Rozprawy doktorskie na temat „Microorganisms”

En aquesta tesi doctoral hem desenvolupat un mètode per la detecció i quantificació múltiple dels microorganismes més comuns que causen infeccions bacterianes amb una velocitat de detecció sense precedents a baix cost i alta sensibilitat. A més a més, fent servir fluids humans reals directament evitant així, els pretractaments tediosos de les mostres. El disseny del sistema està basat en augments d'intensitat del senyal obtingut per espectroscòpia SERS. Això s'aconsegueix utilitzant nanopartícules plasmòniques codificades i funcionalitzades amb elements de reconeixement biològics. D'aquesta manera, quan una mostra conté el patogen a identificar interactua amb els elements de reconeixement units a les nanopartícules, induint la seva acumulació en la superfície del microorganisme. Aquesta agregació de partícules a la membrana dels bacteris produeix espais molt petits entre les partícules fent que el senyal Raman s'amplifiqui en diversos ordres de magnitud respecte a les partícules soltes. Permetent així, la identificació de múltiples microorganismes a la vegada. La quantificació d'aquests, s'aconsegueix passant la mostra a través d'un dispositiu de micro-fluids amb una finestra de recol•lecció on un làser interroga i classifica els agregats a temps real. A més a més, també hem investigat els avantatges de fer servir aptàmers en lloc d'anticossos com a elements de reconeixement biològic. Aquest nou sistema de detecció de patògens obre interessants perspectives per al diagnòstic ràpid i econòmic d'infeccions bacterianes.
En esta tesis doctoral hemos desarrollado un método para la detección y cuantificación múltiple de los microorganismos más comunes que causan infecciones bacterianas con una velocidad de detección sin precedentes a bajo coste y alta sensibilidad. Utilizando además, fluidos humanos reales directamente evitando así, pre-tratamientos tediosos de las muestras. El diseño del sistema está basado en aumentos de intensidad de la señal obtenida por espectroscopia SERS. Esto se logra utilizando nanopartículas plasmónicas codificadas y funcionalizadas con elementos de reconocimiento biológico. De esta manera, cuando una muestra que contiene el patógeno a identificar interactúa con los elementos de reconocimiento unidos a las nanopartículas, induce su acumulación en la superficie del microorganismo. Esta agregación de partículas en las membranas de las bacterias produce espaciados muy pequeños entre las partículas haciendo que la señal Raman se amplifique en varios órdenes de magnitud con respecto a las partículas sueltas. Permitiendo así la identificación de múltiples microorganismos a la vez. La cuantificación de los mismos, se logra pasando la muestra a través de un dispositivo de micro-fluidos con una ventana de recolección donde un láser interroga y clasifica los agregados en tiempo real. Además, también hemos investigado las ventajas de usar aptámeros frente a anticuerpos como elementos de reconocimiento biológico. Este nuevo sistema de detección de patógenos abre interesantes perspectivas para el diagnóstico rápido y barato de las infecciones bacterianas.
This doctoral thesis intended to develop and optimize a method for multiplex detection and quantification of the most common microorganisms causing bacterial infections. This detection approach envisions to directly use different real human fluids avoiding thus, tedious pre-treatments of the samples with an unprecedented speed, low cost, and sensitivity. The design of the system is based on variations in the SERS intensity. This is accomplished using encoded plasmonic nanoparticles functionalized with bio-recognition elements. Consequently, when a sample containing the biological target to be identified interacts with the recognition elements attached to the nanoparticle, will induce an accumulation of them at the surface of the targeted microorganism. This particle aggregation on the bacteria membranes renders a dense array of inter-particle gaps in which the Raman signal is amplified by several orders of magnitude relative to the dispersed particles, enabling a multiplexed deterministic identification of the microorganisms. Quantification is achieved by passing the sample through a microfluidic device with a collection window where a laser interrogates and classifies the bacteria–nanoparticle aggregates in real time. Additionally, a comparison between two of the most common bio-recognition elements (antibodies and aptamers) was performed. This new pathogen detection system opens exciting prospects for fast inexpensive diagnosis of bacterial infections.

