Orientador: Mônica Alonso Cotta / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-09-02T14:12:01Z (GMT). No. of bitstreams: 1 Munar_DuberMarcelMurillo_D.pdf: 4529701 bytes, checksum: 857481ff034498f82f42ab7dc98536b3 (MD5) Previous issue date: 2017 / Resumo: Neste trabalho estudamos características físico-químicas nos diferentes estágios de formação de biofilmes, principalmente no estágio inicial da adesão de bactérias planctônicas em superfícies. Usamos como modelo a bactéria fitopatogênica Xylella fastidiosa, que vive exclusivamente no xilema de diversas plantas lenhosas e é transmitido por diferentes insetos da ordem Hemíptera. As amostras analisadas neste estudo foram todas cultivadas sobre superfícies inorgânicas e após diferentes tempos de cultivo foram lavadas e secas antes de serem analisadas. Para estudar o processo integral de formação de biofilmes, examinamos amostras cultivadas sobre superfícies de vidro e Silício recoberto com ouro, em um meio de cultura complexo, chamado Periwinkle wilt. Através de técnicas como citoquímica, imunomarcação, microscopia e espectroscopia, analisamos os elementos produzidos pelas bactérias ou presentes na membrana celular durante cada estágio da formação de biofilmes. Conseguimos identificar espaço-temporalmente a aparição de pili, vesículas e três tipos diferentes de sustâncias poliméricas extracelulares, assim como algumas moléculas específicas que compõem estes elementos. Posteriormente, focalizamos nossos esforços no processo de adesão inicial em superfícies. Estudaram-se os três principais componentes envolvidos neste processo: adesinas, meio líquido e superfície. Primeiramente, se analisaram amostras de bactérias aderidas em superfícies de Silício. Nestas amostras aplicamos diferentes reagentes com a finalidade de investigar que tipo de ligação química está presente na interface adesina-superfície. Determinou-se que uma das ligações presentes nesta interface é a ligação glicosídica e que provavelmente está relacionada com as moléculas de lipopolissacarídeo localizadas na membrana bacteriana. A complexidade do meio de cultura fez que o estudo dos seus componentes fosse o elemento mais complicado de analisar. Inicialmente, deveram-se procurar as condições mínimas nas quais as bactérias sobrevivem, porém não conseguem aderir em superfícies. Assim, um meio de cultura complexo e definido, chamado Xylella fastidiosa Medium, foi modificado eliminando as componentes não relacionadas diretamente com a adesão bacteriana. Logo, adicionamos sistematicamente diferentes tipos de moléculas e/ou íons no meio de cultura simplificado identificando as condições que favorecem o processo de adesão. Encontramos uma dependência da adesão bacteriana com o pH do meio e a presença de cátions bivalentes com baixa energia de ionização. Como último parâmetro, estudamos a participação da superfície neste processo. Neste caso, além das superfícies de Silício, também usamos superfícies de Fosfeto de Índio para estudar o processo de adesão como função do pH do meio de cultura. A adesão nestas superfícies só apresentou diferenças significativas em um intervalo específico de pH. Analisando o conjunto de evidências obtidas em nossos experimentos, conseguiu-se formular um modelo que descreve este fenômeno tendo em conta todos os parâmetros estudados. Descreve-se o processo de adesão como a interação entre concentrações de duas espécies de moléculas diferentes alusivas à adesina e à superfície. Sugerimos que o processo de adesão pode ser tratado como um mecanismo puramente físico-químico e não biológico, razão pela qual nosso modelo poderia ser em teoria extrapolado a qualquer tipo de superfície e qualquer tipo de bactéria / Abstract: In this work we study physico-chemical characteristics in the different stages of biofilm formation, particularly in the early stages of adhesion of planktonic bacteria to surfaces. We use as model the phytopathogenic bacterium Xylella fastidiosa, which lives exclusively in the xylem of several woody plants and is transmitted by different insects of the order Hemiptera. The samples analyzed in this study were all grown on inorganic surfaces and after different culture times were washed and dried before being analyzed. To study the entire biofilm formation process, we examined samples grown on glass and silicon covered with gold surfaces in a complex culture medium called Periwinkle wilt. Through techniques such as cytochemistry, immunostaining, microscopy and spectroscopy, we analyze the elements produced by the bacteria or present in the cell membrane during each stage of biofilm formation. We have been able to identify space-temporally the appearance of pili, vesicles and three different types of extracellular polymeric substances, as well as some specific molecules that compose these elements. Subsequently, we focused our efforts on the initial cell binding process on surfaces. The three main components that intervene in this process were studied: adhesins, liquid medium and surface. First, samples of bacteria adhered to silicon surfaces were analyzed. In these samples, we applied different reagents to investigate the type of chemical bond present at the adhesin-surface interface. It has been determined that one of the bonds present at this interface is the glycosidic bond, which is probably related to the lipopolysaccharide molecules located in the bacterial membrane. The complexity of the culture medium made the study of its components the most complicated element to analyze. Initially, it was necessary to look for the minimum conditions in which the bacteria survive, but they cannot adhere to surfaces. Thus, a complex and defined culture