Sinais clínicos de dispepsia são frequentemente observados em pequenos animais, recomendando-se nesses pacientes a pesquisa de Helicobacter sp., pois esse agente pode estar associado com a ocorrência desses distúrbios. Há poucos relatos de isolamento dessa bactéria em animais no Brasil. Portanto, o objetivo do estudo foi investigar a presença de Helicobacter sp. em pacientes com indicação de endoscopia digestiva alta, atendidos no Hospital de Clínicas Veterinárias da Universidade Federal do Rio Grande do Sul, empregando os métodos diagnósticos mais comumente adotados para sua detecção. Foram colhidos fragmentos de biópsia gástrica de um total de 30 cães e gatos submetidos à endoscopia, que não faziam uso de medicação antimicrobiana e inibidores de bomba de prótons no período mínimo de quatro semanas anteriores à coleta. Os fragmentos foram submetidos ao teste rápido da ureia (URT), colorações histológicas (H&E, Warthin Starry-WS), imunohistoquímica (IHQ), semi-nested PCR, tendo como alvo o gene 16SrRNA e cultura. Foram considerados positivos para Helicobacter sp. os pacientes que apresentaram resultado positivo em pelo menos dois métodos diagnósticos. Em apenas três animais foram detectadas lesões macroscópicas durante a endoscopia e 17 apresentaram lesões histopatológicas na mucosa do estômago. O URT foi positivo em 25 amostras e Helicobacter foi isolado de oito animais. Lâminas coradas por H&E e WS monstraram bactérias com morfologia compatível em 20 e 26 amostras, respectivamente. As técnicas de IHQ e semi-nested PCR resultaram positivas em 27 e 28 amostras, respectivamente. Ao todo, 28 pacientes foram considerados positivos, sendo que IHQ e semi-nested PCR detectaram 96,4% e 100% desses, respectivamente. Dessa forma, conclui-se que ambas as técnicas podem ser recomendadas para a detecção de Helicobacter em pequenos animais. A presença de Helicobacter sp. pode ter causado os sinais gastrintestinais apresentados por cães e gatos incluídos no estudo, porém em muitos pacientes sintomáticos houve presença da bactéria na ausência de lesões microscópicas detectáveis. / Dyspepsia signs are frequently observed in small animals. In these patients, Helicobacter sp. investigation is recommended, since this pathogen may be associated with the occurrence of this kind of disorders. In Brazil there are few reports of Helicobacter sp. detection in small animals. Therefore, the aim of the study was to investigate the presence of Helicobacter sp. by the most common methods adopted for diagnosis in patients referred for upper endoscopy at the Veterinary Hospital of the Federal University of Rio Grande do Sul. Thirty patients, which had not been using any antimicrobial drugs and proton pump inhibitors for at least four weeks prior the endoscopy, were selected for this study. The biopsy fragments were submitted to rapid urea test (URT), histological staining (H&E, Warthin Starry- WS), immunohistochemistry (IHC), semi-nested PCR targeting the 16S rRNA gene and culture. Patients that tested positive in at least two diagnostic methods were considered positive for Helicobacter sp. Macroscopic lesions were detected in only three animals during endoscopy, and seventeen had histopathological lesions in the stomach mucous. The URT resulted in 25 positive samples, while Helicobacter sp. was isolated from only eight patients. Histological staining (H&E and WS) showed bacteria with typical morphology in 20 and 26 samples, respectively. The IHC and semi-nested PCR were positive in 27 and 28 samples, respectively. A total of 28 patients were considered positive; among them IHC and semi-nested PCR resulted positive in 96.4% and 100%, respectively. In conclusion, both techniques can be recommended for Helicobacter sp. detection in small animals. Helicobacter infection may have been the cause of gastrointestinal signs detected in patients; however many symptomatic animals without microscopic lesions were also diagnosed positive.

