La connaissance des effets et modes d’action des QTL (Quantitative Trait Loci) de résistance des plantes aux maladies est un enjeu majeur pour l’exploitation durable des résistances génétiques en agriculture. Dans le pathosystème pois/Aphanomyces euteiches, présentant une importance économique majeure, l’objectif de la thèse a consisté à valider les effets de QTL de résistance, seuls ou en combinaison, et à connaitre leurs modes d’action sur les étapes du cycle de l’agent pathogène. Un total de 157 NILs (Near Isogenic Lines) de pois issues d’un programme de Back-cross Assisté par Marqueurs, porteuses d’aucun, un, deux ou trois des sept principaux QTL de résistance préalablement identifiés, a été génotypé à l’aide d’une puce 15K SNPs et évalué pour la résistance. Les NILs porteuses de QTL à effets majeurs, seuls ou en combinaison avec des QTL à effets mineurs,ont présenté des niveaux accrus de résistance partielle en conditions contrôlées et au champ par rapport aux NILs dépourvues de QTL, dans différents fonds génétiques. Certaines NILs comportant des QTL individuels ou combinés à effets mineurs ont également montré un niveau réduit de sévérité de la maladie dans l’une ou les deux conditions de test. La plupart des QTL a présenté des effets significatifs sur le ralentissement de l’infection et/ou de la quantité d’ADN pathogène ayant colonisé la racine pendant sept jours après inoculation. Cette thèse fournit des outils et éléments de choix de QTL à combiner en sélection pour augmenter l’efficacité de la résistance partielle à A. euteiches dans les futures variétés de pois / Knowledge of the effects and action modes of resistance QTL to plant diseases is a major challenge for the durable use of genetic resistances in agriculture. In the pea/Aphanomyces euteiches pathosystem, which has a major economic importance, the aim of this study was to validate the single or combined effects of main resistance QTL, and study their action modes on steps of the pathogen life cycle. A total of 157 pea NILs (Near Isogenic Lines) created by Marker-Assisted Back-crossing and carrying no, one, two or three of the seven main resistance QTL previously identified, was genotyped using a 15K SNPs array and evaluated for resistance. The NILs carrying major-effect QTL, individually or in combination with minor-effect QTL, had increased levels of partial resistance in controlledconditions and in the field compared to NILs without QTL, in different genetic backgrounds. Several NILs carrying single or multiple minor-effect QTL also showed reduced levels of disease severity in one or the two test conditions. Most of the QTL had significant effects on slowing down infection and/or pathogen DNA quantity which had colonized the root for seven days after inoculation. This study gives tools and information for the choice of resistance QTL to use in pyramiding breeding strategies for increasing partial levels of resistance to A. euteiches in future pea varieties.

To maintain the quality of receiving water bodies, it is desirable to remove total phosphorus (TP) in stormwater runoff. Many media filtration technologies have been developed to achieve TP and soluble reactive phosphorus (SRP) removal. Efficient media adsorption is essential to insure control of stormwater phosphorus inputs to the receiving water body. This project develops and analyzes a functionalized Biosorption Media (BAM) to remove phosphorus species from stormwater runoff. One goal of this project is to find the BAM values for coefficients such as maximum adsorption capacity (QM: 4.35E-05) for the media through SRP isotherm equilibrium experiments using the Langmuir and Freundlich models. In addition, an upflow column experiment was also performed to study BAM nutrient removal from stormwater runoff. Finally, the information from the isotherm and the column experiments are used to estimate the life expectancy or quantity required of the media, and to define the effectiveness of BAM in phosphorus removal. The result of this study shows that BAM is a feasible stormwater treatment that can remove 60% SRP and > 40% TP at temperature between 21-23°C. The media is adequately modeled by both the Langmuir and the Freundlich models over the concentration range of interest in stormwater.

No description available.

