Avaliou-se prospectivamente 19 pacientes com obstrução infravesical por hiperplasia prostática benigna selecionados pra prostatectomia transvesical. Obteve-se um fragmento da parede da bexiga e quantificou-se o colágeno no epimísio, perimísio e endomísio do detrusor. Amostra vesicais de oito doadores cadavéricos de órgãos serviram como controles. Observou-se padrão focal de aumento do colágeno na camada muscular da bexiga dos pacientes com obstrução. Demonstrou-se que a idade correlaciona-se positivamente com o aumento da quantidade de colágeno com a diminuição da complacência vesical, o aumento da prevalência de hiperatividade detrusora e a maior chance de retenção urinária / We prospectively evaluated 19 patients with bladder outlet obstruction by benign prostatic hyperplasia selected for transvesical prostatectomy. A sample of the bladder wall was obtained and the collagen was measured at the level of epimysium, premysium and endomysium. Bladder samples from eight cadaveric organ donors were used as controls. A focal pattern of collagen increase was observed at the bladder smooth muscle layer of obstructed patients. Age was shown to correlate with increased collagen quantity. Increased collagen quantity also correlated with decreased bladder compliance, higher prevalence of detrusor overactivity and of urinary retention

A disseminação metastática de células tumorais malignas envolve múltiplas etapas e é uma das principais causas de mortalidade. O microambiente tumoral apresenta um importante papel no processo de tumorigênese. A laminina é um componente do microambiente que pode ser clivado em peptídeos bioativos, como o peptídeo AG73 (domínio globular da cadeia 1) que aumenta a formação/atividade de invadopódios. Invadopódios são protrusões de membrana ricas em actina com atividade proteolítica da matriz pericelular. Além da actina, os invadopódios exibem outras proteínas importantes, como a cortactina e o MT1-MMP. Recentemente, descobriu-se que a podoplanina pode desempenhar um papel importante na atividade dos invadopódios. A podoplanina é uma glicoproteína transmembrana do tipo I, intimamente associada à progressão maligna do câncer. A podoplanina e o peptídeo AG73 influenciam a biologia do câncer. Tal particularidade levou-nos a investigar o papel do peptídeo AG73 na regulação da da podoplanina e invadopódios em linhagem celular derivada de carcinoma de células escamosas oral (Cal 27). As células foram cultivadas em DMEM com SFB e tratadas com o peptídeo AG73. As células tratadas por peptídeo de sequência embaralhada serviram como controles. Imunofluorescência foi realizada para analisar os níveis de proteína de podoplanina, cortactina e MT1-MMP. Os resultados mostraram que o peptídeo AG73 diminuiu os níveis de podoplanina nas células Cal27 em comparação aos controles. Nenhuma alteração foi observada em relação à cortactina e MT1-MMP. Estes resultados foram confirmados por imunofluorescência. As células tratadas com AG73 diminuíram a distribuição da podoplanina em todo o citoplasma em comparação com os controles. Nossos resultados sugerem que o peptídeo derivado da laminina AG73 inibe a podoplanina, uma molécula reguladora do câncer, em células malignas humanas. / The metastatic spread of malignant tumor cells involves multiple steps, and is one of the major causes of mortality. The tumor microenvironment has an important role in tumorigenesis process. Laminin is a component of the microenvironment that can be cleaved into bioactive peptides, such as peptide AG73 (globular domain of 1 chain) that increase invadopodia formation/activity. Invadopodia are actin-rich membrane protrusions with proteolytic activity of peri-cellular matrix. In addition to actin invadopodia exhibit other key proteins such as cortactin and MT1-MMP. Recently it has been discovered that podoplanin may play an important role in the invadopodia activity. Podoplanin is a type I transmembrane glycoprotein, closely associated with the malignant progression of cancer. Podoplanin and the peptide AG73 influence cancer biology. This prompted us to investigate the role of peptide AG73 in the regulation of podoplanin and invadopodia formation in a cell line derived from oral squamous cell carcinoma (Cal 27). Cells were cultured in DMEM with FBS and treated with peptide AG73. Cells treated by scrambled peptide served as controls. Immunoblot was carried out to analyze protein levels of podoplanin, cortactin and MT1-MMP. Results showed that the peptide AG73 decreased podoplanin levels in Cal27 cells compared to controls. No alterations were observed with regard to cortactin and MT1-MMP. These results were confirmed by immunofluorescence. Cells treated by AG73 decreased podoplanin distribution throughout the cytoplasm compared to controls. Our results suggest that the laminin-derived peptide AG73 inhibits podoplanin, a cancer regulator molecule, in human malignant cells.