medium, called Xylella fastidiosa Medium, was modified eliminating components not directly related to bacterial adhesion. Then, we systematically add different types of molecules and / or ions in the simplified culture medium, identifying the conditions that favor the adhesion process. We found a dependence of bacterial adhesion with the pH of the medium and the presence of bivalent cations with low ionization energy. As a final parameter, we studied the participation of the surface in this process. In this case, in addition to the Silicon surfaces, we also used Indium Phosphide surfaces to study the adhesion process as a function of the pH of the culture medium. Adhesion on these surfaces only showed significant differences over a specific pH range. Analyzing the set of evidences obtained in our experiments, we were able to formulate a model that describes this phenomenon taking into account all the parameters studied. The adhesion process is described as the interaction between concentrations of two different species of molecules alluding to adhesin and to the surface. We suggest that the adhesion process can be treated as a purely physico-chemical and non-biological mechanism, so that our model could in theory be extrapolated to any type of surface and any type of bacteria / Doutorado / Física / Doutor em Ciências / 1060584 / 2010/51748-7 / 479486/2012-3 / CAPES / FAPESP / CNPQ

Les processus rhizosphériques sont reconnus comme une des stratégies majeures élaborées par les plantes afin d'augmenter la disponibilité en phosphore (P) et ainsi améliorer leur nutrition phosphatée. Pourtant, l'effet exact de ces processus est encore mal caractérisé et quantifié. L'objectif de ces travaux a été d e déterminer le rôle exercé par les modifications chimiques induites par les racines, particulièrement la modification de pH, dans les changements de disponibilité en P dans la rhizosphère. Pour ce faire, nous avons utilisé des modèles mécanistes géochimiques (« triple plane », échange d'ion et Nica-Donnan) en considérant une approche additive pour simuler l'effet de l'activité racinaire sur la disponibilité en P. Dans une première étape, nous avons caractérisé l'effet du pH sur la disponibilité en P dans plusieurs sols, un Cambisol et un Luvisol. Le Luvisol présentait deux concentrations en P inorganique contrastées en raison d'un essai de fertilisation phosphatée longue durée. Dans la rhizosphère du blé dur (Triticum turgidum durum L.) cultivé sur les mêmes sols, nous avons caractérisé qu'en plus de l'alcalinisation, le prélèvement en P et surtout en calcium (Ca) sont les processus rhizosphériques responsables du changement de disponibilit é en P observé. Le prélèvement du Ca favorise l'augmentation de la disponibilité en P dans la rhizosphère, en diminuant l'effet promoteur du processus d'adsorption-désorption du Ca sur celui du P. L'influence relative de ces trois processus rhizosphériques dépend toutefois de la composition chimique de la solution du sol (concentration en Ca et pH en particulier). Nos simulations mettent également en évidence la relation entre les changements de disponibilité en P est la distribution du P adsorbé sur les différentes phases minérales. La minéralogie du sol, spécialement l'abondance relative d'illite vs. les oxydes de fer, contrôle l'influence des processus rhizosphériques en déterminant les minéraux impliqués dans l'adsorption du P. A travers l'identification d'un nouveau processus rhizosphérique découlant du prélèvement en Ca et de ses effets sur la disponibilité en P, nos résultats démontrent la validité des modèles géochimiques pour prédire l'influence des processus rhizosphériques déterminant la disponibilité en P. / Root-induced chemical processes are recognized as a major strategy developed by plants to enhance phosphorus (P) availability and thus to promote P acquisition. However, the exact influence of these root-induced chemical processes is still poorly understood and quantified. The present study aimed at investigating the influence of root-induced chemical processes, especially root-induced pH changes, on P availability in the rhizosphere. In this work, we used a set of mechanistic adsorption models (« 1-pK triple plane », ion-exchange and Nica-Donnan) within the framework of the component additive approach in order to simulate the effects of root activity on P availability. First, we described the effects of pH on P availability in several soils unaffected by roots, a Chromic Cambisol and a Luvisol. The Luvisol showed different concentrations in inorganic P because of a long-term fertilisation trial. In the rhizosphere of durum wheat (Triticum tu rgidum durum L.) grown on these two soils, we found that calcium (Ca) uptake, in addition to P uptake and root-induced alkalisation, controlled to various extents the changes of soil P availability. Calcium uptake markedly increased P availability by decreasing the promoting effect of Ca adsorption on P adsorption. The relative influence of these three root processes depended on the solution composition (especially concentration of Ca and pH). Our simulations showed the relationship between changes in P availability and the speciation of adsorbed P onto the different soil minerals. Soil mineralogy, especially the relative abundance of illite vs. Fe oxides, controlled the influence of root processes by regulating the contribution of soil minerals to P adsorption. By identifying a novel root-induced processes, namely the Ca uptake, and describing its influence on P availability, our results demonstrate the ability of surface complexation models to predict the effects of root-i nduced processes on P availability in soils.