<p>The β-barrel assembly machinery (BAM) is responsible for the biogenesis of outer membrane proteins (OMPs) into the outer membranes of Gram-negative bacteria. These OMPs have a membrane-embedded domain consisting of a β-barrel fold which can vary from 8 to 36 β-strands, with each serving an important role in the cell such as nutrient uptake and virulence. BAM was first identified nearly two decades ago, but only recently has the molecular structure of the full complex been reported. Together with many years of functional characterization, we have a significantly clearer depiction of BAM's structure, the intra-complex interactions, conformational changes that BAM may undergo during OMP biogenesis, and the role chaperones may play. But still, despite advances over the past two decades, the mechanism for BAM-mediated OMP biogenesis has remained elusive. Over the years, several theories have been proposed that have varying degrees of support from the literature, but none has of yet been conclusive enough to be widely accepted as the sole mechanism. Here we present our recent work on the structures of BAM in its near native environment, characterized by cryo-EM, and study its interaction with OMP substrates. Specifically, we focused on the role of BAM-mediated EspP biogenesis, and structurally characterized crosslinked intermediates to atomic resolution, allowing for a more complete understanding of BAM-mediated OMP biogenesis. We also characterized BAM-mediated OmpT and OmpA biogenesis, which further supports a BamA-budding model for OMP biogenesis. Given its essential role in Gram-negative bacteria, BAM is an attractive target for antibiotics, and we contributed to characterizing a novel antibiotic designed against BAM called darobactin, which binds to the lateral gate of BAM, thereby disrupting OMP biogenesis and leading to programmed bacterial lysis.</p>

A investigação dos mecanismos de interação dos vírus com as células do hospedeiro trazem informações relevantes para a identificação de alvos no desenvolvimento de drogas para impedir a penetração e/ou desenvolvimento dos vírus. Peptídeos desenhados a partir de proteínas virais foram desenvolvidos e testados quanto as suas capacidades de inibir o processo de fusão do vírus com a célula do hospedeiro. Alguns se encontram em fase de avaliação clínica. Anticorpos contra a proteína p24 do HIV-1 foram detectados no soro de pacientes HIV-positivos, e estes reconhecem pequenas seqüências peptídicas desta proteína. Neste trabalho foi analisada a interação entre uma seqüência peptídica correspondente aos aminoácidos 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) da proteína p24, denominado p24- 1, com sistemas biomiméticos. Os sistemas utilizados foram filmes de Langmuir (monocamadas) de dipalmitoil fosfatidil colina (DPPC) e dipalmitoil fosfatidil glicerol (DPPG) e vesículas multilamelares (MLVs) de DPPC. O p24-1 encontra-se desorganizado em solução aquosa, mas com a interação com as MLVs de DPPC teve induzido uma conformação hélice ?, de acordo com o espectro de dicroísmo circular (CD). Esta característica foi confirmada pela predição de hélice a seguida por uma estrutura não ordenada contendo 11 resíduos do p24-1. As isotermas de pressão e potencial de superfície das monocamadas de DPPC foram afetadas com a presença de 0,05% mo1 de p24-1, com uma expansão de aproximadamente 5%. Para concentrações acima de 0,5% mo1 de p24-1 a expansão foi de 20%, com saturação do efeito da concentração. O efeito de expansão foi acompanhado por uma alteração na morfologia das monocamadas, estudados com microscopia no ângulo de Brewster (BAM). A incorporação do p24-1 impede a formação de grandes domínios de DPPC. O efeito cooperativo causado na monocamada de fosfolipídios pelo p24-1 sugere que esse tem um potencial na atividade antiviral por participar da expansão da membrana da célula hospedeira. / The investigation of the interaction mechanisms between the viruses and the host cells brings relevant information for the identification of targets on the development of drugs to prevent the penetration and/or development of the viruses. Peptides designed from viral proteins have been developed and tested on its capacities of inhibiting the merging process of the virus with the host cell. Some of them are in clinical evaluation. Antibodies against the protein p24 of the HIV-1 have been detected in the serum of HIV-positive patients, and they are able to recognize short peptide sequences of this protein. In this work, it was analyzed the interaction between a peptide sequence corresponding to amino acids 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) of the protein p24, called p24- 1, and biomimetic systems. The systems used were Langmuir films (monolayers) of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) and multilamelar vesicles (MLVs) of DPPC. p24-1 is found disorganized in watery solution, but with the interaction with the MLVs of DPPC it had induced a conformation ?-helix, according to the circular dichoism spectra (CD). This characteristic was confirmed by the prediction of ?-helix followed by an unordered structure with 11 residues of p24-1. The isotherms of pressure and potential of surface of the DPPC monolayers were affected by the presence of 0,05% mo1 of p24-1, with an expansion of approximately 5%. For concentrations above 0,5% mol of p24-1 the expansion was 20%, with saturation of the concentration effect. The expansion effect was followed by a morphologic alteration of the monolayers, studied with microscopy of the Brewster angle (BAM). The incorporation of p24-1 prevents the formation of large domains of DPPC. The cooperative effect caused in the phospholipid monolayer by p24-1 suggests that this peptide has a potential in the antiviral activity, once its participates on the expansion of the host cell membrane.