El diagnosi de moltes malalties és de vital importància per proporcionar el tractament adequat i per tant per controlar les necessitats de salut públiques. Els mètodes estàndard que es fan servir per confirmar la presència de microorganismes consisteixen típicament en l’ús de mètodes de cultiu específics per multiplicar, separar, identificar i comptar les bactèries. La durada d’aquests processos depèn del microorganisme en concret, però en molts casos un resultat confirmatori pot tardar entre uns pocs dies o inclús vàries setmanes. Un dels principals objectius en aquesta àrea és la detecció ràpida de microorganismes, d’una forma acurada i barata. Els polímers d’impremta molecular (PIMs) ofereixen una alternativa robusta i econòmica als anticossos naturals, però encara es requereix el seu desenvolupament pel reconeixement de molècules de gran mida. En aquesta tesi presentem diferents polímers d’impremta molecular amb l’objectiu de desenvolupar una nova aproximació per detectar proteïnes de la superfície de bactèries i microorganismes, aproximació basada en anticossos artificials utilitzats en la construcció de dispositius portàtils i econòmics. Aquests objectius generals s’assoleixen implementant una sèrie d’objectius específics: i. desenvolupament d’un camí simple per la construcció d’anticossos artificials utilitzant processos d’impremta molecular, ii. aplicació d’impedimetria, voltametria d’ona quadrada i potenciometria com a tècniques de detecció conjuntament amb una capa sensora formada per polímers d’impremta molecular, iii. ús d’elèctrodes comercials i de fabricació casolana per la detecció electroquímica en la cerca de dispositius portables i d’un sol ús, iv. impressió molecular i detecció de proteïnes de superfície de bactèries i/o microorganismes.
La diagnosis de muchas enfermedades es de vital importancia para proporcionar el tratamiento adecuado y por lo tanto para el control de las necesidades de salud públicas. Los métodos estándar utilizados en la confirmación de la presencia de microorganismos consisten típicamente en el uso de métodos de cultivo específicos para multiplicar, separar, identificar y contar las bacterias. La durada de estos procesos depende del microorganismo en concreto, pero en muchos casos se necesitan entre pocos días o incluso varias semanas para tener una confirmación del resultado. Uno de los principales objetivos en esta área es la detección rápida de microorganismos, de una forma fiable y barata. Los polímeros de impronta molecular (PIMs) ofrecen una alternativa robusta y económica a los anticuerpos naturales, pero aún se requiere su desarrollo para el reconocimiento de moléculas de elevado tamaño. En esta tesis presentamos diferentes polímeros de impronta molecular con el objetivo de desarrollar una nueva aplicación para detectar proteínas de la superficie de bacterias y microorganismos, aproximación basada en anticuerpos artificiales utilizados en la construcción de dispositivos portátiles y económicos. Estos objetivos generales se consiguen implementando una serie de objetivos específicos: i. desarrollo de un camino simple para la construcción de anticuerpos artificiales utilizando procesos de impronta molecular, ii. aplicación de impedimetría, voltamperometría de onda cuadrada y potenciometría como técnicas de detección conjuntamente con una capa sensora formada por polímeros de impronta molecular, iii. uso de electrodos comerciales y de fabricación casera para la detección electroquímica en la búsqueda de dispositivos portátiles y de un solo uso, iv. impresión molecular y detección de proteínas de superficie de bacterias y/o microorganismos.
The diagnosis of most illnesses is of vital importance for providing the appropriate cure and hence controlling public health concerns. The standard methods that are used to confirm the presence of microorganisms typically consist of specific enrichment media to multiply, separate, identify and count bacterial cells. The duration of the process depends on the microorganism, but in most cases a confirmatory result can take from a few days to even weeks. One of the major objectives in this area is to detect microorganisms quickly, accurately and cheaply. Molecularly imprinted polymers (MIPs) offer in principle a robust, cost-efficient alternative to natural antibodies, but it is still a challenge to develop such materials for large molecule recognition. In this thesis we present a variety of molecular imprinting approaches with an aim to develop a new approach for detecting bacterial surface proteins and microorganisms based on artificial antibodies for the construction of label-free and cost-effective portable devices. These general objectives are achieved by implementing a series of specific objectives: i. development of an easy pathway to make artificial antibodies by molecular imprinting process, ii. application of impedimetry, square wave voltammetry and potentiometry as detection techniques using molecularly imprinting polymers as the sensing layer, iii. use of homemade and commercially available screen-printed electrodes for the electrochemical detection of targets in the search for disposable and portable devices iv. electrochemical imprinting and detection of bacterial surface proteins and/or microorganisms.

