfênico na cisteína peroxidásica (Cys-47). Assim, conseguimos elucidar o papel catalítico dessas proteínas, bem como identificar seus sistemas redutores, obtendo informações que podem ser relevantes para o entendimento do mecanismo da patogenicidade da X. fastidiosa. Os resultados apresentados neste trabalho podem contribuir para o desenvolvimento de novas técnicas de controle de praga para a doença CVC em citrus e outras que envolvam a bactéria X. fastidiosa. / The phytopathogenic bacterium Xylella fastidiosa is the etiological agent of Citrus Variegated Chlorosis (CVC) that causes losses of about 100 millions dollars per year in Brazil. During infection, reactive oxygen species play a central role in plant pathogen defense. To survive under oxidative stress imposed by the host, microorganisms express antioxidant proteins, including the peroxiredoxins alkyl hydroperoxide reductase subunit C (AhpC) and bacterioferritin comigratory protein (Bcp). Peroxiredoxins are peroxidases, which rely on an activated cysteine residue to catalyze the reduction of hydroperoxides. By proteome analysis, Smolka et al. (2003) identified the products of ahpc and bcp genes present in whole cell extract of X. fastidiosa. To characterize the function and structure of AhpC and Bcp protein, their genes were cloned in Escherichia coli and the corresponding proteins purified by nickel affinity chromatography. Recombinant proteins presented thiol-dependent peroxidase activity against hydrogen peroxide and organic hydroperoxides. AhpC and Bcp peroxidase activities are dependent on alkyl hydroperoxide reductase subunit F (AhpF), and on thioredoxin system, respectively. Paradoxically, AhpF flavoenzyme possesses hydrogen peroxide-forming oxidase activity. Contrary to classical assumptions, competitive kinetics employing horseradish peroxidase assays showed that the second-order rate constants of AhpC and Bcp reaction with hydrogen peroxide are in the order of 107 M-1.s-1, as fast as the activity of selenium-dependent glutathione peroxidases and catalases. Non-reducing SDS-PAGE and cysteine quantification using DTNB indicated different peroxidasic mechanisms: AhpC is a typical 2-Cys peroxiredoxin (with intermolecular disulfide bond formation), while Bcp is an atypical 2-Cys peroxiredoxin (with intramolecular disulfide bond formation). In contrast to the well-conserved AhpC cysteines responsible for the peroxidase activity (Cys-47 and Cys-165), only through site-specific mutagenesis and mass spectrometry we could identified the cysteine residues involved in the Bcp peroxidase activity (Cys-47 and Cys-83). Structural characterization by size exclusion chromatography and dynamic light scattering revealed that AhpC native protein forms stable and redox state independent decamers. The crystal structure of Bcp C47S, the first 2-Cys Prx with a 35-residue between the active cysteines ever characterized, shows that protein contains the common fold of peroxiredoxins and that active cysteines lies ~12.4 Å away one from the other. Based on circular dichroism, we presented data indicating that disulfide bond formation may require significant conformational changes, which probably is triggered by the peroxidatic cysteine oxidation to sulfenic acid. In conclusion, we elucidated the catalytic mechanisms and reduction systems of AhpC and Bcp proteins that may help to understand the pathogenicity mechanism of X. fastidiosa. These results can contribute to the development of plague control methods against X. fastidiosa.

Leukemia is the most common malignant disease in children patients. In our laboratory (CLIP) a novel subtype of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL) with lineage switch during early phase of treatment towards myeloid lineage (swALL) was recently documented. SwALL incidence is almost 4 % of all BCP-ALLs (Slámová et al., 2014). DNA methylation (presence of 5-methylcytosine) is together with post-translational histone modifications and non- coding RNAs an epigenetic mechanism which regulates gene expression without changes of genetic code. DNA methylation is easily detected by bisulphite conversion and subsequent sequencing. The aim of this work was to compare genome-wide DNA methylation patterns between patients with swALL and control BCP-ALLs. The first step in achieving that was revision and improvement of bioinformatic processing protocol for eRRBS data from massive parallel sequencing. To improve the sequence adapter trimming I tested four bioinformatic tools - FAR, cutadapt, Trimmomatic and fastx_clipper. I implemented the fastest and most effective - Trimmomatic into the processing protocol. As a next step I analysed the data with improved protocol and extended the analysis in R programming environment where the comparison of studied groups was performed. The comparison of...