A investigação dos mecanismos de interação dos vírus com as células do hospedeiro trazem informações relevantes para a identificação de alvos no desenvolvimento de drogas para impedir a penetração e/ou desenvolvimento dos vírus. Peptídeos desenhados a partir de proteínas virais foram desenvolvidos e testados quanto as suas capacidades de inibir o processo de fusão do vírus com a célula do hospedeiro. Alguns se encontram em fase de avaliação clínica. Anticorpos contra a proteína p24 do HIV-1 foram detectados no soro de pacientes HIV-positivos, e estes reconhecem pequenas seqüências peptídicas desta proteína. Neste trabalho foi analisada a interação entre uma seqüência peptídica correspondente aos aminoácidos 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) da proteína p24, denominado p24- 1, com sistemas biomiméticos. Os sistemas utilizados foram filmes de Langmuir (monocamadas) de dipalmitoil fosfatidil colina (DPPC) e dipalmitoil fosfatidil glicerol (DPPG) e vesículas multilamelares (MLVs) de DPPC. O p24-1 encontra-se desorganizado em solução aquosa, mas com a interação com as MLVs de DPPC teve induzido uma conformação hélice ?, de acordo com o espectro de dicroísmo circular (CD). Esta característica foi confirmada pela predição de hélice a seguida por uma estrutura não ordenada contendo 11 resíduos do p24-1. As isotermas de pressão e potencial de superfície das monocamadas de DPPC foram afetadas com a presença de 0,05% mo1 de p24-1, com uma expansão de aproximadamente 5%. Para concentrações acima de 0,5% mo1 de p24-1 a expansão foi de 20%, com saturação do efeito da concentração. O efeito de expansão foi acompanhado por uma alteração na morfologia das monocamadas, estudados com microscopia no ângulo de Brewster (BAM). A incorporação do p24-1 impede a formação de grandes domínios de DPPC. O efeito cooperativo causado na monocamada de fosfolipídios pelo p24-1 sugere que esse tem um potencial na atividade antiviral por participar da expansão da membrana da célula hospedeira. / The investigation of the interaction mechanisms between the viruses and the host cells brings relevant information for the identification of targets on the development of drugs to prevent the penetration and/or development of the viruses. Peptides designed from viral proteins have been developed and tested on its capacities of inhibiting the merging process of the virus with the host cell. Some of them are in clinical evaluation. Antibodies against the protein p24 of the HIV-1 have been detected in the serum of HIV-positive patients, and they are able to recognize short peptide sequences of this protein. In this work, it was analyzed the interaction between a peptide sequence corresponding to amino acids 196-224 (AAMQMLKETINEEAAEWDRVHPVHAGPIA) of the protein p24, called p24- 1, and biomimetic systems. The systems used were Langmuir films (monolayers) of dipalmitoyl phosphatidyl choline (DPPC) and dipalmitoyl phosphatidyl glycerol (DPPG) and multilamelar vesicles (MLVs) of DPPC. p24-1 is found disorganized in watery solution, but with the interaction with the MLVs of DPPC it had induced a conformation ?-helix, according to the circular dichoism spectra (CD). This characteristic was confirmed by the prediction of ?-helix followed by an unordered structure with 11 residues of p24-1. The isotherms of pressure and potential of surface of the DPPC monolayers were affected by the presence of 0,05% mo1 of p24-1, with an expansion of approximately 5%. For concentrations above 0,5% mol of p24-1 the expansion was 20%, with saturation of the concentration effect. The expansion effect was followed by a morphologic alteration of the monolayers, studied with microscopy of the Brewster angle (BAM). The incorporation of p24-1 prevents the formation of large domains of DPPC. The cooperative effect caused in the phospholipid monolayer by p24-1 suggests that this peptide has a potential in the antiviral activity, once its participates on the expansion of the host cell membrane.