Estressores múltiplos, decorrentes de atividades antropogênicas, podem gerar efeitos combinados em organismos marinhos. Efeitos da acidificação oceânica associados a outros impactos antrópicos são pouco compreendidos, especialmente em zonas costeiras. Nestas regiões, o sombreamento causado por infraestruturas, como portos, pode potencialmente interagir com a redução do pH da água do mar e afetar populações de crustáceos. O objetivo foi avaliar os efeitos de pH reduzido e sombreamento na mortalidade, crescimento, calcificação e respostas comportamentais de deslocamento a odores de predadores vivos (sinal de perigo) e gastrópodes mortos (sinal de recurso disponível) usando juvenis do ermitão Pagurus criniticornis, coletados na Baía do Araçá (São Paulo, SE Brasil). Após 98 dias de experimento, os estressores apresentaram interação significativa sobre a mortalidade dos organismos, além de efeito aditivo sobre o crescimento. A calcificação não diferiu entre tratamentos, indicando que indivíduos foram capazes de manter a calcificação em condições de pH reduzido. Quando expostos a odor de predadores, o deslocamento dos ermitões foi afetado somente pelo sombreamento. Entretanto, uma interação entre ambos os estressores foi observada para o odor de gastrópodes, causando a redução do deslocamento. Este estudo mostra como impactos locais podem amplificar efeitos de mudanças ambientais globais em populações de crustáceos do entremarés. / Multiple stressors due to anthropogenic activities may cause combined impacts in marine organisms. Effects of ocean acidification associated with other anthropogenic impacts are poorly understood, especially in coastal regions. In these areas, shading caused by infrastructure development, such as harbor construction, may potentially interact with CO2-induced pH reduction and affect invertebrate populations. Here we evaluated reduced pH and shading effects on mortality, growth, calcification and displacement behavior to live predator (danger signal) and dead gastropod (resource availability signal) odors using juveniles of the hermit crab Pagurus criniticornis collected in Araçá Bay (São Paulo state, SE Brazil). After a 98 days experiment period, both stressors had a significant interaction on mortality and an additive effect on total growth. No difference in calcification was recorded among treatments, indicating that individuals were able to maintain calcification under reduced pH. When exposed to odor of live predators, crabs\' responses were affected only by shading. However, an interactive effect between both stressors was observed in response to gastropod odor, leading to reduced displacement behavior. This study shows how local disturbance impacts may enhance effects of global environmental changes on intertidal crustacean populations.

Les suspensions minérales de particules fines sont connues pour leur comportement non-Newtonien pendant l'écoulement. Les interactions particule-particule dans de tels systèmes (c’est-à-dire, suspensions minérales) ne sont pas limitées au contact physique, e.g. collision et frottement. La capacité des minéraux de développer une charge dans l'environnement aqueux justifie des comportements différents des systèmes similaires de première vue. C’est à dire qu’étant caractérisées avec la même fraction volumétrique, composition chimique et granulométrie des solides et densité de dispersant, deux suspensions peuvent montrer un comportement rhéologique différent en raison de la chimie de la solution. Dans ce cas, la composition ionique du dispersant définira la charge des particules, et donc le degré d'agglomération/dispersion dans la suspension. Les argiles phyllosilicates sont connues pour être particulièrement problématiques dans les processus de valorisation des minéraux. L’origine de ces minéraux phyllosilicates implique leur inhomogénéité chimique spatiale, ce qui signifie que le bord et la face de la particule montrent des propriétés chimiques et physiques différentes. La présence de tels minéraux dans les dispositifs d'agitation (réservoirs d'agitation, cellules de flottation) est souvent caractérisée par la coexistence de volumes de suspension stagnants et agités, ce qui a un impact négatif sur l'efficacité de l'agitation. Dans ce travail, les suspensions aqueuses diluées de Na-bentonite ont été examinées par vélocimétrie par imagerie par résonance magnétique afin d'étudier l'influence du pH et du type d'électrolyte monovalent sur leur comportement rhéologique local. Les résultats ont montré que les suspensions contenant 0,1% de solide en volume peuvent présenter une bande de cisaillement, une localisation de cisaillement ou aucun phénomène local en fonction de la chimie du milieu de suspension. Il a été suggéré que l’existence d’une «master curve» (ou courbe d’écoulement globale) pour les suspensions diluées dépendait de l’organisation des particules de bentonite dans la suspension, cette organisation est influencée par la chimie de solution et l’historique des contraintes précédentes. Dans l'étape suivante, une seconde et troisieme phases minérales (hématite et quartz) ont été ajoutées dans la matrice de bentonite. L’intérêt dans tels systèmes est lié au comportement sous écoulement des matrices formées avec des types de contacts différents entre particules. Les types de contacts établis dans les suspensions avec une chimie différente du milieu ont été discutés, ainsi que leurs propriétés d’écoulement / Fine particle mineral slurries are known to exhibit non-Newtonian behavior under the load. The particle-particle interactions in such suspensions go beyond physical contact due to the collision and friction. An ability of minerals to gain the charge in the aqueous environment justifies different behaviors of the similar systems. Being characterized with the same volumetric fraction, chemistry and particle size distribution of solids, and specific gravity of dispersing media, two suspensions can possess different rheological behavior due to the chemistry of the solution. In this case, the ionic composition of the media defines particle charging, and thus the degree of agglomeration/dispersion in the suspension. Phyllosilicate clays are known to be particularly problematic in the mineral beneficiation processes. Their nature leads spatial chemical inhomogeneity, meaning that the particle edge and face possess different chemical and physical properties. The presence of such minerals in the stirring devices (stirring tanks, flotation cells) is often characterized with coexistence of stagnant and agitated volumes of slurry, which negatively impacts the efficiency of stirring. In this work, the dilute aqueous Na-bentonite suspensions were examined via magnetic resonance imaging velocimetry to investigate the influence of pH and type of monovalent electrolyte on their local rheological behavior. The results indicated that suspensions with 0.1 vol.