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Universidade Estadual Paulista (UNESP)
O tratamento adequado da água nas redes de abastecimento é de grande importância, para que doenças sejam evitadas. O processo tradicional mais utilizado para desinfecção de água é a cloração, porém esta vem causando sérios problemas, já que subprodutos são formados. Entre eles estão os trihalometanos, prejudiciais à saúde humana, por serem cancerígenos e mutagênicos. Busca-se então, métodos alternativos de desinfecção da água para abastecimento público. Processos Oxidativos Avançados (POAs) são as chamadas “tecnologias limpas” e vêm sendo estudados para este fim, pois consistem na produção de radicais altamente oxidantes, que provocam a morte de microrganismos, sem deixar resíduos na água. Dentre os POAs estão os processos fotocatalíticos, que através de luz UV e um eletrodo semicondutor, produzem radicais hidroxila, capazes de inativar uma série de microrganismos. No presente trabalho testamos o processo fotocatalítico, utilizando luz UV-A e eletrodos de TiO2 para verificar a eficiência da fotocatálise sobre a inativação das bactérias Escherichia coli e Staphylococcus aureus. Foi verificado que o processo é eficaz, sendo que sua eficiência pode ser significativamente melhorada quando o eletrodo é dopado com íons prata (aceptor de elétrons), promovendo a desinfecção total da água
The disinfection of water has great importance because illnesses transmitted by water are prevented by killing of pathogenic microorganisms. The traditional process used for water disinfection is the chlorination. However, the chlorination produces some problems when chlorine reacts with organic matters forming trialomethanes. They are harmful to the health human and are carcinogenic and mutagenic. Thus, alternative methods for water disinfection such as: the advanced oxidative processes (AOP), such as the photocatalytic can be considered a “clean technology” because it consists of the highly oxidative substances like the hydroxyl radicals that provoke the death of microorganisms without leaving chemicals residues in the water. In the present work we tested the photocatalytic process using thermal TiO2 electrodes that works a semiconductor surface to producing hydroxyl radicals when UV light incises on the surface. The radical are capable to inactivate many kinds of microorganisms. We tested two bacteria, Staphylococcus aureus and Escherichia coli. It was verified that the process is efficient to kill bacteria and its efficiency can significantly be improved when the electrode was doped with silver ions (aceptor of electrons) promoting a total disinfection of water

As sequências genômicas carregam uma ampla gama de informações sobre os organismos que a compõem. Obviamente, devido à grande semelhança destas informações e funções, espera-se que uma determinada sequência possa pertencer a muitos organismos, com probabilidades semelhantes. Entretanto, cada genoma carrega dentro de si certas peculiaridades que podem ser extraídas utilizando as ferramentas adequadas. Neste contexto, este trabalho propõe um processo de análise de sequências genômicas de bactérias, utilizando algumas medidas que são particularmente importantes: a entropia de triples (Sn), a quantificação da periodicidade 3 (P3) em uma sequência, o coeficiente de clusterização (D) e o percentual de GC. O processo aqui proposto nos permite inferir a qual organismo uma determinada sequência genômica pode pertencer, mostrando-se viável a sua utilização em metagenômica. Os resultados neste trabalho demonstram a eficácia deste método. Foram identificados 100% dos organismos presentes nas amostras estudadas (VP). Por outro lado, foi encontrado um grande número de organismos não pertencentes às amostras (FP), o que indica a grande similaridade de determinadas sequências, corroborando com alguns estudos que indicam que o genoma carrega consigo sequências órtologas, comuns a inúmeros organismos.
