Submitted by Biblioteca da Faculdade de Farmácia (bff@ndc.uff.br) on 2017-04-19T18:55:50Z No. of bitstreams: 1 Ferreira, Thaís Gonçalves [Dissertação, 2012].pdf: 3520619 bytes, checksum: 0a743346b244c2aef9fcdf90c62f99ae (MD5) / Made available in DSpace on 2017-04-19T18:55:50Z (GMT). No. of bitstreams: 1 Ferreira, Thaís Gonçalves [Dissertação, 2012].pdf: 3520619 bytes, checksum: 0a743346b244c2aef9fcdf90c62f99ae (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro / Ministério da Ciência e Tecnologia (MTC/Pronex) / Ministério da Saúde do Brasil / INTRODUÇÃO: Clostridium difficile é um importante patógeno entérico e o agente etiológico da diarreia associada ao C. difficile (CDAD). Pacientes com CDAD excretam uma grande quantidade de células vegetativas e esporos em suas fezes, levando a contaminação do ambiente hospitalar e propagação deste patógeno. Este fato pode ser explicado pela resistência dos esporos a muitos desinfetantes utilizados na rotina de desinfecção hospitalar. O objetivo deste estudo foi avaliar a atividade de desinfetantes hospitalares contra os esporos e células vegetativas de C. difficile. Além disso, foram analisados os perfis de proteínas totais das células vegetativas das cepas de C. difficile, tratadas com os mesmos desinfetantes. MÉTODOS: Os agentes de limpeza hospitalar Virkon® (peroxigênio), Cloro-Rio® (agente liberador de cloro ativo), Peresal® (peroxigênio), Riohex® (biguanida), e Cidex Opa® (aldeído), comumente utilizados em hospitais brasileiros, foram testados contra os esporos das cepas de C. difficile HU17- ribotipo 133 e SJ1-ribotipo 135, ambos ribotipos encontrados exclusivamente no Brasil. A cepa hipervirulenta de C. difficile, BI/NAP1/027, foi utilizada para comparação. Os testes foram realizados de acordo com o método padrão para teste da atividade esporocida de desinfetantes dos Estados Unidos, ASTM E2414-05. Para a análise do perfil de proteínas totais, as mesmas cepas de C. difficile, incluindo também a ATCC 9689, foram crescidas na presença e na ausência de concentração subinibitória dos desinfetantes citados e submetidas a eletroforese em gel de SDS-PAGE. RESULTADOS: Os agentes Cloro-Rio® e Cidex Opa® eliminaram completamente os esporos de todas as cepas testadas, enquanto o Riohex® não exibiu qualquer redução significante. Por outro lado, o Virkon® reduziu significativamente a concentração de esporos para as cepas HU17-133 e BI/NAP1/027, mas o mesmo não foi observado com a cepa SJ1-135. O agente Peresal® eliminou completamente os esporos da cepa HU17-133, mas apenas reduziu a concentração inicial dos esporos para as cepas C. difficile SJ1-135 e BI/NAP1/027. Os desinfetantes Cloro- Rio® e Riohex® foram os que mais afetaram a expressão de proteínas em todas as cepas avaliadas. CONCLUSÕES: Em conjunto, nossos resultados mostraram que o Cloro-Rio® e Cidex Opa® foram os desinfetantes mais eficazes para eliminação dos esporos de C. difficile. O estudo das proteínas afetadas pelos desinfetantes poderia ajudar a elucidar a resistência do C. difficile frente a alguns agentes de limpeza e criar novas alternativas para eliminar e/ou diminuir a contaminação do ambiente hospitalar por esta bactéria / Introduction: Clostridium difficile is an important enteric pathogen and the etiological agent of C. difficile-associated diarrhea (CDAD). Patients with CDAD, excrete a large amount of vegetative cells and spores in their stools, leading to contamination of the hospital environment and spread of the pathogen. This fact can be explained by the resistance of spores to many disinfectants used in hospital routine. The aim of this study was to evaluate the activity of hospital disinfectants against C. difficile spores and vegetative cells. Furthermore were analyzed the whole proteins profiles of the vegetative cells of these strains treated with the same disinfectant. METHODS: The hospital cleaning agents Virkon® (peroxygen), Cloro-Rio® (chlorine), Peresal® (peroxygen), Riohex® (biguanide), and Cidex Opa® (aldehyde), usually used in Brazilian hospitals, were tested against C. difficile strains spores HU17-ribotype 133 and SJ1-ribotype 135, both exclusively Brazilian. BI/NAP1/ribotype 027 strain was also used for comparison. The tests were performed in accordance with the standard method for testing the sporicidal activity of disinfectants of the United States, ASTM E2414-05. For the analysis of whole proteins profile, the strains were grown in the presence and absence of sub inhibitory concentrations of the disinfectants and applied on SDS-PAGE gel. RESULTS: Cloro-Rio® and Cidex Opa® completely eliminated spores of all strains tested, while Riohex® did not show any significant reduction. On the other hand, Virkon® significantly reduced HU17 and BI/NAP1/027 spores, but the same was not observed with SJ1 strain. The agent Peresal® completely eliminate the spores of strain HU17-133, but only reduced the initial concentration of spores for strains SJ1-135 and BI/NAP1/027. The disinfectants Cloro-Rio® and Riohex® were the most affected protein expression in all strains evaluated. CONCLUSIONS: Taken together, our results show that Cloro-Rio® and Cidex Opa® are the most remarkable agents for eliminating spores. The study of proteins affected by the disinfectant might help to elucidate the resistance of C. difficile against some cleaning agents and create new alternatives to eliminate and/or reduce the contamination of the hospital by this bacterium