A hemoglobina extracelular de Glossoscolex paulistus tem uma massa molecular de 3,6 MDa, determinada por ultracentrifugação analítica. Esta proteína possui uma estrutura oligomérica composta por 144 cadeias globínicas e 36 cadeias sem o grupo heme, denominadas linkers. A HbGp é caracterizada por apresentar uma alta resistência a auto-oxidação e uma alta estabilidade oligomérica quando submetida a condições de estresse, tais como, variações de temperatura, pH e adição de agentes químicos (ureia, GuHCl e surfactantes). A primeira parte dos resultados desse trabalho descreve a estabilidade oligomérica da oxi-HbGp na presença de ureia, no pH 7,0 monitorada pela sonda de fluorescência fluoresceína isotiocianato (FITC). Este estudo foi desenvolvido através do uso de várias técnicas espectroscópicas tais como: absorção óptica no UV-VIS, emissão de fluorescência estática e resolvida no tempo, anisotropia estática e decaimento de anisotropia. Os tempos de vida de emissão de fluorescência dos triptofanos e da sonda FITC foram obtidos. Os decaimentos dos triptofanos são multi-exponenciais com quatro tempos de vida, onde dois estão na faixa de picosegundos e dois na faixa de nanosegundos. Os decaimentos de emissão dos triptofanos da oxi-HbGp pura e da oxi-HbGp modificada com FITC são bastante similares. Na ausência do desnaturante e na presença de até 2,5 mol/L de ureia os tempos curtos são predominantes. Em 3,5 e 6,0 mol/L de ureia as contribuições dos tempos longos aumentam significativamente. O processo de desenovelamento da oxi-HbGp induzido pela ureia é caracterizado pela dissociação oligomérica da proteína e desnaturação das subunidades dissociadas. Os decaimentos de emissão da sonda para o sistema FITC-HbGp são também multi-exponenciais com três tempos de vida, onde um dos tempos, na faixa de nanosegundos, é semelhante ao da sonda livre em tampão de 3,9 ns. Com o aumento da concentração de ureia, as contribuições dos tempos longos aumentam implicando a remoção da supressão observada para a emissão da sonda no sistema HbGp-FITC. Por outro lado, os decaimentos de anisotropia são caracterizados por dois tempos de correlação rotacional, associados, respectivamente, ao movimento residual da sonda em relação à proteína. Na segunda parte desse trabalho a forma apo-HbGp, ou seja, a oxi-HbGp sem os grupos hemes foi estudada através de um conjunto de técnicas: eletroforese, ultracentrifugação analítica (AUC), espalhamento dinâmico de luz (DLS), absorção óptica no UV-Vis, dicroísmo circular (CD), fluorescência estática e resolvida no tempo, na ausência e na presença da sonda 1-Anilino-8-naftaleno-sulfonato (1,8-ANS). Além disso, a concentração da apo-HbGp foi determinada pelo método do ácido bicinconínico (BCA). A partir dessa concentração o coeficiente de extinção molar foi calculado utilizando a lei de Beer-Lambert. Os dados de AUC mostraram duas espécies em solução, correspondendo ao monômero d e trímero abc, com coeficientes de sedimentação em torno de 2,0 e 3,5 S, respectivamente. Pelos dados de DLS percebe-se que a forma apo-HbGp monitorada por longos períodos de tempo é muito instável quando comparada à oxi-HbGp. No estudo de fluorescência resolvida no tempo da apo-HbGp foi observado à predominância de tempos longos, uma vez que os grupos hemes que promovem a supressão da emissão de fluorescência dos triptofanos foram removidos. Três tempos de vida são observados sendo dois longos na faixa de nano-segundos e um na faixa de sub-nano-segundos. / The extracellular hemogobin of Glossoscolex paulistus (HbGp) has a molecular mass of 3.6 MDa, determined by analytical ultracentrifugation. This protein has an oligomeric structure composed by 144 globin chains, and 36 additional chains lacking the heme group, named linkers. This class of proteins has a high resistance to oxidation and high oligomeric stability when subjected to stressful conditions such as temperature variation, pH and addition of chemical agents (urea, GuHCl, and surfactants). The first part of the results of this work describes the oxy-HbGp oligomeric stability, in the presence of urea, at pH 7.0, monitored by fluorescein isothiocyanate (FITC) fluorescence probe. This study was developed through the use of several spectroscopic techniques, such as, UV-VIS optical absorption, static and time-resolved fluorescence (time-correlated single photon counting TCSPC), static anisotropy and time-resolved anisotropy decay. Fluorescence lifetimes were monitored for both tryptophans and FITC and the corresponding emission decays were obtained. Tryptophan decays are multi-exponential with four characteristic lifetimes: two in the picosecond and two in the nanosecond time ranges. The tryptophan emission decays for pure HbGp and HbGp-FITC systems are quite similar. In the absence of denaturant, and up to 2.5 mol/L of urea, the shorter lifetimes predominate in the decay. At 3.5 and 6.0 mol/L of urea, the longer lifetimes increase significantly their contribution. Urea-induced unfolding process is characterized by protein oligomeric dissociation and denaturation of dissociated subunits. FITC emission decays for FITC-HbGp system are also multi-exponential with three lifetimes: two in the sub-nanosecond and one in the nanosecond range with a value quite similar to the free probe in buffer of 3.9 ns. Increase of urea concentration leads to increase of the longer lifetime contribution, implying the removal of the quenching of the probe emission observed for the native HbGp-FITC system. On the other hand, anisotropy decays are characterized by two rotational correlation times associated to the bound probe, and are due to some residual motion of the probe relative to the protein. In