L'objectif de cette thèse est de créer un nouveau système qui est capable de gérer des auto-formations intelligentes, interactives et adaptatives aux besoins, styles d'apprentissage et à l'état des connaissances de chaque apprenant. Ce système est nommé IWEBISE (Interactive and Intelligent System for Education). Il est le résultat de l'accouplement entre cinq grands domaines : l'ingénierie des connaissances, l'interaction homme-machine, la psychologie cognitive, l'intelligence artificielle et la psychopédagogie. IWEBISE est constitué de cinq parties :<br> Modèle de l'apprenant : Cette partie détaille comment le style d'apprentissage des apprenants est modélisé selon le modèle Felder-Silverman, qui dépend de nombreux paramètres : Nombre d'exemples, nombre d'exercices, le lieu des exemples avant ou après le contenu et le lieu des exercices avant ou après le contenu. Les connaissances des étudiants sont exprimées dans le système en utilisant le modèle de recouvrement (Overlay), qui les considère comme une partie du domaine de la connaissance. L' IWEBISE utilise également un modèle ouvert de l'apprenant . Celui-ci permet aux étudiants de changer par eux-mêmes leurs états de connaissances relatives à chaque concept, ce qui leur permet d'étudier à grands pas sans se sentir bloqués par leurs processus d'apprentissage. Le modèle de l'apprenant est composé de deux parties: La partie statique qui stocke les renseignements personnels de l'apprenant. La partie dynamique qui garde les interactions des apprenants avec le système. Elles sont présentées par plusieurs paramètres: nombre de réponses correctes (NCA), nombre de réponses incorrectes (NICA), temps passé à résoudre une question (TSSQ), temps consacré à la lecture ou à l'interaction avec un concept spécifique (TSR) et le nombre de tentatives pour répondre à une question (NAAQ). Une fois qu'un apprenant a passé une séance de pré-test, la partie dynamique est lancée en utilisant ces paramètres. Six méthodes sont employées pour symboliser l'état des connaissances des apprenants dans six niveaux (excellent, très bien, bien, plutôt bien, faibles et très faibles) dans le but de déterminer la meilleure pour être utilisée plus tard dans le nouveau IWEBISE. Ces méthodes sont: FBAM, ART2, Fuzzy-ART2, HMM et NN / HMM. F-mesure métrique est employé pour mesurer la performance des méthodes mentionnées. Les résultats montrent que Fuzzy-ART2 donne la meilleure qualité de catégorisation (0.281 ), qui est considérée comme un facteur très important pour s''assurer qu'une carte de concepts appropriée est affichée à l'apprenant en fonction de son état de connaissances. <br> Modèle de tuteur : Cette partie détaille la façon de modéliser les stratégies pédagogiques utilisées par les enseignants pendant la présentation du contenu des cours aux étudiants. Elles sont modélisées par une table composée de neuf champs qui permettent de stocker les couleurs utilisées pour présenter l'état des connaissances des apprenants dans le plan du parcours et la possibilité de montrer ou de cacher un concept d'apprentissage. Le modèle de tuteur se concentre également sur un algorithme de prédiction pour prévoir les concepts suivants qui pourraient être visités par les apprenants. Le processus de prédiction est réalisé en suivant trois phases: Phase d'initialisation: Pour chaque apprenant un HMM (λ) est construit à base de sa précédente séquence d'accès aux concepts. Phase d'ajustement: Étant donnée une nouvelle séquence observée et un HMM (λ), l'algorithme de Baum-Welch est utilisé pour ajuster les HMM initialisés et de maximiser la nouvelle séquence observée. Phase de prédiction: l'algorithme Forward est appliqué pour déterminer la distribution de probabilité de chaque concept dans le cours. La plus haute valeur représente le concept suivant qui sera visité par l'apprenant. L'exactitude de prédiction est évaluée par deux critères, la Sensibilité et la Précision. La sensibilité est définie comme le nombre de concepts prédits correctement (vrais positifs) divisé par le nombre de concepts visités (positifs réels). La précision est le pourcentage de prédictions positives qui sont correctes. Les résultats montrent que HMM génère une plus grande précision en utilisant une séquence de concepts plus large qui varie de 20% à 99% et quand la précision est élevée, la sensibilité est élevée aussi.