Les spores ou les conidies fongiques sont responsables de la dissémination des champignons filamenteux dans l'environnement (air, eau, sol,…). Ensuite les spores fongiques peuvent se déposer sur les équipements dans les ateliers de fabrication, sur les matières premières agricoles et sur les aliments. Au laboratoire, les spores sont obtenues en cultivant les champignons filamenteux en conditions optimales en termes de température, activité de l'eau, nutriments, de manière à obtenir le matériel biologique le plus rapidement possible. Or naturellement, lors de la sporulation, les champignons sont soumis à différents stress, notamment hydrique, ce qui entraîne des différences notables dans l'état physiologique de la spore. Ainsi notre objectif durant cette thèse est d’évaluer l’état physiologique des spores lorsqu’elles sont soumises à certaines conditions. Une première partie de la thèse vise à établir un nouveau modèle pour une meilleure détermination du temps de germination. L’étape suivante présente l’évaluation de l’influence du stress hydrique de la sporogénèse à la germination des spores. Les deux dernières parties présentent enfin l’évaluation des conditions de stockage sur la germination des spores. L’état physiologique est un facteur clé dans le processus de germination, il serait opportun de l’intégrer dans les modèles prédictifs de la germination. / Fungal spores or conidia are responsible for filamentous fungi spread in environment (air, water, soil…). Then, they can be found on several environments including foods. In laboratory spores are obtained under favorable conditions. However, these conditions are not real, spores are subject to various stress including water stress after their formation. These conditions can make some interactions with their physiological state. Thus, our aim consists in evaluating spores physiological state after their exposition to various conditions of storage. First part of this thesis is about definition of a new model of germination for improving germination time determination. Next step concerns evaluation of water stress during spore’s germination process. The last two parts are finally dedicated to evaluation of storage condtions on spore’s germination time. Physiological state is a key factor in the germination process. It would be appropriate to include this factor in predictive models.