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Genomic sequences carry a wide range of information on organism that compose it. Obviously, by reason that great similarity of this information and functions, it is expected that each sequence can belong to many organisms with a similar probability. However, each genome carries within itself certain peculiarities that can be extracted using appropriate tools. In this context , this paper proposes a methodology for the analysis of genomic sequences of bacteria , using some measures that are particularly important : The entropy of triples ( Sn ) , the quantification of frequency 3 (P3) in a sequence , the clustering coefficient ( D ) and the percentage of GC . The method proposed here allows us to infer which a particular organism genome sequence may belong, being feasible for use in Metagenomics. The results of this study demonstrate the effectiveness of this method, 100 % of the organisms were identified in the samples studied (VP). On the other hand, a large number of bodies which did not belong samples were found (FP), which indicates the high similarity of certain sequences, corroborating some studies indicate that the genome carries ortholog sequences, common to countless organisms.

Em biorreatores de agitação mecânica (STR), a circulação e a mistura do fluido são influenciadas pela configuração do equipamento e pela disposição dos impelidores e aspersores de gás. Já em biorreatores airlift, sua geometria e, principalmente, o tipo e forma de aspersão de oxigênio, têm primordial efeito tanto na transferência de oxigênio quanto no crescimento microbiano e na formação de produtos. Para o cultivo de Aspergillus oryzae IPT-301, o suprimento de oxigênio é um parâmetro fundamental, em razão do metabolismo unicamente aeróbio deste microrganismo. Neste contexto, analisou-se o transporte de massa gasosa em ambos os equipamentos, contendo fluidos com viscosidades distintas: água destilada e diferentes concentrações de soluções de pectina. Com estudos de mecânica dos fluidos, correlações matemáticas empíricas para determinação do coeficiente volumétrico de transferência de oxigênio (KLa) foram utilizadas para relacionar os resultados experimentais com os calculados. A produção de pectinases também foi avaliada nesses equipamentos. O meio de cultivo continha sais nutrientes, extrato de levedura, glicose e pectina cítrica. Avaliaram-se diferentes configurações de impelidores Rushton e pitched blade, além de várias geometrias de aspersores de gás, tais como ferradura de aço inoxidável, pedra sinterizada, aeradores de aquário e de latão e funis de vidro sinterizado. Em STR, a análise fatorial mostrou que os maiores incrementos de KLa foram com a combinação de impelidores Rushton, em água, com aspersor do tipo aquário a 700rpm e 1,71L/L/min; em airlift, com o aspersor aquário, alocado na região externa do tubo interno, e com o aspersor pedra sinterizada. O modelo proposto por Miller (1974) foi o mais adequado para determinar a potência requerida pelos fluidos deste trabalho, em STR e sob aeração. O modelo de Wang et al. (1979), com adaptações, ajustou-se aos dados com água; e para as soluções de pectina, a correlação descrita por Badino Jr. et al. (2001) foi melhor ajustada. Três ensaios para produção enzimática foram processados em STR: ambos com três impelidores Rushton, aspersores dos tipos ferradura (A), aquário (B) e aquário com meio com pectina desesterificada (C). Em airlift, foram testadas as condições produtivas com os aspersores do tipo aquário (externo) (A), pedra sinterizada (B) e aquário (externo), com pectina desesterificada (C). Em STR, o melhor resultado de KLa, em meio isento de células, foi na condição C (29,88h-1), bem como vantagens econômicas como o menor tempo de permanência da máxima frequência dos agitadores (tf,máx) (A: 23h; B: 8,5h; C:5h). Porém, resultado superior de máxima produção de pectinases foi na condição B (A: 24,60U/mL; B: 25,53U/mL; C: 13,36U/mL) em tf,máx inferior à condição A. Em airlift, o transporte de oxigênio, em meio isento de células, foi mais favorecido em A (21,96h-1), bem como o menor tempo máximo para manter a máxima vazão específica do gás (A: 29h; B: 72h; C: 57h). Além disso, a máxima atividade enzimática foi superior na mesma condição (A: 24,61U/mL; B: 21,94U/mL; C: 2,29U/mL). Assim, conclui-se que, desde que planejadas as condições operacionais e de processo de produção de pectinases de A. oryzae, ambos os biorreatores podem ser aplicados na produção de pectinases fúngicas.