the second part of this work the apo-HbGp (oxy-HbGp without heme groups) was studied through a set of techniques: Sodium dodecyl sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Analytical Ultracentrifugation (AUC), Dynamic Light Scattering (DLS), UV-VIS optical absorption, Circular Dichroism (CD), static and time-resolved fluorescence, in the absence and in the presence of 8-anilino-1-naphtalene-sulfonic acid (ANS) probe. Moreover, the apo-HbGp concentration was determined by Bicinchoninic Acid (BCA) method. Based on these protein concentration results, it was possible to determine spectrophotometrically the apo-HbGp molar extinction coefficient employing the Lambert-Beer law. AUC results showed two species in solution, corresponding to the monomeric and trimeric species, with sedimentation coefficients around 2.0 and 3.5 S, respectively. The DLS data showed that the apo-HbGp form is very unstable when monitored for long times as compared to the HbGp oxy-form. In the time-resolved fluorescence study of apo-HbGp the predominance of long lifetimes was observed. This occurs because the heme groups that promote the tryptophan quenching of fluorescence in the native HbGp were removed. Three lifetimes are observed, two long in the nanosecond and one in the sub-nanosecond time ranges.

Les vésicules extracellulaires (EVs) participent à la communication intercellulaire. Dans la littérature actuelle, elles sont divisées en deux classes principales selon leur origine intracellulaire. En premier lieu, les exosomes sont formés à l'intérieur des endosomes multivésiculaires et sont libérés lors de la fusion de ces compartiments avec la membrane plasmique (MP). La taille des exosomes est contrôlée au cours de leur biogenèse et varie de 50 à 150 nm. Deuxièmement, les EVs sont formées par bourgeonnement direct et sécrétion à partir de la MP. Ces EVs sont plus hétérogènes et leur taille varie de 50 à 1000 nm. Malgré le fait que la nature hétérogène de EVs soit clairement documentée dans la littérature, la composition en protéines et les mécanismes exacts de la biogenèse des différentes EVs restent un sujet de débat en cours. Le but principal de ce travail était de redéfinir autant de sous-types différents d’EVs que possible, en trouvant des marqueurs protéiques spécifiques, et d'étudier les outils possibles pour affecter spécifiquement leur sécrétion. Dans ce projet, nous avons mis en place plusieurs outils utiles pour la caractérisation d’EVs. Tout d'abord, mes principaux efforts ont été concentrés sur la mise en place de plusieurs protocoles d'isolation et d'analyse d’EVs. Cela a conduit à la production d'une cartographie des protéines vésiculaires, qui si elle est appliquée pour caractériser les EVs, permettra de mieux les identifier par leur composition. Deuxièmement, j'ai étudié la façon dont la sécrétion de ces sous-populations d’EVs peut être modulée par l'inhibition de quelques protéines de la famille RAB et par certaines drogues. Enfin, grâce à une collaboration établie au sein de l'unité, j'ai eu l'occasion de participer à une comparaison des propriétés fonctionnelles entre les EVs et les virus sécrétés simultanément par les cellules infectées. Mes résultats confirment l'hypothèse selon laquelle l'origine intracellulaire des EVs sera reflétée dans leur composition. Les résultats présentés confirment la coexistence de plusieurs classes d'EVs et donnent un aperçu sur les moyens de les caractériser dans une préparation d’EVs donnée. En outre, nous fournissons un exemple de l'application de notre ensemble de protéines dans les études portant sur la biogenèse des EVs. / Extracellular vesicles (EVs) are participating in intercellular communication. Classically, in the current literature, they are divided into two main classes depending on their intracellular origin. Firstly, exosomes are formed within multivesicular endosomes and released upon fusion of these compartments with plasma membrane. The size of exosomes is controlled during their biogenesis and ranges from 50 to 150 nm. Secondly, EVs are formed by direct budding and pinching off from the plasma membrane. These EVs are more heterogeneous and their size varies from 50 to 1000 nm. Despite the fact that a heterogeneous nature of EVs is clearly documented in the literature, the exact protein content and biogenesis mechanisms of different EVs remain a matter of on-going debate. The principal goal of this work was to re-define as many different subtypes of EVs as possible, by finding specific protein markers, and investigate possible tools to affect specifically their secretion. In this project, we set up several tools useful for EV characterization. Firstly, my main efforts were concentrated on establishment of several protocols to isolate and analyse EVs. This led to the foundation of a vesicle protein cartography, which if applied to characterize EVs, will allow better understanding of the composition of the studied EVs. Secondly, I investigated how secretion of these EV subpopulations might be modulated by inhibition of a few RAB proteins and by some drugs. Finally, thanks to a collaboration established within the unit, I had the opportunity to participate in a comparison of the functional properties between EVs and viruses secreted simultaneously by infected cells. My results confirmed the hypothesis that the intracellular origin of EVs will be reflected in their composition. The results presented in this study point at the coexistence of several EV classes and provide insights on how to demonstrate their presence in a given EV preparation. In addition, we provide an example of the application of our set of proteins in studies addressing EV biogenesis.

Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

A cirrose é caracterizada por fibrose e por nódulos regenerativos que resultam na desorganização da arquitetura tecidual, sendo considerado um estágio irreversível. A administração de fatores hepatotróficos exógenos (FHE) poderia estimular a proliferação celular de células hepáticas e reduzir a cirrose induzida em ratos. Deste modo, os FHE atuariam na remodelação da matriz extracelular (ECM). Os objetivos desse trabalho foram avaliar os efeitos dos FHE em ratos cirróticos induzidos experimentalmente por tioacetamida. Foram realizadas análises histopatológicas, imunoistoquímica para BrdU, mensuração de colágeno, microscopia eletrônica de varredura, análises bioquímicas de função hepática e avaliação da expressão gênica de colágeno α1, TGFβ1, TIMP I, MMP 2 e Plau, por PCR em tempo real. Após administração de FHE obteve-se diminuição da expressão gênica dos genes fibrogênicos: colágenoα1, TGFβ1, TIMP I e MMP 2. Durante a remodelação da ECM foram observadas melhoras morfofuncionais, com diminuição do número de nódulos regenerativos parenquimais, diminuição da espessura dos septos fibrosos e reaparecimento de veias centrolobulares. Foi observado aumento do peso e volume do fígado dos ratos, assim como aumento na relação fígado/carcaça. Os animais tratados com FHE apresentaram redução de 29,62% do colágeno parenquimal total, quando comparados com sua própria biópsia antes do tratamento. Animais não submetidos ao tratamento com FHE tiveram um acréscimo de 8,7% de colágeno. Os índices de função hepática revelaram decréscimo significante nos níveis de gamaglutamiltranspeptida (GGT), alanina aminotransferase e aspartato aminotransferase. Desta forma, os FHE atuaram na remodelação da matriz extracelular hepática em fígados cirróticos pela diminuição da expressão de genes fibrogênicos e não necessariamente pelo aumento da expressão de genes fibrolíticos. Ademais, animais cirróticos apresentaram em seu fígado e intestino progranulina, um novo fator de crescimento. Propomos, dessa forma, a utilizalção da progranulina como um possível marcador clínico e alvo terapêutico para doenças hepáticas. / Cirrhosis is characterized by fibrosis and regenerative nodules, which result in the disorganization of the hepatic architecture, being considered an irreversible situation. The administration of exogenous hepatotrophic factors (EHF) could stimulate hepatic regeneration in hepatocytes cells and reduce the cirrhosis induced in rats. The aim of this work was to evaluate the effect of EHF in rat cirrhosis induced experimentally by thioacetamide. It was carried out histopathologics analysis; BrdU imunoperoxidase, collagen measurement, scanning electron microscopy; biochemical analysis for hepatic function and analysis on genic expression for collagen α1, TGFβ 1, TIMP I, MMP 2, and Plau by real time PCR. After the EHF administration, it was observed a reduction in the expression of fibrogenics genes as: collagenα1, TGFβ1, TIMP I and MMP 2. During the extracellular matrix (ECM) remodelation morphofuntional improvements were observed, with decrease of regenerative nodules and fibrous septs thickness as well as reappearance of central vein. It was observed increase in liver weight, volume and in the relation liver/carcass. The animals treated with EHF had a reduction of 29.62% in the total collagen when compared with their own biopsy before treatment, while the non-treated animals had an increase of 8,7%. The index of hepatic functions had significant improvement in the levels of gamaglutamiltranspeptida (GGT), alanine aminotransferase and aspartate aminotransferase. Therefore, the EHF acts on the extracellular matrix remodelation though reduction of fibrogenics gene expression and not necessarily by fibrolitics genes expression increase. In addiction, we have found progranulin, a new growth factor, in liver and intestine of cirrhotic animals. Hence, we propose the utilization of progranulin as a clinical marker and a therapeutic target for hepatic deseases.