A análise microbiológica atua como ferramenta fundamental para a obtenção de dados sobre a qualidade, sanidade, higiene e segurança na produção de alimentos; desta forma, tem sido adotada na indústria alimentícia para o controle de qualidade. Por sua composição completa e balanceada, o leite é um substrato ideal para o desenvolvimento de diversos grupos de microrganismos. Com o objetivo de pesquisar bactérias da espécie Bacillus cereus em amostras de leite fluido, bem como a capacidade de germinação de esporos e a multiplicação dessa bactéria após processo de fervura, com manutenção das amostras à temperatura ambiente e à temperatura de refrigeração por períodos de 1, 2, 4, 6, 8, 10 e 12 horas, foram analisadas 75 amostras de leite, conforme as metodologias recomendadas por Silva et al. (2007). Destas, 46 amostras (61,3%) mostraram-se com algum grau de contaminação pela bactéria antes de serem submetidas à fervura. Por sua vez, as amostras mantidas à temperatura ambiente após a fervura, tiveram suas contagens bacterianas, principalmente a partir da 8a hora, superiores à contagem inicial, inclusive atingindo níveis capazes de desencadear uma toxinfecção alimentar, demonstrando a ocorrência da germinação dos esporos e a multiplicação das células vegetativas. Por outro lado, alíquotas dessas mesmas amostras mantidas sob refrigeração (7ºC) não atingiram populações preocupantes, enfatizando, desse modo, a importância da necessidade da refrigeração do leite após a fervura. / The microbiological analysis acts as basic tool for the attainment of data on the quality, health, hygiene and security in the food production, in such a way, she has been adopted in the nourishing industry for the quality control. For its complete and balanced composition, milk is an ideal substratum for the development of diverse groups of microorganisms. With the objective to search cereus bacteria of the Bacillus species in fluid milk samples, as well as the capacity of germination of spores and the multiplication of this bacterium after boil process, with maintenance of the samples to the ambient temperature and the temperature of refrigeration for periods of 1, 2, 4, 6, 8, 10 and 12 hours, 75 milk samples had been analyzed, as the methodologies recommended for Silva et al. (2007). Of these, 46 samples (61.3%) had revealed with some degree of contamination for the bacterium before being submitted to the boil. In turn, the samples kept to the ambient temperature after the boil, had its bacterial countings, mainly from 8a hour, superiors to the initial counting, also reaching levels capable to unchain an alimentary toxinfection, demonstrating to the occurrence of the germination of the spores and the multiplication of the vegetative cells. On the other hand, aliquot of these same samples kept under refrigeration (7ºC) had not reached preoccupying populations, emphasizing, in this manner, the importance of the necessity of the refrigeration of milk after the boil.

No description available.

O S. mutans é o agente etiológico da cárie dental humana, uma doença com ampla distribuição mundial. A adesão à superfície dental depende da interação entre a proteína de superfície P1 e a aglutinina salivar (SAG) adsorvida ao dente. A região N-terminal da P1 é um alvo vacinal importante que está diretamente associada às funções de adesão e agregação. Este trabalho teve o objetivo de avaliar estratégias vacinais contra o S. mutans baseadas na proteína P1 usando linhagens recombinantes de B. subtilis. O B. subtilis é uma bactéria gram positiva, formadora de esporos, não patogênica, empregada como sistema de expressão de proteínas heterólogas e como veículo vacinal administrado por vias de mucosas. Empregamos o B. subtilis para expressar e purificar a proteína P139-512 derivada da proteína P1 de S. mutans UA159. O antígeno P139-512 apresentou epítopos lineares e conformacionais semelhantes aos presentes na proteína P1 nativa. O sítio de ligação à SAG está preservado nessa proteína assim como suas propriedades imunogênicas. A coadministração parenteral do antígeno com adjuvantes vacinais promoveu resposta sistêmica específica com anticorpos eficazes no bloqueio da adesão de S. mutans. Por fim, usamos esporos de B. subtilis como veículo de entrega de mucosa para o antígeno alvo de S. mutans. Esporos de B. subtilis foram modificados para expressar na superfície adesinas bacterianas (SlpA, InvA ou Inv600) com capacidade de ligação ao epitélio intestinal e, quando no estágio de célula vegetativa, expressar intracelularmente o antígeno P139-512. A imunização oral com os esporos adesivos induziu altas concentrações de anticorpos sistêmicos e de mucosa. A imunização nasal ou sublingual com os esporos recombinantes induziu níveis de anticorpos sistêmicos maiores do que aqueles obtidos após a imunização oral. Além disso, esses anticorpos foram mais eficientes em bloquear a adesão de S. mutans à SAG imobilizada, sem interferir com a agregação. Em conclusão, os resultados obtidos abrem perspectivas interessantes para o desenvolvimento de vacinas anti-cárie baseadas em linhagens de B. subtilis. / S. mutans is the major etiologic agent of human dental caries, a disease with worldwide distribution. The adhesion to the tooth surface is dependent on the interaction of the P1 surface protein and salivary agglutinin (SAG) adsorbed to the tooth. The N-terminal region of P1 is an important vaccine target that is directly associated with adhesion and aggregation functions. This study aimed to evaluate vaccination strategies against S. mutans based on the P1 protein using recombinant B. subtilis strains. B. subtilis is a gram positive, spore-forming, non-pathogenic bacterium used as expression system for heterologous proteins and as a vaccine vehicle administered by mucosal routes. Inicially, we employed a recombinant B. subtilis strain to express and purify the P139-512 protein derived from the S. mutans UA159 P1 protein. The P139-512 antigen showed important conformational and linear epitopes similar to those present in the native P1 protein. The SAG-binding site is preserved in P139-512 as well as immunological properties. The parenteral co-administration of antigen with vaccine adjuvants stimulated systemic antibodies effective in blocking adhesion of S. mutans to SAG. Lastly, we used B. subtilis spores as a mucosal delivery vehicle for antigen targeting. B. subtilis endospores were modified to display bacterial adhesins (SlpA, InvA or Inv600), capable to bind to the intestinal epithelium, on the spore surface and to express intracellularly the P139-512 antigen during the vegetative cell stage. Oral immunization with adhesives spores induced high systemic and mucosal specific antibodies levels. The nasal or sublingual immunization with B. subtilis recombinant spores induced higher amounts of systemic antibodies than the oral immunization. Furthermore, the specific antibodies were highly effective in blocking the adherence of S. mutans to immobilized SAG, without interfering with aggregation. In conclusion, the results open interesting perspectives for the development of anti-caries vaccines based on B. subtilis strains.