% solid can exhibit shear banding, shear localization or no local phenomenon as a function of chemistry of the suspending media. It was suggested that the existence of master curve (or global flow curve) for dilute suspensions was dependent on the bentonite particle organization in the suspension, which was influenced by the chemistry of the environment and the previous flow history. In the next step, second mineral phase (hematite or quartz) was added to the bentonite matrix. The interest in examination of such systems is related to the flow behavior of matrix formed with different kinds of inter-particle contacts. For example, at pH 4 the resulting electrostatic interaction between positively charged bentonite edge and negatively charged quartz is attractive, whereas at the same pH it is repulsive with the positively charged hematite. These electrostatic interactions result in different organization of matrix particles around another mineral phase. In the system with solely repulsive interactions between all sites of all mineral phases (e.g., quartz and bentonite, pH 10) the deviation from Newtonian behavior is justified by the shear-induced particle rearrangements, collision and friction. The difference in the arrangement of bentonite particle aggregates around the hematite or quartz particles was observed using SEM. As a next step the third mineral phase was added. The types of contacts established in the suspensions with different chemistry of the media were discussed along with their flow propperties

L'incidence des candidoses invasives a considérablement augmenté au cours des dernières décennies, parallèlement à l'augmentation du nombre de patients à risque. Ces pathologies sont associées à une morbi-mortalité élevée. Ce pronostic peut toutefois être amélioré par l'instauration d'un traitement antifongique précoce. Quatre classes de molécules antifongiques sont aujourd’hui disponibles pour la prévention et le traitement des candidoses invasives : échinocandines, antifongiques azolés, polyènes et pyrimidines. Du fait d’une efficacité importante et d’une meilleure tolérance, les échinocandines et les antifongiques azolés sont les plus largement utilisés. L'utilisation massive de ces molécules a conduit à une modification de l'épidémiologie des candidoses invasives, avec l'émergence d'espèces non-albicans intrinsèquement moins sensibles à ces antifongiques, à l'exemple de C. glabrata ou C. parapsilosis. De plus, des souches résistantes voire multi-résistantes aux échinocandines et aux antifongiques azolés sont de plus en plus fréquemment isolées, et associées à des échecs thérapeutiques. L'activité de ces antifongiques est également limitée par le phénomène de tolérance, résultant de la capacité d'adaptation des levures aux stress membranaires et pariétaux induits par ces molécules.Pour toutes ces raisons, l'identification et le développement de nouvelles stratégies antifongiques sont nécessaires. Ce travail de thèse s'inscrit dans cette optique, avec pour objectifs l'étude de la tolérance et de la résistance aux antifongiques chez Candida spp. et la caractérisation de nouvelles cibles thérapeutiques impliquées dans ces processus.La première partie de ce travail a permis de démontrer l'intérêt du séquençage nouvelle génération et d'une approche multigénique pour l'étude des mécanismes impliqués dans la résistance aux antifongiques azolés et aux échinocandines chez Candida spp. Par ce biais, il a été possible d'appréhender de nouveaux mécanismes potentiellement impliqués dans la résistance aux antifongiques, qui nécessitent toutefois d'être confirmés.Dans un deuxième temps, ce travail a permis de mettre en évidence que l'ensemble des protéines de la voie de signalisation du pH ou voie Rim chez les levures est impliqué dans la tolérance aux antifongiques chez C. albicans, l'espèce la plus fréquemment isolée en pathologie humaine. De plus, de nouveaux gènes Rim-dépendants ont été identifiés par RNA-sequencing, à l'exemple de HSP90, codant pour une protéine chaperone responsable de la régulation de multiples processus biologiques, et IPT1, responsable de la biosynthèse du principal sphingolipide membranaire. Ces deux gènes ont précédemment été impliqués dans la tolérance aux antifongiques azolés et aux échinocandines chez C. albicans et pourraient participer à la tolérance aux antifongiques médiée par la voie Rim. Ces résultats ouvrent des perspectives intéressantes : en effet, en ciblant la voie Rim, il pourrait être possible de potentialiser l'activité des molécules antifongiques actuellement commercialisées et de cibler indirectement Hsp90 tout en s'affranchissant des problèmes de toxicité car cette voie de signalisation est spécifique du règne fongique.Enfin, une dernière partie de ce travail réalisée dans le cadre du partenariat de l'ANR FungiBET a permis de montrer que la protéine BET (Bromodomain and Extra-Terminal) Bdf1 de C. glabrata, impliquée dans la régulation épigénétique de la transcription, est essentielle à la croissance in vitro. Plus précisément, l'intégrité des deux bromodomaines BD1 et BD2 de Bdf1, responsables de la liaison de cette protéine aux histones, est essentielle chez cette espèce, qui est la deuxième la plus fréquemment isolée dans les candidoses invasives en Europe et aux Etats-Unis. Ce résultat confirme les premières données obtenues chez C. albicans, et l'intérêt de l'inhibition des protéines BET fongiques comme nouvelle stratégie antifongique / The incidence of invasive candidiasis (IC) has dramatically increased over the past decades, partly due to the increasing number of at-risk patients. IC is associated with high mortality rates: however, its prognosis can be improved by early treatment. Four antifungal classes are available today for the prevention and treatment of IC: echinocandins, azoles, polyenes and pyrimidines. Due to their high efficacy and interesting safety profile, echinocandins and azoles