Submitted by Erika Demachki (erikademachki@gmail.com) on 2017-03-14T19:03:25Z No. of bitstreams: 2 Dissertação - Lana OHara Souza Silva - 2017.pdf: 3124489 bytes, checksum: 38e1e83b1c39e6954300e0cd3e709f2a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2017-03-20T12:27:41Z (GMT) No. of bitstreams: 2 Dissertação - Lana OHara Souza Silva - 2017.pdf: 3124489 bytes, checksum: 38e1e83b1c39e6954300e0cd3e709f2a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2017-03-20T12:27:41Z (GMT). No. of bitstreams: 2 Dissertação - Lana OHara Souza Silva - 2017.pdf: 3124489 bytes, checksum: 38e1e83b1c39e6954300e0cd3e709f2a (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-02-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The Paracoccidioides genus is composed of thermodimorphic fungus that causes paracoccidioidomycosis (PCM), an endemic human systemic mycosis in Latin America. These organisms grow as mycelium in temperatures below 28 °C and as yeast form in temperatures above 37 °C. Nitrogen is an important element in this microorganism’s nutrition that participates in the synthesis of proteins, nucleic acids and others biomolecules. In this regard, nitrogen uptake and metabolism are essential to growth and fungal establishment. When nitrogen levels and sources such as glutamine and ammonia concentration are limited, pathogenic fungus use a regulation system called Nitrogen Catabolic Repression that induces the expression of genes encoding permeases and enzymes required for the catabolism of secondary nitrogen sources, such as formamidase, gamma-glutamiltranspeptidase and urease. Gamma-glutamiltranspeptidase is an enzyme that catalyzes the first reaction of glutationa degradation and it has been the target of several studies about nitrogen starvation in various fungi. It has been observed that the expression of the gene encoding this enzyme was induced in limiting conditions of nitrogen and was repressed when the availability of nitrogen was high. Urease is an enzyme that catalyzes the degradation of urea in ammonia and carbonic acid. This enzyme is already known as a virulence factor in fungi such as Cryptococcus. neoformans, and also has been the target of studies about nitrogen starvation. In this study we expressed gamma-GT and urease proteins from Paracoccidioides brasiliensis, isolate Pb18, in Escherichia coli. The gene coding for Ggt and Ure were cloned in pET32a expression vector, and used for E. coli pLysS transformation. The recombinant proteins produced were shown to be catalytically active. Together, data obtained in this work could add knowledge about the role of gamma-GT and urease and can be used as a foundation for complementary experiments regarding nitrogen metabolism regulation, as well as in Paracoccidioides spp pathogenesis. / Resumo: O gênero Paracoccidioides é composto por fungos termodimórficos que causam a paracoccidioidomicose (PCM), uma micose sistêmica humana endêmica na América Latina. Quando cultivados em temperaturas menores que 28 °C o fungo cresce como micélio e em temperaturas em torno de 37 °C, como levedura. O nitrogênio é um importante nutriente para os micro-organismos, pois participa da síntese de proteínas, ácidos nucléicos e outras biomoléculas. Nesse sentido, a captação e o metabolismo de nitrogênio são essenciais para o crescimento e o estabelecimento do fungo no hospedeiro. Quando os níveis de nitrogênio e fontes como glutamina e amônia estão em concentrações limitantes, os fungos patogênicos utilizam um sistema de regulação chamado Repressão Catabólica de Nitrogênio que induz a expressão de genes que codificam permeases e enzimas necessárias para o catabolismo de fontes secundárias de nitrogênio como a formamidase, a gama-glutamil transpeptidase e a urease. A gama-glutamil transpeptidase é uma enzima que catalisa a primeira reação da degradação da glutationa. Ela tem sido alvo de estudos de privação de nitrogênio em diversos fungos, nos quais foi observada uma alta expressão do gene codificador dessa enzima em condições limitantes de nitrogênio, enquanto que, em alta disponibilidade de nitrogênio a sua expressão era reprimida. A urease é uma enzima que degrada uréia em amônia e ácido carbônico. Ela já é conhecida por ser um fator de virulência em alguns fungos, como Cryptococcus neoformans, e também tem sido alvo de estudos de privação de nitrogênio. Neste estudo nós expressamos as proteínas gama-GT e urease de Paracoccidioides brasiliensis, isolado Pb18, em sistema heterólogo bacteriano de Escherichia coli. O fragmento dos genes codificadores de Ggt e Ure foram clonados em vetor de expressão pET32a e os respectivos clones foram utilizados na transformação de células de E. coli pLySs. As proteínas recombinantes produzidas mostraram estar cataliticamente ativas. Os dados obtidos neste trabalho puderam acrescentar conhecimentos sobre as enzimas gama-GT e urease e podem ser usados como base para experimentos complementares em relação à regulação do metabolismo de nitrogênio bem como na patogênese de Paracoccidioides spp.