Bacillus cereus is a ubiquitous organism and a potential foodborne pathogen that can cause two types of gastrointestinal diseases: emesis and diarrhea. The emetic syndrome is caused by a heat and acid stable peptide toxin that is pre-formed in food, while the diarrheal syndrome is associated to two 3-protein, heat labile enterotoxin complexes that are formed in the intestine after ingestion of the organism. There are many reports on the isolation and characterization of Bacillus cereus from various foods, however there are no studies on the levels, toxigenicity and physical characteristics of B. cereus isolated from U.S. retail spices. A huge part of spices sold in the U.S. are imported from developing nations. Developing nations lack hygienic practices during processing and packaging of spices, due to which there is a high chance of imported spices being contaminated with B. cereus. Therefore, the main objective of this thesis work was to characterize B. cereus spores from U.S. retail spices. Levels of aerobic spores and B. cereus spores were determined. B. cereus spores were further analyzed for their enterotoxigenic ability, growth characteristics and physical spore characteristics. In the 247 spice samples analyzed 77 were found to contain B. cereus, while 11 were positive for B. thuringiensis. Eighty four of the 88 spices tested possessed either one of the enterotoxin genes. None of the isolates tested positive for the emetic toxin (ces) gene. Seventy five of the B. cereus isolates grew at 12 °C, although only two isolates grew well at 9 °C. Seven selected diarrheal B. cereus spore strains had D95-values ranging from 0.64-3.53 min while the two emetic strains had D95-values of 7.04 min and 6.64 min. B. cereus grew well in pre-cooked rice. After 48 h, counts of 1.26 X 107 and 3.8 X 107 B. cereus/ 10 g were obtained in pre-cooked rice maintained at 17 °C and 20 °C respectively. At 12 °C, counts did not reach 104 CFU/ 10g even after 48 h of incubation. The aerobic mesophilic bacterial population and B. cereus population of 0.1% crushed pepper in pre-cooked rice over a period of 48h at temperature 20 °C and 17 °C were also analyzed. Counts of B. cereus in pepper rice samples reached a maximum of 1600 MPN/ 10 g and 1100 MPN/ 10 g at 20 °C and 17 °C respectively while the aerobic mesophilic counts per 10 g were 2.4 X 108 and 4.4 X 106 at these temperatures. The low B. cereus counts and high aerobic mesophilic population indicates competition of nutrients in cooked rice by background flora other than B. cereus. The physical spore characteristics of five B. cereus and 3 B. thuringiensis strains were studied using transmission electron microscopy (TEM). Tubular, whip-like appendages were present in four B. cereus and two B. thuringiensis, while all seven isolates possessed exosporia.