are more commonly used. Massive use of these compounds has led to epidemiological changes in IC, with the emergence of non-albicans species which are intrinsically less susceptible to these antifungals, such as C. glabrata or C. parapsilosis. In addition, Candida spp. strains resistant, or even multiresistant, to azoles and echinocandins are increasingly isolated and associated with therapeutic failures. Antifungal activity is also limited by tolerance, a reversible phenomenon resulting from the yeast's adaptation to membrane and cell wall stresses caused by these molecules.For these reasons, identification and development of new antifungal strategies are needed. This work aimed at studying antifungal resistance and tolerance in Candida spp. and characterizing new therapeutic targets involved in these process.First, this work showed the interest of next-generation sequencing and multigene approaches to study mechanisms involved in resistance to echinocandins and azoles antifungals in Candida spp. It notably allowed to identify new mechanisms involved in antifungal resistance, which still need to be confirmed.In a second part, this work allowed to show that all the proteins of the pH-signaling pathway, known as the Rim pathway in yeasts, are involved in antifungal tolerance in C. albicans, the most frequent species responsible for IC in humans. In addition, new Rim-dependent genes were identified through RNA-sequencing, such as HSP90, coding for a major chaperone involved in the regulation of multiple cellular process, as well as IPT1, responsible for the synthesis of the main membrane sphingolipid.Both genes were previously shown to be involved in azoles and echinocandins tolerance in C. albicans, and could therefore play a role in antifungal tolerance mediated by the Rim pathway. These results offer great perspectives. Indeed, targeting the Rim pathway would allow to enhance the activity of commercially available antifungals and to indirectly target Hsp90, with no or limited toxicity as this signaling pathway is fungal-specific.Thirdly, a last part of this work performed in the context of the ANR FungiBET consortium, allowed to show that the BET protein Bdf1 in C. glabrata, which is involved in epigenetic regulation of transcription, is required for in vitro growth. More precisely, the integrity of both BD1 and BD2 Bdf1 bromodomains are essential in this species, which ranks second among causes of IC. This result confirms the first data obtained in C. albicans and the interest of inhibiting fungal BET proteins as a new antifungal strategy

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-05T15:41:23Z No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-04-06T12:12:45Z (GMT) No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-04-06T12:12:45Z (GMT). No. of bitstreams: 2 Dissertação - Bianca Silva Vieira de Souza - 2018.pdf: 1725073 bytes, checksum: 7c51424462ee34f86259c875516fd2ef (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-03-08 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Staphylococcus saprophyticus is a coagulase negative bacteria that is part of the human microbiota and may also be present in surfaces, food and the environment. It can act as a pathogen causing urinary tract infections (UTIs) in humans. The ability to capture micro and macro nutrients is related to the ability to survive and establish infection in pathogenic microorganisms. One of the micronutrients is iron, which can be acquired by microorganisms through secret siderophores or iron-reducing system on the cell surface. When S. saprophyticus infection is initiated the bacteria causes changes in the medium, resulting in a change in the pH of the urine. Iron can be found in soluble form (Fe 2+) in higher concentration at acid pH, and insoluble (Fe 3+) in higher concentration at basic pH. In this study, the proteomic profile of S. saprophyticus when grown at acidic and basic pH was evaluated. The results demonstrate that S. saprophyticus respond to the instances of modulating the pH of proteins related to iron metabolism. A siderophores production capacity by S. saprophyticus was also investigated after culturing in minimal SSD medium without iron. The results demonstrate that this bacteria produces siderophores of the carboxylate type when cultivated in the absence of iron. Phagocytosis assays in macrophages demonstrate that S. saprophyticus is more susceptible to death after infection when they are deprived of iron, demonstrating that this element is important to ensure infection. The present study increased the knowledge of the proteomic and metabolic flexibility of S. saprophyticus in response to extracellular iron levels. / Staphylococcus saprophyticus é uma bactéria coagulase negativa que faz parte da microbiota humana, podendo tambémser encontrado em superfícies, alimentos e meio ambiente. Pode atuar como patógeno causando infecções de trato-geniturinário (ITU) em seres humanos. A capacidade de captar micro e macro nutrientes está relacionada com a habilidade de sobrevivência e estabelecimento de infecção em microrganismos patogênicos. Uma desses micronutrientes é o ferro, que pode ser adquirido por microrganismos através de sideróforos secretados ou sistema redutor de ferro na superfície celular. Quando a infecção por S. saprophyticus é iniciada a bactéria ocasiona mudanças no meio, tendo como consequência a variação do pH da urina. O ferro pode ser encontrado na forma solúvel (Fe 2+) em maior concentração em pH ácido, e insolúvel (Fe3+) em maior concentração em pH básico. Neste estudo o perfil proteômico de S. saprophyticus quando cultivado em pH ácido e básico foi avaliado. Os resultados demonstram que S. saprophyticus responde às alterações de pH modulando proteínas relacionadas ao metabolismo de ferro. Foi investigada também a capacidade de produção de sideróforos por S. saprophyticus após cultivo em meio mínimo SSD sem ferro. Os resultados demonstram que esta bactéria produz sideróforos do tipo carboxilato quando cultivado na ausência de ferro. Ensaios de fagocitose em macrófagos demonstram que S. saprophyticus é mais suscetível à morte após infecção quando previamente privado de ferro, demonstrando que este elemento é importante para garantir a infecção. O presente estudo amplia o conhecimento da flexibilidade proteômica e metabólica de S. saprophyticus em resposta às alterações de níveis de ferro extracelular.