Le travail de la thèse présente une série de (bio)capteurs conductimétriques, à base de la clinoptilolite, pour la détermination de l’ammonium, de l’urée et de la L-arginine. La clinoptilolite, le matériau nanométrique, possédant des propriétés de la sorption intrinsèque et une capacité d’échange cationique vis-à-vis des espèces ammonium, a été d’abord utilisée pour la réalisation d’un microcapteur conductimétrique sélectif à NH4+. Ci-après, une application de ce nanomatériau dans les biocapteurs est favorable pour le fonctionnement dans les solutions tampons multicomposants. Parmi plusieurs variantes de biocapteurs à l’urée à base de la zéolite, la plus intéressante est le biocapteur, dans lequel la couche de la clinoptilolite, déposée sur le transducteur, a été recouverte par le dépôt de la couche de l’uréase et de la zéolite. Pour l’élaboration d’un biocapteur conductimétrique hautement sensible pour la détermination de la L-arginine, l’arginase et l’uréase ont été co-réticulées sur le transducteur. Une détermination quantitative de la L-arginine dans une solution buvable « Arginine Veyron » a montré un fort accord avec les données fournies par le producteur. Une procédure détaillée de l’optimisation du biocapteur conductimétrique pour la détection de la L-arginine dans le sérum bovin a été proposée. La clinoptilolite a été également appliquée comme un modificateur dans la co-immobilisation de l’arginase et l’uréase pour améliorer les caractéristiques analytiques de biocapteur conductimétriques pour la détermination de la L-arginine / Currentwork presents a serie of conductometric (bio)sensors based on clinoptilolite, for ammonium, urea and L-arginine determination. Clinoptilolite, a nanoscale material possessing exceptional sorption and cation-exchange properties toward ammonium species, was initially used for the development of NH4+-selective conductometric microsensor. The clinoptilolite-based microsensor was selective toward ammonium in the presence of interferences that are commonly found along with ammonium in natural waters. Hereafter, an application of this nanomaterial in biosensors is favorable for operation in multicomponent buffer solutions. Among the several variants of the urea biosensors based on zeolite, considerably better characteristics were obtained for the biosensor comprising a clinoptilolite adlayer and an upper layer of immobilized urease and zeolite. In the work, for first time was developed a highly sensitive conductometric biosensor for L-arginine determination based on arginase and urease co-immobilized in a single membrane. The results of a quantitative determination of L-arginine in a drinkable solution “Arginine Veyron”, obtained by the biosensor, were in high correlation with the data provided by the producer. The L-arginine conductometric biosensor was optimized for the serum analysis. Clinoptilolite was also applied as a modifier in co-immobilization of arginase and urease for the improvement of analytical characteristics of the conductometric biosensor for L-arginine determination

Ohne zuverlässige Chemo- und Biosensoren wären beispielsweise die Überwachung von Prozessparametern in der chemischen und biotechnologischen Industrie, die Detektion von geringsten Analytkonzentrationen in der biomedizinischen Analytik oder die Spurenanalyse von Schadstoffen undenkbar. Neue Sensormaterialien wie stimuliresponsive Hydrogele spielen bei der Entwicklung neuer chemischer und biochemischer Sensoren eine immer größere Rolle. Hydrogele sind „intelligente“ hydrophile Polymernetzwerke, die in Abhängigkeit von spezifischen Stimuli quellen und entquellen können. In Kombination mit piezoresistiven Drucksensoren wird dann der resultierende Quelldruck in eine messbare Ausgangsspannung umgewandelt. In dieser Arbeit werden sowohl neuartige stimuliresponsive Hydrogele für die Detektion von Ethanol in alkoholischen Getränken als auch zum Nachweis von gelöstem Ammoniak und Harnstoff für biotechnologische Prozesse vorgestellt. Nach der gezielten Synthese und Funktionalisierung der Gele werden zunächst die Quelleigenschaften in freier Quellung untersucht. Im Mittelpunkt der Charakterisierung stehen dabei sensorisch relevante Eigenschaften wie beispielsweise das Quellverhalten in Abhängigkeit vom jeweiligen Stimulus. Im nächsten Schritt werden piezoresistive Hydrogelsensoren aufgebaut und vermessen. Dabei werden wichtige Sensoreigenschaften wie der sensitive Messbereich, Nachweisgrenzen oder Querempfindlichkeiten detailliert untersucht und die Sensorkonzepte hinsichtlich ihres Anwendungspotenzials bewertet.