<br> Modèle de domaine : Le contenu des cours est organisé en un réseau de concepts pour représenter les objectifs d'apprentissage. Un objectif d'apprentissage concerne plusieurs concepts qui sont classés en trois types: des concepts principaux, des concepts pré-requis et des sous-concepts. Chaque nœud interne dans le réseau représente un concept, tandis que les nœuds externes dans le niveau le plus bas symbolisent plusieurs types d'unités d'enseignement, qui sont sous la forme de fichiers multimédia Flash interactive, images, vidéos, textes, exercices, des exemples. Trois différentes méthodes sont utilisées pour décrire le modèle de domaine : HBAM (Hirarchical Bidirectional Associative Memory), une base de données MySql, et XML, Dans la première méthode, le domaine de connaissances est conçu et modélisé en utilisant un réseau de neurones BAM hiérarchique. Le premier BAM-1 associe les objectifs d'apprentissage avec les concepts, mais le second BAM-2 est utilisé pour attribuer des unités d'enseignement à chaque concept. La couche de sortie de BAM-1 est la couche d'entrée du BAM-2, qui peut être vu comme une couche intermédiaire de toute l'architecture. Nombre de nœuds de couche d'entrée, de milieu et de sortie représentent le nombre d'objectifs d'apprentissage, les concepts et les unités d'enseignement respectivement . Dans la seconde méthode une base de données relationnelle est utilisée pour représenter le domaine de connaissance. Il est composé d'onze tableaux (les catégories principales, les sous-catégories, les sujets, les objectifs d'apprentissage, les objectifs d'apprentissage pré-requis, les concepts, les concepts pré-requis, la relation d'un sous-concepts avec un concept, le contenu et les questions pour les post-tests et pré-tests). Un Document Type Définition (DTD) du fichier est construit dans la troisième méthode pour déterminer un ensemble de règles pour définir et décrire l'organisation des connaissances dans un fichier XML. La méthode de base de données relationnelle est sélectionnée pour être utilisée au sein de notre nouveau système IWEBISE car d'une part, certains concepteurs de cours préfèrent avoir leur contenu des cours confidentiels et protégés et de l'autre part, XML n'est pas capable de traiter le contenu de cours énormes et tous les types de données tels que des images et la vidéo. <br> Le moteur d'adaptation : Il est considéré comme l'une des parties les plus importantes de IWEBISE grâce aux trois raisons suivantes: Il relie les différentes parties du système, il génère la page d'un concept selon le style d'apprentissage de chaque apprenant et il adapte la carte de concepts en fonction de l'état des connaissances de chaque apprenants aussi. <br> L'interface utilisateur représente les moyens d'interaction disponible sur le système IWEBISE. Il est classé en quatre niveaux : Administrateur: Il permet aux administrateurs de créer une catégorie de cours, sous-catégorie, gérer les utilisateurs et les processus d'abonnement. Concepteur: Il permet aux concepteurs de cours de gérer les objectifs d'apprentissage, les concepts, les sous-concepts, les contenu des concepts et les questions des tests. Elle leur permet également d'exporter leurs cours sous la norme SCORM. Tuteur: Il permet aux enseignants de gérer leurs stratégies d'enseignement et de donner des conseils appropriés aux apprenants. Apprenant: Il permet aux apprenants de compléter leur processus d'apprentissage en utilisant le pré-test, les post-tests, le questionnaire «Index des styles d'apprentissage", les glossaires, le chat et le forum. Le nouveau système IWEBISE est évalué par des concepteurs de cours et par des étudiants, dans le but d'optimiser ses performances au cours de l'enseignement et les processus d'apprentissage. Douze critères sont utilisés pour l'évaluer: la Cohérence, l'évidence, la prévisibilité, la richesse, l'exhaustivité, la motivation, la structure d'Hypertext, l'autonomie, la facilité d'utilisation, l'esthétique, la collaboration et l'interactivité.<br> L'originalité de cette thèse est basée sur : 1.L'utilisation d'une nouvelle architecture d'un réseau de neurones appelé HBAM pour modéliser le domaine des connaissances d'un cours. Ce nouveau réseau peut être utilisé dans de nombreux autres domaines tels que: la reconnaissance des formes ; 2.L 'utilisation d'un nouvel algorithme hybride qui utilise un réseau de neurones (Fuzzy-ART2) et une méthode statistique (HMM) pour la modélisation des connaissances des apprenants ; 3.L 'utilisation de nombreux algorithmes d'apprentissage tels que: FBAM, ART2, Fuzzy-ART2 et une structure hybride Fuzzy-ART2/HMM, qui sont utilisés pour classer la réflexion des apprenants et leurs raisonnements en six niveaux ; 4.L'utilisation d'un HMM pour prédire le prochain concept, basé sur l'histoire des concepts visités par un apprenant ; 5.La définition des styles d'apprentissage des apprenants à l' Institut Supérieur des Langues (Université d'Alep) par rapport à l'apprentissage d'une langue. Ceci est fait en utilisant le modèle Felder et Silverman ; 6.Le comparaison d'IWEBISE avec d'autres systèmes éducatifs. 