O efeito do caroço de algodão moído (CAM) na alimentação de cordeiros(as) foi avaliado em três experimentos. As dietas foram isonitrogenadas (16% PB) e composta por 90% de concentrado e 10% de feno de \"coastcross\". Experimento 1: Quarenta e cinco cordeiros Dorper x Santa Inês (PC inicial de 21,3±3,7 kg e 79±6 dias) foram distribuídos em delineamento de blocos completos casualizados. Os tratamentos foram definidos de acordo com os teores de CAM na dieta: 0, 7, 14, 21 ou 28% na MS. Na 3ª e 6a semanas do confinamento foi avaliado o comportamento ingestivo dos cordeiros. Amostras do músculo L. dorsi foram usadas para a avaliação da carne e determinação do perfil de ácidos graxos. Houve redução linear no peso final dos animais e no CMS. Na avaliação do comportamento ingestivo houve redução linear na ruminação em Min/g de FDN. Com a redução no peso final, as características da carcaça também reduziram linearmente. Houve alteração no perfil de ácidos graxos com redução linear para palmitoleico, oleico e monoinsaturados, enquanto o ácido linoleico aumentou. Foi observado efeito quadrático para esteárico, vacênico, linolênico e insaturados. A inclusão do CAM afetou negativamente o desempenho e as características da carcaça e demostrou baixa capacidade em estimular a ruminação. Experimento 2: Cinco machos inteiros, Dorper x Santa Inês, canulados no rúmen, foram distribuídos em delineamento experimental quadrado latino 5x5 para avaliar os efeitos da inclusão de CAM na digestibilidade das dietas, metabolismo de nitrogênio e parâmetros ruminais. As dietas experimentais foram as mesmas do Exp. 1. No último dia de colheita de amostras, o conteúdo ruminal foi amostrado. A digestibilidade da matéria seca, matéria orgânica, proteína bruta e carboidratos não fibrosos apresentaram redução linear. Entretanto, a digestibilidade do EE apresentou efeito quadrático e a FDN não foi afetada. Houve efeito quadrático para AGCC totais e propionato, redução linear no acetato e aumento linear do pH ruminal. O nitrogênio amoniacal não foi afetado. Experimento 3: Vinte e uma cordeiras White Dorper x Santa Inês (PC inicial de 23,6±1,2 kg e 87,7±4,5 dias) foram utilizadas em delineamento de blocos completos casualizados. As dietas experimentais foram: CA0 - controle, sem caroço de algodão; CAI21 - com 21% de caroço de algodão inteiro; e CAM21 - com 21% de caroço de algodão moído. Amostras do músculo L. dorsi foram coletadas para as análises físico-químicas e avaliação sensorial. O painel sensorial foi composto por 80 provadores não treinados. A cor da carne apresentou diferença quando comparado o tratamento controle com o grupo que recebeu caroço de algodão (CA21=CAI21+CAM21) (L:29,6vs36,7; a*:9,0vs7,5; b*:11,0vs8,7). Na avaliação sensorial o tratamento controle apresentou melhores notas para textura (6.5vs5.9), suculência (6.8vs6.1), aceitação global (6.2vs5.6), e sabor (6.0vs5.5) quando comparado com o CA21. Não foi observado efeito da moagem do CA em nenhuma das características avaliadas. A adição de caroço de algodão inteiro ou moído na alimentação de cordeiras em confinamento influenciou negativamente a qualidade sensorial da carne mostrando que seu uso deve ser restrito. / The effects of feeding ground linted cottonseed (GCS) to feedlot lambs were determined in three trials. The diets were isonitrogenous (16% CP) and composed of 90% concentrate and 10% hay \"coastcross\". Experiment 1: Forty-five ram lambs, Dorper x Santa Inês (initial BW of 21.3±3.7 kg and 79±6 days) were distributed in a randomized complete block design. Experimental treatments were defined according to the level of GCS in the diet: 0, 7, 14, 21 or 28% in the DM. In the 3rd and 6th weeks of the feedlot was evaluated the ingestive behavior of the lambs. L. dorsi muscle samples were used for the evaluation of meat and determination of fatty acid profile. There was a linear decrease in final weight of the animals and in DMI. There was a linear decrease in rumination Min/g of NDF. With the reduction in final weight, carcass characteristics also decreased linearly. There was a change in fatty acid profile with a linear reduction of palmitoleic acid, oleic and monounsaturated fatty acids, while linoleic acid increased. There was a quadratic effect for stearic, vaccenic, linolenic acid and total unsaturated. The inclusion of GCS negatively affected the performance and carcass characteristics and demonstrated low capacity to stimulate rumination. Experiment 2: Five ram lambs, Dorper x Santa Inês (45.2 ± 0.8 kg at the beginning of the trial), cannulated in the rumen, were distributed in a 5x5 Latin square design to determine the effects of adding GCS in the diet digestibility, nitrogen metabolism and rumen constituents. Each experimental period lasted 22 days, 17 days for adaptation and 5 days for sampling. The treatments were the same of Exp. 1. The digestibility of dry matter, organic matter, crude protein and non-fiber carbohydrates decrease linearly. However, the digestibility of EE had a quadratic effect and NDF was not affected. There was a quadratic effect for total SCFA and propionate, and a linear decrease for acetate. Ruminal pH increased linearly. Ammonia nitrogen was not affected. Experiment 3: The objective of this experiment was to determine the effects of feeding high-concentrate diets with whole or ground cottonseed on growth, carcass characteristics, sensory evaluation and meat quality of ewe lambs. Twenty-one White Dorper x Santa Inês ewe lambs (initial BW = 23.6 ± 1.2 kg and 87.7± 4.5 days) were used in a randomized complete block design according to initial BW and age. The experimental diets were: 1) CS0: control, without cottonseed; 2) WCS21: with 21% of whole cottonseed; and 3) GCS21: with 21% of ground cottonseed. Samples of longissimus muscle were used for meat quality analysis and sensory evaluation. Sensory test was performed by 80 untrained panelists. Color was different when the control diet was compared with the cottonseed (CS21=WC21+GC21) groups (L:29.6vs36.7; a*:9.0vs7.5; b*:11.0vs8.7). The control group had greater tenderness (6.5vs5.9), succulence (6.8vs6.1), acceptability (6.2vs5.6), and flavor (6.0vs5.5) values compared with CS21. In conclusion, whole or ground cottonseed fed to feedlot ewe lambs had a detrimental effect on sensory evaluation showing that its use should be restricted.