The effect of organic and mineral fertilizers on biological activity of different genesis soils in long-term crop rotation trials was determined. Biological activity was diverse in the soils of different genesis and it activity correlated with some soil chemical properties. Organic and mineral fertilizers and their combinations more increased biological activity in the soil than only mineral fertilizers. Mineral fertilizers suppressed dehydrogenase and alkaline phosphatase activity (180 kg ha-1) with phosphorus and potassium fertilizers. The relationship between the crops grown, their yield and enzyme activity and respiration intensity in the soil was investigated.

Les biotechnologies sont un outil puissant permettant d’emprunter les circuits biologiques pour produire des composés aux applications multiples (médecine, alimentation, industries…). Les cyanobactéries possèdent des propriétés génétiques et trophiques précieuses pour réduire les coûts et l’empreinte environnementale de ces procédés (photosynthèse, fixation du CO₂, sources d’azote assimilables...). Elles produisent aussi naturellement certaines molécules énergétiques comme le H₂ dont pourraient émerger de nouvelles filières propres de biocarburants. Cependant, une compréhension globale et approfondie de leur physiologie est nécessaire pour concevoir un châssis biologique performant à partir de ces organismes. Elles sont aisément manipulables génétiquement mais présentent une versatilité favorisant la fixation de mutations bénéfiques mais aussi délétères pour leur exploitation à grande échelle. Au cours de ma thèse, j’ai construit et étudié des mutants d’un régulateur de l’assimilation du CO₂ dont l’activation est liée à la photosynthèse. J’ai montré que l’activité du cycle de Calvin synchronise les flux du carbone et le statut rédox de Synechocystis et que sa dérégulation se répercute de manière pléiotropique sur son métabolisme. Plus spécifiquement, je me suis intéressé au déséquilibre carbone/azote dans cette espèce et à son métabolisme de l’urée qui présente un intérêt biotechnologique considérable. J’ai démontré que ce dernier était en compétition avec l’hydrogénase pour l’insertion du nickel dans leurs centres catalytiques respectifs. L’insuffisance de ce métal a permis de sélectionner des mutants de l’uréase tolérant une exposition prolongée à l’urée et conservant une forte capacité de production de H₂ en présence de ce substrat azoté. L’ensemble de ces résultats montre que le métabolisme de Synechocystis peut être détourné au profit de certains processus cellulaires. Les approches « omiques » permettent d’identifier globalement les réponses physiologiques induites ainsi que les leviers biologiques de compensation. Ces travaux sont discutés au regard des implications biotechnologiques de l’instabilité génétique et de la nécessité de renforcer notre compréhension de la plasticité métabolique et génomique des cyanobactéries. / Biotechnology is a powerful tool allowing exploitation of biological circuits to produce compounds with multiple uses (medicine, nutrition, industrial…). Cyanobacteria have valuable genetic and trophic properties which could reduce the costs and the environmental footprint of these processes (photosynthesis, CO₂ fixation, assimilation of diverse nitrogen sources…). They also naturally produce energetic molecules such as H₂ from which new and sustainable biofuels sectors may rise. However, a global and fine understanding of their physiology is required in order to design an efficient biological chassis with these organisms. They are genetically manipulable but also exhibit a strong versatility favoring fixation of mutations that can be either beneficial or harmful to their large-scale cultivation. Over the course of my PhD, I constructed and studied mutants of a CO₂ fixation regulator whose activation is linked to photosynthesis. I showed that the Calvin cycle activity synchronizes carbon fluxes and redox status in Synechocystis and that its deregulation affects the metabolism in a pleiotropic manner. I was specifically interested into the carbon/nitrogen balance in this species and its urea metabolism which is of prime interest in biotechnology. I demonstrated that the latter was in competition with the hydrogenase for the insertion of nickel into their respective catalytic centers. Scarcity of this metal leads to selection of mutants thriving upon prolonged exposure to urea that retained a high capacity of H₂ production in presence of this nitrogenic substrate. This work shows that the metabolism of Synechocystis can be altered in favor of other cellular processes. Omics approaches allow global identification of the physiological responses induced as well as the biological compensation mechanisms. These observations are discussed with regards to biotechnological implications of genetic instability and the need to strengthen our understanding of metabolic and genetic plasticity in cyanobacteria.

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