This document refers to research whose main objective was to investigate the relationship among hydraulic, chemical and electrical signals in poor tomato plants ABA production. The document is organized into three chapters: The first chapter presents a detailed extracellular measurement protocol of electrical signals in plants and how to associate these measurements to determine gas exchange using the Infrared gas analyzer (IRGA) and turgor pressure using the patch clamp pressure probe (ZIM-probe). The second chapter refers to recording of the action potential generated spontaneously and evoked by electrical stimulation in ABA-deficient tomato, mutant sitiens. The final chapter presents the results for turgor pressure, gas exchange and electrical signals measurements in mutants notabilis and sitiens in the re-irrigated after a period of drought. The possible role of electrical signals in the plant signalling under stress conditions is discussed. The main conclusions related to chapters 1, 2 and 3 were: Measurement of extracellular electrical signals can be performed with gas exchange and turgor pressure measurements using IRGA ZIM-probe equipment; Electrical signals generated spontaneously in sitiens mutants propagate with amplitude and speed higher than in wild plants. Mutant is less responsive to electrical stimulation showing higher excitation threshold and longer refractory period than wild plants; The mutant plants are more responsive electrically to re-irrigation after drought than wild plants. The electrical signals precede changes in gas exchange in all genotypes, post irrigation. The ZIM-probe was not efficient to evaluate the turgor pressure in mutant plants under stress conditions, but is a promising tool for studies involving hydraulic and electrical signalling in wild plants. / O presente documento refere-se a pesquisa cujo principal objetivo foi investigar a relação entre sinais hidráulicos, químicos e elétricos em plantas de tomate deficientes da produção de ABA. O documento foi organizado em três capítulos: O primeiro capítulo apresenta um detalhado protocolo de medição extracelular de sinais elétricos em plantas e como associar estas medições a determinação de trocas gasosas usando o Analisador de gás por infravermelho (IRGA) e pressão de turgor usando a sonda de pressão (ZIM-probe). O segundo capítulo refere-se ao registro de potenciais de ação gerados espontaneamente e evocados por estímulo elétrico em tomateiro deficiente na produção de ABA, mutante sitiens. O último capítulo apresenta os resultados referentes a medições de pressão de turgor, trocas gasosas e sinais elétricos nos mutantes notabilis e sitiens, deficientes na produção de ABA reirrigadas após um período de déficit hídrico. O possível papel dos sinais elétricos na sinalização em plantas em condições de estresse é discutido. As principais conclusões referentes aos capítulos 1, 2 e 3 foram, respectivamente: As plantas mutantes são mais responsivas eletricamente a re-irrigação, após déficit hídrico com que as plantas selvagens. Medições extracelulares de sinais elétricos podem ser realizadas com medidas de trocas gasosas e pressão de turgor utilizando os equipamentos IRGA e ZIM-probe; Sinais elétricos gerados espontaneamente nos mutantes sitiens se propagam com amplitude e velocidade maiores do que nas plantas selvagens. sitiens é menos responsivo a estímulo elétrico do que plantas selvagens apresentando maior limiar de excitação e período refratário; Os sinais elétricos precedem as alterações nas trocas gasosas pós irrigação em todos os genótipos estudados. A sonda ZIM-probe não se mostrou eficiente para avaliar a pressão de turgor em plantas mutantes sob condições de estresse, mas para as plantas selvagens é uma ferramenta promissora para estudos envolvendo sinalização hidráulica e elétrica.