O objetivo deste estudo foi avaliar o pH e a liberação de íons cálcio dos cimentos AH Plus, MTA Fillapex e Sealapex, em diferentes períodos, quando empregados em obturações de canais radiculares, bem como, a qualidade das obturações, após 1 ano de realizadas. O pH e a liberação de íons cálcio foram avaliados por meio de um peagômetro e espectrofotômetro de absorção atômica, respectivamente. A qualidade das obturações foi avaliada por meio de um estereomicroscópio. Cento e vinte e quatro pré-molares inferiores humanos extraídos, unirradiculados, foram divididos em 01 grupo controle e 02 grupos experimentais, de acordo com a padronização do diâmetro do forame apical. Em seguida, os 02 grupos experimentais foram divididos em 4 subgrupos, considerando as técnicas de instrumentação e obturação empregadas. Após o preparo dos canais radiculares cada subgrupo teve o número de dentes divididos por dois, formando novos subgrupos, de acordo com os cimentos obturadores utilizados. Imediatamente após a obturação, a raiz de cada dente foi imersa em 15 mL de água ultrapura para avaliação do pH e liberação de íons cálcio nos seguintes períodos: 1 h, 1, 3, 7, 30, 180 e 365 dias, após a obturação. Após o período de 1 ano, os dentes foram seccionados transversalmente a 2, 4, 6 mm do forame apical para análise da qualidade das obturações, analisando-se a porcentagem de cimento, guta-percha e espaços vazios na massa obturadora. Os cimentos apresentaram pH neutro, porém, aos 180 dias, os valores mostraram um ligeiro aumento quando utilizados em canais com forames de diâmetro 0,25 mm. Quando utilizados em canais com forames de diâmetro 0,45 mm, o pH mostrou-se com valores maiores para todos os períodos sem distinção do cimento. Todos os cimentos liberaram íons cálcio, com menores valores para o AH Plus. Na estereomicroscopia, observaram-se variações nas porcentagens de cimento e guta-percha, considerando-se os diâmetros foraminais e técnicas obturadoras, e porcentagem insignificante de espaços vazios. / The objective of this study was to evaluate the pH and calcium ion release of AH Plus, MTA Fillapex and Sealapex cements at different time periods when used in root canal obturations, as well as the quality of the obturations one year after being performed. The pH and calcium ion release were evaluated by means of a pH meter and an atomic absorption spectrophotometer, respectively. The quality of the obturations was evaluated using a stereomicroscope. One hundred and twenty-four extracted human uniradicular mandibular premolars were divided into 1 control and 2 experimental groups, according to the standardization of the apical foramen diameter. Next, the 2 experimental groups were divided into 4 subgroups, considering the instrumentation techniques and obturations employed. After the preparation of the root canals, each subgroup had the number of teeth divided by two, forming new subgroups, according to the sealers used. Immediately after the obturation, the root of each tooth was immersed in 15 mL of ultrapure water for the evaluation of the pH and calcium ion release in the following time periods: 1 hr, 1, 3, 7, 30, 180 and 365 days after the obturation. After the period of one year, the teeth were transversely sectioned at 2, 4 and 6 mm from the root apex to examine the quality of the obturations, analyzing the percentage of cement, gutta-percha and voids in the obturator mass. The cements presented a neutral pH, which showed slightly increase as of 180 days when used in canals with foramens of 0.25 mm diameters. When used in canals with foramens with diameters of 0.45 mm, the pH presented higher values in all periods for all cements. All cements released calcium ions, with lower values for the AH Plus. In the stereomicroscopy, there were variations in the percentages of cement and gutta-percha, considering the foraminal diameters and obturation techniques and an insignificant percentage of voids.