7.Le capacité d'l'IWEBISE à l'exportation et la réalisation des cours conformément à la norme SCORM avec l'objectif de les réutiliser dans d'autres plates-formes d'enseignement telle que: Moodle ; 8.La construction et la mise en œuvre d'un nouveau système intelligent et adaptatif pour l'éducation basé sur le Web pour l'enseignement de la grammaire anglaise.

In an era of acute concern about airline safety, bird strikes are still one of the major hazards to aviation worldwide. The severity of the problem is such that it is mandatory in all developed countries to include bird management as part of airport safety management programs. In Australia, there are approximately 500 bird aircraft strikes per year (Bailey 2000). Brisbane airport has a relatively high occurrence of strikes, with an average of 77 recorded every year (2002-2004). Given the severity of the problem, a variety of techniques have been employed by airports to reduce bird strikes. Scare devices, repellents, continuous patrols for bird hazing, use of raptors to clear airspace of birds and depredation are used by many airports. Even given the diversity of control methods available, it is accepted that habitat management is the most effective long term way to control birds in and around the airport space. Experimental studies have shown that habitat manipulation and active scaring measures (shooting, scaring etc), can reduce bird numbers to an acceptable level. The current study investigated bird populations in six major vegetation habitat types identified within the operational and surrounding areas of Brisbane airport. In order to determine areas where greater bird control and management should be focused, bird abundance, distribution, and activity were recorded and habitats that pose the greatest bird strike risk to aircraft were identified. Secondly, species with high hazard potential were identified and ranked according to their hazard potential to aircraft. This study also investigated the effectiveness of different vegetation management options to reduce bird species abundance within operational areas of Brisbane airport. Four different management options were compared. Each management option was assessed for grass structural complexity and potential food resources available to hazardous bird species. Analysis of recorded data showed that of the habitats compared within the Brisbane airport boundaries, grasslands surrounding runways, taxiways and aprons possess the greatest richness and abundance of bird species that pose the greatest potential hazard to aircraft. Ibis and the Australian kestrel were identified as the bird species that pose the greatest risk to aircraft at Brisbane airport, and both were found in greatest numbers within the managed grasslands surrounding operational areas at the airport. An improved reporting process that allows correct identification of all individual bird species involved in bird strikes will not only increase the accuracy of risk assessments, but will also allow implementation of more effective control strategies at Brisbane airport. Compared with current grassland management practice, a vegetation management option of maintaining grass height at 30-50cm reduced total bird utilisation by 89% while utilisation of grassland by potentially hazardous birds was also reduced by 85%. Maintaining grass height within the 30-50cm range also resulted in a 45% reduction in the number of manipulations required per year (11 to 6), when compared with current management practices, and a 64% reduction in annual maintenance cost per hectare. When extrapolated to the entire maintained grass area at Brisbane airport, this resulted in a saving of over $60 000 annually. Optimisation of potential hazard reduction will rely on future studies that investigate the effect of particular vegetation species that could replace the existing mix of grasses used at Brisbane airport and an understanding of the relative importance of vegetation structure and food supply in determining utilisation by potentially hazardous bird species.