The diploma thesis revises ten selected species of sphenopterid ferns of Carboniferous age deposited in the National Museum in Prague, the West-Bohemian Museum in Pilsen and the British Geological Survey in Keyworth, England. Sphenopterid ferns were studied based on reproductive organs aquired by maceration of coalified plant remains preserved as compressions. Sporangia and their annulus are important diagnostic features for individual genera and species of sphenopterid ferns and for selected species have not been described yet. Following species were studied: Boweria schatzlarensis, Myriotheca anglica, Renaultia crépini, Sturia amoena, Oligocarpia gutbiery, Zeilleria hymenophylloides, Zeilleria avoldensis, Discopteris sp. ("doubravensis"), Scolecopteris elegans a Waldenburgia corynepteroides. With the exception of Waldenburgia corynepteroides, Scolecopteris elegans, Zeilleria hymenophylloides and Zeilleria avoldensis species type material was studied. Maceration of sporangia of Boweria schatzlarensis showed presence of lateral annulus while in case of Myriotheca anglica the annulus is lateral or more likely of a special type, placed on both sides of the sporangia. Annulus type determination of Renaultia crépini also made possible to assign this species within the range of the genus Tenchovia and...

The Pneumocystis carinii carinii DNA content in nuclei of trophic forms and cysts (spore cases) containing 2, 4, or 8 intracystic bodies, were compared using quantitative fluorescence image analysis. The nuclear DNA content was found to be lower than the theoretical limits of Feulgen cytophotometry. Several fluorescent DNA dyes provide brighter staining, but these techniques suffer from nonspecific binding to other cellular components, such as RNA. It was demonstrated that the thick glycocalyx surfaces of trophic forms and the cyst walls of P. carinii organisms, as well as the cell wall of S. cerevisiae, bound all fluorescent dyes tested to varying degrees. Hence in this study, measurements were performed on cells in which the outer surfaces of organisms were first removed with lyticase. Two stains that appeared most specific for DNA, DB181 and 4',6-diamidino-2-phenylindole (DAPI), were used for quantitations; lower deviations of fluorescence intensities were observed with DB181. Haploid wild type Saccharomyces cerevisiae and cdc-28 temperature-sensitive mutant cells, accumulated at the restrictive temperature (37°C), were used as quantitative internal standards for estimating the absolute nuclear DNA content of P. carinii. Haploid wild type and mutant nuclei stained with DAPI had the same relative fluorescence intensities. The P. carinii nuclear DNA content of trophic forms and individual intracystic bodies (spores), regardless of life cycle stage, were not different. The mean values obtained were 6.9 and 6.7 fg DNA/nucleus with DB181 and DAPI, respectively (approximately 9.26 and 8.99 Mbp nucleotides, respectively). Since these would include 2C (G-2 phase) and S-phase nuclei, a 1C population of nuclei was selected by histogram distributions of DB181-stained nuclei. Almost all nuclei analyzed in all life cycle stages fell within this population. The 1C mean of 6.55 fg DNA/nucleus (median, 6.62 fg DNA/nucleus) was estimated as representing 8.79 Mbp nucleotides, assuming only A-T binding of the dye and taking into account the G+C content of S. cerevisiae and P. carinii. A 4C (G-2-phase diploid nuclei) population was not detected in histograms of DB181- or DAPI-stained nuclei. The P. carinii nuclear DNA content values obtained in this study were similar to those independently obtained by calculating the total DNA in the organism's chromosomes resolved by electrophoretic techniques. Together, the data on total chromosome numbers and the estimated DNA content of those chromosomes, with our quantitation of nuclear DNA content of different life-cycle stages demonstrate that P. carinii carinii isolated from infected rat lungs are haploid organisms.

No description available.

Image-charge detection is an analytical technique in which a highly-charged particle is detected by the magnitude of the image current that it generates in a detecting electrode. This current is represented as a voltage between the charged particle and the sensing electrode. It is a single particle detection method, ideal for the analysis of large, variable mass particles such as biological cells. Some of the physical properties of Bacillus subtilis spores were explored using different applications of image-charge detection. B. subtilis is a gram-negative spore-forming bacteria that has been shown to exhibit extremophile behavior. The particular extremophile behavior that was investigated in this study is the resistance to extreme mechanical stress. The effects of high-velocity impacts upon these spores were studied using image-charge detection. The elastic properties of these spores as well as spore survivability to high-velocity impacts were investigated. Spores were shown to survive impacts at velocities up to 299 ± 28 m/s. The average kinetic energy loss experienced by impacting spores, regardless of velocity at impact, was between 71 and 72%. Both conventional and novel image-charge detection techniques were used for these studies. The novel version of a charge detector that was demonstrated was fabricated using patterned metal electrodes on printed circuit boards. The simplicity and versatility of this method was demonstrated with a multi-stage charge detector, a unique bouncing detector, and charge-detection mass spectrometry detector which is capable of measuring the absolute mass of a single highly-charged particle.