Foram estudados a composição e o pH do meio de cultivo para a produção do fragmento de anticorpo (scFv) anti-LDL(-), expresso em Pichia pastoris recombinante. Os experimentos que definiram a composição e pH do meio assim como a concentração inicial de células na fase de indução foram realizados em agitador orbital a 250 rpm, com temperatura de 30 ºC na fase de crescimento e 20 ºC na fase de indução, durante 72 horas, com adição diária de 1% (v/v) de metanol. Para modificação do meio foi realizado um planejamento experimental empregando como variáveis independentes: extrato de soja, casaminoácidos e ureia, os quais substituíram o YNB e a biotina presentes no meio padrão (BMMY). Apesar de haver maior produção no meio com extrato de soja, o meio contendo 10 g.L-1 de casaminoácidos foi selecionado, uma vez que este favoreceu a etapa de purificação. A partir da faixa de pH estudada entre 3,0 e 8,0, determinou-se que o pH 8,0 no início da fase de indução favorece a maior produção. Finalmente, o meio BMMY-CA (pH 8,0) foi utilizado para cultivo em biorreator com volume de trabalho de 10L e a partir deste cultivo foram calculados os parâmetro cinéticos (velocidades de crescimento, de consumo de substrato e de produção, bem como produtividade). Diante do conjunto de experimentos realizados, foi possível otimizar a composição do meio de cultivo e as condições operacionais, que possibilitaram um aumento do rendimento bem como aumento do volume de produção do scFv anti-LDL(-) em biorreator. / Composition and pH culture medium for the production of anti- LDL(-) antibody fragment (scFv) were studied in recombinant yeast Pichia pastoris. The experiments that defined medium composition, pH and the initial cell concentration in the induction phase were carried out in baffled shaker flasks at 250 rpm, with temperature at 30°C for growth phase and 20°C for induction phase, during 72 hours with daily addition of 1% (v/v) methanol. A design of experiments employing for medium modification with independent variables: soy extract, casamino acids and urea, which replaced the YNB and biotin present in the standard medium (BMMY) was conducted for medium formulation. Even though, there was an increase in medium production with soy extract, the medium containing 10 g.L-1 casamino acids was selected since it favors the purification step. Through a pH range study (3.0 to 8.0), it was determined that pH 8.0 during induction phase offered higher levels. Then, the best results from shaker flask cultivation (BMMY-CA with casamino acids and pH 8.0) were carried out in 1L bioreactor, showing a biomass and a scFv production increase compared to the standard, BMMY (pH 6.0). Finally, the medium BMMY-CA (pH 8,0) was submitted to a volume scale-up into a 10L bioreactor, which was also used to evaluate kinetic parameters (rates of growth, substrate consumption and production as well as productivity). In conclusion, by optimizing culture medium and operating conditions it was possible to increase yield and scale-up the production of scFv anti-LDL(-) in bioreactor.

Made available in DSpace on 2016-04-28T14:59:47Z (GMT). No. of bitstreams: 1 2009 - Stela Gomes Pelegrino.pdf: 247744 bytes, checksum: edcaaef4e983d4522513a9fcb967acdc (MD5) Previous issue date: 2008-04-29 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / The search of better quality of the milk induces that changes and advances should be reached trought manipulation of de diet of the animals. The evaluation of the food absorption processes have been a very important in the investigation of the animal nutrition. The objective was to evaluate the on ruminal fermentation parameters in high yielding dairy cows fed total mixed ration (TMR). Eighteen Holstein cows in initial third of lactation were distributed to three groups. Cows were fed ad libitum thirty a day. The diet containing corn silage, barley, cottonseed and commercial ration, with forage:concentrate ratio of 45:55. Ruminal fluid was used for sampling at 0, 2, 4 and 6 hours after first meal for determination of ammonia, pH and VFA. The daily average intakes of nutrients, coefficients digestibility and milk production was evaluated how index of the production. There was no effect for averages of molar ruminal concentration ammonia. Was different, 4 hour after fed for the parameters: pH, VFA, acetic, butyric acids evidencing intense ruminal fermentative activity. Total VFA and pH was different in 4 hour after fed. The values of the propionic acid had followed the same trend of the AGV but they had not presented significant difference, the relation acetate: propionato was remained above of 3, keeping adjusted the text of fat of milk. This study demonstrates that the use of total diet provided a ruminal environment with adequate conditions of fermentation without large oscillations, what it diminishes the incidence of metabolic riots in cows of high production. / As melhores mudan?as na produ??o do leite podem ser atingidas atrav?s da manipula??o da dieta dos animais. Avaliar os processos de absor??o de alimentos direcionados para a s?ntese do leite tem sido de grande import?ncia nas pesquisas de nutri??o animal O objetivo deste trabalho foi avaliar os par?metros ruminais de vacas de alta produ??o alimentadas com dieta total. Dezoito animais da ra?a Holandesa, no primeiro ter?o de lacta??o, foram divididos em tr?s grupos, por ordem de pari??o. Os animais receberam dieta total ? vontade (ad libitum) tr?s vezes ao dia composta por silagem de milho, cevada ?mida, caro?o de algod?o e ra??o comercial, com a rela??o volumoso:concentrado de 45:55. Amostras de l?quido ruminal foram coletadas nos hor?rios de 0, 2, 4 e 6 horas, ap?s a alimenta??o, no per?odo da manh?, sendo utilizadas para determina??es das concentra??es de am?nia, pH e AGV do l?quido ruminal. Os valores de consumo m?dio di?rio, coeficiente de digestibilidade, produ??o de leite foram avaliados como indicadores da produ??o. N?o foi observado efeito significante nas concentra??es m?dias ruminais de am?nia. Na quarta hora ap?s a alimenta??o foi observada diferen?a significativa dos par?metros: pH, AGV total, ?cidos ac?tico e but?rico, evidenciando a intensa atividade fermentativa ruminal. Os valores m?dios de ?cido propi?nico seguiram a mesma tend?ncia dos AGV mas n?o apresentaram diferen?a significativa, a rela??o acetato:propionato manteve-se acima de 3, mantendo adequado o teor de gordura do leite. Este trabalho demonstra que o uso de dieta total proporcionou um ambiente ruminal com condi??es adequadas de fermenta??o sem grandes oscila??es, o que diminui a incid?ncia de dist?rbios metab?licos em vacas de alta produ??o.