Submitted by Mateus Dassie Maximino (mateusmaximino23@gmail.com) on 2018-04-23T13:55:51Z No. of bitstreams: 1 Dissertação Mestrado - Mateus Dassie Maximino.pdf: 5933036 bytes, checksum: 96040f746ca01578e5036f01f3d89e6e (MD5) / Approved for entry into archive by Claudia Adriana Spindola null (claudia@fct.unesp.br) on 2018-04-23T14:37:07Z (GMT) No. of bitstreams: 1 maximino_md_me_prud.pdf: 5933036 bytes, checksum: 96040f746ca01578e5036f01f3d89e6e (MD5) / Made available in DSpace on 2018-04-23T14:37:07Z (GMT). No. of bitstreams: 1 maximino_md_me_prud.pdf: 5933036 bytes, checksum: 96040f746ca01578e5036f01f3d89e6e (MD5) Previous issue date: 2018-02-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O presente trabalho trata do estudo da interação entre os poluentes emergentes amoxicilina (AMX) e azul de metileno (AM) com modelos-simples de membrana biológica compostas de fosfolipídios zwiteriônicos (DPPC e DOPC), através de filmes de Langmuir e vesículas unilamelares gigantes (GUVs). As membranas foram expostas aos poluentes de forma individual e também misturados (MIX) para que se aproximasse de uma situação real, neste caso, o chamado efeito coquetel, no qual os poluentes são encontrados misturados no ambiente. O objetivo principal foi investigar as possíveis interações entre os poluentes e os fosfolipídios e os consequentes efeitos nas membranas. Nos filmes de Langmuir, as isotermas π-A (pressão superficial por área molecular média) de DPPC revelaram que a AMX e o AM são expulsos da interface ar-água conforme a fase condensada é atingida. O DOPC demonstrou ser menos afetado pela AMX, uma vez que a isoterma não apresentou deslocamento. Contudo, o DOPC se mostrou mais susceptível a interagir com o AM, visto que um deslocamento significativo foi observado. Para ambos os lipídios a MIX causou maiores efeitos, evidenciados por deslocamentos maiores nas isotermas. Análises de módulo compressional mostraram um pequeno aumento na fluidez do filme de DPPC, não observado para o DOPC. As medidas de microscopia de ângulo de Brewster (BAM) na presença dos poluentes revelaram mudanças na morfologia dos filmes, afetando a formação dos domínios de DPPC e impedindo a formação dos mesmos nas monocamadas de DOPC. Os espectros de PMIRRAS para o DPPC indicaram possíveis interações eletrostáticas dos poluentes com a cabeça polar do lipídio, assim como uma desordem na região da sua cauda apolar. A monocamada de DOPC foi bastante afetada pois ocorreram mudanças nas vibrações de quase todos os grupos químicos do lipídio, apresentando inclusive mudanças na região da cauda do lipídio. Os espectros de emissão da sonda LAURDAN nas LUVs revelaram um aumento da rigidez para ambos os lipídios, diferentemente do observado pelas análises de elasticidade da monocamada de Langmuir. As GUVs para ambos os fosfolipídios, ao contrário das monocamadas, apresentaram maior efeito sobre os poluentes aplicados de forma individual, visto que para os poluentes avaliados individualmente foram observados a perda de contraste de fase para os lipídios na presença do AM, assim como mudanças na estabilidade para as GUVs de DOPC em AMX. Tais efeitos não foram observados para a MIX, devido as diferenças estruturais dos modelos de membrana. / The present work is about the interaction between the emerging pollutants amoxicillin (AMX) and methylene blue (MB), with biological membrane models of zwitterionic phospholipids (DPPC and DOPC), through Langmuir films and giant unilamellar vesicles (GUVs). The membrane was exposed to the presence of the pollutants individually, still being performed the analysis with the mixture (MIX) of both in order to get closer to a real situation, in which the pollutants are found mixed in the environment. The main goal was to investigate the possible interactions between the pollutants and phospholipids. The π-A (surface pressure by mean molecular area) isotherms of DPPC reveal that in the condensed phase the lipid expelled the AMX and MB of the air-water interface. The DOPC showed to be less affected by the AMX, once the isotherm did not present a displacement. However, the DOPC showed to be more susceptible to interact with the MB, since significant displacements were observed. In both lipids, the MIX showed to be more effective, causing