A utilização descontrolada de pesticidas tem provocado no decorrer dos anos a intoxicação de milhares de pessoas no mundo, uma vez que, seus resíduos têm sido depositados em alimentos, em solos e em ambientes aquáticos. Assim, a construção de duas novas plataformas sensoras para a detecção de pesticidas é o objetivo desse trabalho. Na primeira plataforma foi utilizado o polieletrólito catiônico polietilenoimina (PEI) em conjunto com o polissacarídeo extracelular algal (PSE) produzido pela microalga criptofícea Cryptomonas tetrapirenoidosa preparados através da técnica de deposição \"spin-coating\". E a segunda plataforma foi produzida por eletrodeposição pulsada, entre um potencial de redução e um de oxidação, utilizando nanoestruturas de óxido de zinco (ZnO). Para caracterizar as plataformas, foram utilizadas as técnicas de microscopia eletrônica de varredura com fonte de emissão de campo (FEG-SEM), difração de raios X (XRD), espectroscopia de absorção ultravioleta-visível (UV-Vis), microscopia de força atômica (AFM) e espectroscopia de reflexão-absorção no Infravermelho com modulação da polarização (PM-IRRAS). Através da imobilização da enzima polifenol oxidase na forma de extrato bruto em sua fonte natural (fruto abacate), as plataformas de PEI/PSE e ZnO, foram avaliadas como biossensores de catecol e do inseticida carbaril. De modo comparativo, as plataformas de PEI/PSE sem a presença imobilizada da enzima também foram estudadas para a detecção do catecol e do carbaril. A simplicidade na formação e na construção dessas plataformas vem qualificá-las como viáveis a serem produzidas em escala industrial e com baixo custo de processamento. E diante dos resultados obtidos no desenvolvimento desses biossensores destaca-se a eficiência e a rapidez de detecção, o que os tornam economicamente promissores e competitivos em termos de aplicações ambientais. / The uncontrolled use of pesticides has resulted over the years the intoxication of thousands of people in the world, since their waste has been deposited in food, in soil and aquatic environments. Thus, the construction of two new sensors platforms for pesticide detection is the objective of this work. At first platform was used cationic polyelectrolyte polyethyleneimine (PEI) along with the extracellular algal polysaccharide (EPS) produced by microalgae criptofícea Cryptomonas tetrapirenoidosa prepared by deposition technique \"spin-coating\". The second platform was produced by pulsed electrodeposition between a reduction and an oxidation potential using nanostructures zinc oxide (ZnO). To characterize the platforms, we used the techniques of field emission gun scanning electron microscopy (FEG-SEM), X-ray diffraction (XRD), ultraviolet visible absorption spectroscopy (UV-Vis), atomic force microscopy (AFM), and polarization modulation infrared reflection-absorption spectroscopy (PM-IRRAS). By immobilization of the polyphenol oxidase enzyme as a crude extract in their natural source (avocado fruit), platforms PEI/PSE and ZnO, they were evaluated as catechol and carbaryl insecticide biosensors. In a comparative way, the platforms PEI/PSE without the presence of immobilized enzyme were also studied for detection of catechol and carbaryl. The simplicity in the formation and construction of these platforms comes qualify them as viable to be produced on an industrial scale and low cost processing. And on the results obtained in the development of such biosensors stand out the efficiency and speed of detection, which make them economically promising and competitive in terms of environmental applications.

A leptospirose é uma zoonose de distribuição mundial causada por espiroquetas patogênicas do gênero Leptospira. Resultados experimentais demonstraram que a patogênese pode estar relacionada com a capacidade destas bactérias em aderir a proteínas da matriz extracelular, escapar da resposta imune do hospedeiro e de produzir toxinas. Este trabalho teve como objetivo identificar proteínas secretadas por Leptospira interrogans sorovar Pomona estirpe Fromm kennewicki (patogênica) e Leptospira biflexa sorovar Patoc estirpe Patoc I (saprófita), através de análise proteômica. As leptospiras foram cultivadas em meio EMJH suplementado com soro de coelho ou albumina bovina. Os sobrenadantes foram filtrados, dialisados e liofilizados para aplicação das tecnologias de análise proteômica utilizando gel bidimensional e análise em solução. A análise dos peptídeos obtidos, nos dois procedimentos, foi