bigger displacements. The analysis of compressional modulus showed a small increase of the fluidity of the DPPC film, which was not observed to the DOPC. The Brewster Angle Microscopy (BAM) measurements in the presence of the pollutants revealed changes in the morphology, affecting the domains formation of DPPC and inhibiting the formation of the same in monolayers. The PM-IRRAS specters to the DPPC indicated possible electrostatic interactions with the head, as well as a disorder in the tail. The DOPC monolayer was quite affected because there have been changes in almost all chemical groups of lipid, including changes in the tail region of the lipid. The emission spectrum from the dye LAURDAN in the LUVs revealed an increase in the rigidity for both lipids, different from the observed in the analysis of elasticity in the Langmuir monolayers. The GUVs for both phospholipids, unlike the monolayers, showed a major effect on the pollutants applied individually since that were observed losses of phase contrast for the lipids in the presence of MB, such as changes in the stability of the DOPC GUVs in AMX. Such effects were not observed in the MIX, due to the structural differences in the membrane models.

<p dir="ltr">The obligate human pathogen, <i>Neisseria gonorrhoeae </i>(Ngo), has continued to acquire widespread antibiotic resistance. Ngo is the causative agent of the sexually transmitted disease gonorrhea, and can cause additional complications such as endocarditis, septicemia, and infertility if left untreated. The Centers for Disease Control and Prevention (CDC) now recommends a treatment option of a single drug of last resort, ceftriaxone, leaving a need for novel therapeutics against this pathogen.</p><p dir="ltr">Like many bacterial pathogens, Ngo is Gram-negative consisting of both an inner membrane (IM) and outer membrane (OM). The transmembrane proteins in the IM have primarily an α-helical fold, while the transmembrane proteins in the OM have a β-barrel fold. These β-barrel outer membrane proteins (OMPs) have essential functions in regulating the homeostasis and nutrient acquisition of the cell, in addition to promoting virulence in pathogenic strains. These OMPs are folded and inserted into the outer membrane by the β-barrel assembly machinery (BAM) complex. In <i>E. coli,</i> BAM consists of five proteins: BamA, an OMP itself, and four lipoproteins, BamB, C, D, and E.</p><p dir="ltr">Here we present our work toward the structural characterization of BAM from Ngo (<i>Ng</i>BAM) using cryo-EM. Ngo lack a homolog of BamB and may function as a four component complex. To better understand the mechanism for how <i>Ng</i>BAM is able to mediate OMP biogenesis despite lacking a component that is critical in <i>E. coli</i>, we determined the cryo-EM structure of <i>Ng</i>BAM, which revealed several distinct features including that the barrel domain of BamA being observed in the inward-open conformation. We also investigated <i>Ng</i>BAM as a therapeutic target, by studying its interaction with a novel broad spectrum antibiotic darobactin. We first showed darobactin is effective against the laboratory strains of NgoFA19 and ATCC-49226. We also show it is effective against the human isolate WHOX, with a comparable MIC to ceftriaxone. To structurally characterize the mechanism of inhibition by darobactin, we used cryo-EM to determine the structures of <i>Ng</i>BAM bound to two darobactin compounds. In these structures, darobactin binding was accompanied by large conformational changes in <i>Ng</i>BamA. To further probe the effects of darobactin on the conformational plasticity of <i>Ng</i>BAM we performed experiments using double electron-electron resonance spectroscopy, which showed distance changes between the engineered site labels consistent with the conformational changes observed in our structural observation. In addition, narrowing of the peak distributions indicated that darobactin binding was reducing the overall conformational heterogeneity of the complex. Taken together, the work presented here contributes to the understanding of how <i>Ng</i>BAM functions in folding and inserting OMPs and provides a foundation for future structure based drug design of darobactin and other potential compounds.</p>