realizada utilizando-se LC/MS/MS. Foi possível a identificação de 159 proteínas diferentes nas amostras de L.interrogans, entre as quais 64 foram positivas em pelo menos uma das ferramentas usadas para a predição. Em L. biflexa, 104 proteínas diferentes foram identificadas, entre elas 43 proteínas foram positivas pela análise in silico. Entre as proteínas identificadas, estão aquelas que possuem peptídeo sinal sec ou tat dependentes. Em outras, a predição da localização celular é desconhecida ou podem ter múltiplos sítios de localização, e ainda, proteínas que não possuem peptídeo sinal e que podem ser secretadas por mecanismos não convencionais. Muitos destas são proteínas hipotéticas sem domínios conservados detectados. No que diz respeito à atividade proteolítica, foi identificada a presença de metaloproteases no secretoma de L.interrogans. Não houve detecção da presença significativa de proteases bacterianas em amostras de L. biflexa. A identificação e a caracterização funcional de proteínas secretadas poderão contribuir para a elucidação dos mecanismos patogênicos e no desenvolvimento de novas estratégias para o tratamento e prevenção de leptospirose. / Leptospirosis is a zoonosis of worldwide distribution caused by pathogenic spirochetes of the genus Leptospira. The mechanisms by which leptospires invade the host and cause the disease are not yet fully understood. Experimental results have shown that the pathogenesis may be related to the ability of these bacteria to bind to extracellular matrix proteins, to escape hosts immune responses and to produce toxins. This work aimed to identify secreted proteins by Leptospira interrogans serovar Pomona strain Fromm kennewicki (pathogenic) and Leptospira biflexa serovar strain Patoc Patoc I (saprophyte) through proteomic analysis. The leptospires were grown in EMJH supplemented with rabbit serum or BSA. Supernatants were filtered, dialyzed and lyophilized to proteomic technology, two-dimensional gel and non-gel. The analysis of the obtained peptides in two procedures was performed using LC/MS/LC. It was possible to identify 159 different proteins in the samples of L.interrogans; among them, 64 were positive proteins in at least one of the tools used for prediction. In L. biflexa, 104 different proteins were identified; among them, 43 positive proteins were positive by in silico analysis. Among the identified proteins are those that possess sec or tat dependent signal peptide. In others, the prediction of the cellular location is unknown or may have multiple sites of localization, and even proteins which have no signal peptide can be secreted by unconventional mechanisms. Many of these are hypothetical proteins with no detected putative conserved domains. The presence of metalloproteases has been identified in the L.interrogans´ secretome, using proteolytic assay. There was no significant detection of the presence of bacterial proteases in samples of L. biflexa. The identification and functional characterization of secreted proteins may contribute to the elucidation of pathogenic mechanisms and in the developing of new strategies for the treatment and prevention of leptospirosis.

Cai Yi Jin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 118-128). / Chapter 1. --- Introduction / Chapter 1.1 --- Importance and Cultivation history of edible mushroom --- p.1 / Chapter 1.2 --- Variety and structure of growth substrates for mushroom --- p.4 / Chapter 1.3 --- Mushroom growth and substrate-degrading enzymes --- p.8 / Chapter 1.4 --- Purpose of study --- p.15 / Chapter 2. --- Methods and Materials / Chapter 2.1 --- Organisms --- p.17 / Chapter 2.2 --- Media --- p.17 / Chapter 2.3 --- Culture conditions --- p.21 / Chapter 2.3.1 --- Growth temperature --- p.21 / Chapter 2.3.2 --- Growth Studies --- p.21 / Chapter 2.3.2.1 --- Effect of pH on mycelial growth --- p.21 / Chapter 2.3.2.2 --- Effect of different carbon sources on mycelial growth --- p.21 / Chapter 2.3.2.3 --- Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth --- p.22 / Chapter 2.3.3 --- Culture conditions for production of extracellular enzymes --- p.23 / Chapter 2.3.3.1 --- Tyrosinase --- p.23 / Chapter 2.3.3.2 --- Laccase --- p.23 / Chapter 2.3.3.3 --- Manganese-dependent Peroxidase and Lignin Peroxidase --- p.23 / Chapter 2.3.3.4 --- Cellulytic and Xylanolytic enzymes --- p.24 / Chapter 2.3.3.5 --- Lipase --- p.25 / Chapter 2.3.4 --- Culture conditions for studying properties of cellulases of V. volvacea --- p.26 / Chapter 2.3.4.1 --- CMCase --- p.26 / Chapter 2.3.4.2 --- "CMCase, FPase and β-Glucosidase" --- p.26 / Chapter 2.3.4.3 --- β-Glucosidase --- p.26 / Chapter 2.4 --- Enzyme assay --- p.27 / Chapter 2.4.1 --- Tyrosinase --- p.27 / Chapter 2.4.2 --- Laccase --- p.27 / Chapter a. --- o-Tolidine Method --- p.27 / Chapter b. --- ABTS Method --- p.28 / Chapter c. --- Syringaldazine Method --- p.28 / Chapter 2.4.3 --- Lignin peroxidase --- p.29 / Chapter 2.4.4 --- Manganese-dependent peroxidase --- p.29 / Chapter 2.4.5 --- Exoglucanase (avicelase) --- p.30 / Chapter 2.4.6 --- Endoglucanase (carboxymethylcellulase or CMCase) --- p.31 / Chapter 2.4.7 --- Filter paper digesting enzyme (FPase) --- p.32 / Chapter 2.4.8 --- P-Glucosidase --- p.32 / Chapter 2.4.9 --- Xylanase --- p.34 / Chapter 2.4.10 --- β-Xylosidase --- p.34 / Chapter 2.4.11 --- Lipase --- p.36 / Chapter 2.5 --- Other analytical methods --- p.36 / Chapter 2.5.1 --- Determination of phenol oxidase activity by the Bavendamm reaction --- p.36 / Chapter 2.5.2 --- Qualitative evaluation of CMCase by Congo red staining --- p.37 / Chapter 2.5.3 --- Effect of phenolic monomers and tannic acid on CMCase activity of V. volvacea --- p.38 / Chapter 2.5.4 --- Protein determination --- p.39 / Chapter 2.5.5 --- Non-denaturing gel electrophoresis pattern of fungal laccases --- p.39 / Chapter 2.6 --- Chemicals --- p.39 / Chapter 3. --- Results / Chapter 3.1 --- Growth and Nutritional characteristics --- p.44 / Chapter 3.1.1 --- Fungal growth on defined and non-defined culture media --- p.44 / Chapter 3.1.2 --- Effect of carbon source on fungal --- p.45 / Chapter 3.1.3 --- Effect of pH on fungal growth --- p.45 / Chapter 3.2 --- Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth --- p.45 / Chapter 3.2.1 --- Effect of lignin-related phenolic monomers on fungal growth --- p.45 / Chapter 3.2.2 --- Effect of tannin derivatives on fungal growth --- p.61 / Chapter 3.3 --- Phenol Oxidase --- p.67 / Chapter 3.3.1 --- Phenol oxidase --- p.67 / Chapter 3.3.1.1 --- Guaiacol-reacting enzyme --- p.67 / Chapter 3.3.1.2 --- o-Anisidine oxidizing enzyme --- p.68 / Chapter 3.3.2 --- Tyrosinase --- p.69 / Chapter 3.3.3 --- Laccase --- p.69 / Chapter 3.3.3.1 --- "Laccase detected by o-Tolidine, ABTS Syringaldazine" --- p.69 / Chapter 3.3.3.2 --- Effect of pH on laccase activity --- p.69 / Chapter 3.4 --- Lignin-Transforming Enzymes --- p.73 / Chapter 3.4.1 --- Lignin peroxidase (LP) --- p.73 / Chapter 3.4.2 --- Manganese-dependent peroxidase (MnP) --- p.74 / Chapter 3.5 --- Cellulases --- p.78 / Chapter 3.5.1. --- Cellulases of V. volvacea --- p.78 / Chapter 3.5.1.1 --- Qualitative estimation of cellulose-degrading enzymes of V. volvacea grown on different substrates --- p.78 / Chapter 3.5.1.2 --- Influence of pH and temperature --- p.79 / Chapter 3.5.1.3 --- Cellulolytic activities in cultures grown on cellulose --- p.83 / Chapter 3.5.1.4 --- Cellulolytic activities in cultures grown on paddy straw --- p.91 / Chapter 3.5.1.5 --- β-Glucosidase activity in cultures grown on cellobiose --- p.91 / Chapter 3.5.1.6 --- Effect of lignin-related phenolic monomers and tannic acid on CMCase of V. volvacea --- p.95 / Chapter 3.5.2 --- Cellulases of P.sajor-caju --- p.96 / Chapter 3.5.3 --- Cellulases of L. edodes --- p.96 / Chapter 3.6 --- Xylanase --- p.96 / Chapter 3.6.1 --- "Xylanase of V. volvacea, strain V34" --- p.96 / Chapter 3.6.2 --- Xylanase of P.sajor-caju --- p.100 / Chapter 3.6.3 --- Xylanase of L. edodes --- p.100 / Chapter 3.7 --- Lipase of V. volvacea --- p.103 / Chapter 4. --- Discussion / Chapter 4.1. --- Carbon nutrition and pH for fungal growth --- p.104 / Chapter 4.1.1 --- Carbon nutrition --- p.104 / Chapter 4.1.2 --- pH --- p.104 / Chapter 4.2 --- "Effect of lignin-related phenolic monomers and tannin derivatives on fungal growth of L. edodes, P. sajor-caju and V, volvacea" --- p.105 / Chapter 4.2.1 --- Lignin-related phenolic monomers --- p.105 / Chapter 4.2.2 --- Tannin derivatives --- p.107 / Chapter 4.3 --- "Production of phenoloxidases by V. volvacea, L. edodes and P. sajor-caju" --- p.108 / Chapter 4.3.1 --- Guaiacol- and Anisidine reacting enzymes and Tyrosinase --- p.108 / Chapter 4.3.2 --- Laccase --- p.109 / Chapter 4.4. --- "Lignin-degrading Enzymes of V. volvacea, P. sajor-caju and L. edodes" --- p.110 / Chapter 4.5. --- "Cellulolytic and Hemicellulolytic Activity of V. volvacea, P.sajor-caju and L. edodes" --- p.113 / References --- p.118 / Appendix1 --- p.129