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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior,CAPES
In stirred tank reactors (STR), circulation and mixing of fluid are influenced by the reactor configuration and by how the impellers and the gas spargers are arranged in them. On the other hand, in airlift bioreactors, their geometry and especially the type and form of oxygen sparging have an effect on both oxygen transfer and on microbial growth and product formation. Oxygen supply for the cultivation of Aspergillus oryzae IPT-301 is a key parameter due to the aerobic metabolism of this microorganism. In this context, oxygen transfer in both equipments was analyzed. They contained fluids with different viscosities: distilled water and different concentrations of pectin solutions. Through the use of fluid mechanics studies, empirical mathematical correlations were used in order to determine the volumetric oxygen transfer coefficient (KLa) to match experimental and calculated results. Pectinase production was also assessed in those devices. The culture medium contained salts, yeast extract, glucose and citrus pectin. Different Rushton and pitched blade impeller configurations were evaluated, as well as various gas sparger geometries, such as stainless steel horseshoe, sintered stone, aquarium and brass spargers, and also sintered glass funnels. In STR, factorial design showed that the largest KLa value was obtained with the combination of Rushton impellers, in water, with the aquarium sparger, at 700rpm and 1.71L/min; in airlift, with the same sparger, put in the outer space of the inner tube and with the sintered stone sparger. The empirical correlation proposed by Miller (1974) was the most suitable one to determine the power requirement by the fluids in this work, in STR and under aeration. The correlation proposed by Wang et al. (1979), with adaptations, was better adjusted to the data set with water; the correlation described by Badino Jr. et al. (2001) was better suited for pectin solutions. Three tests for enzyme production were processed in STR: with three Rushton impellers, in all of them, and horseshoe (A), aquarium (B) and aquarium with non-esterified pectin medium (C) spargers. In airlift, enzyme production was tested with aquarium (external) (A), sintered stone (B) and aquarium (external) with non-esterified pectin (C) spargers. In STR, the best result of KLa in cell-free medium was provided in condition C (29.88h-1), as well as economic advantages such as shorter length of maintenance of the maximum impeller speed (tf,máx) (A: 23h; B: 8.5h; C: 5h). Nonetheless, higher pectinase production was obtained in condition B (A: 24.60U/mL; B: 25.53U/mL, C: 13.36U/mL) in tf,máx shorter than in condition A. In airlift, higher oxygen transfer in cell-free medium was obtained in condition A (21.96h-1), as well as the lowest length of maintenance of the maximum specific gas flow rate (A: 29h; B: 72h; C: 57h). Furthermore, maximum enzyme activity was higher in the same condition (A: 24.61U/mL; B: 21.94U/mL, C: 2.29U/mL). Thus, we conclude that if the operational conditions for pectinase production by A. oryzae are well planned, both bioreactors can be applied for the production of fungal pectinases.

Concentration of microorganisms from a sample volume would increase the limits of detection of samples used for rapid-detection methods. Rapid detection methods are is advantageous for the food industry to rapidly test for bacteria in order release products on a timely basis. Ultrasonic concentration was considered a promising method for manipulation of microorganisms. An ultrasonic chamber consisting of parallel piezoceramic discs with a reticulated polyurethane foam mesh was used to concentrate Saccharomyces cerevisiae yeast and Escherichia coli bacteria. The concentration of yeast was seen to increase by 200% (from 8.0 x 104 cells mL-1 to 2.4 x 105 cells mL-1) while almost zero concentration of bacteria was observed. The poor concentration effect seen with the smaller microorganisms was explained by the volume dependent acoustic radiation force exerted on the particles; the concentration forces are 1,000 times smaller for a 1 μm bacteria cell versus a 10 μm yeast cell.

Phosphoglycerate mutase catalyses the interconversion of 2-phosphoglycerate and 3-phosphoglycerate in the glycolytic/gluconeogenic pathways. There are two main types of phosphoglycerate mutase: 2,3-bisphosphoglycerate dependent and 2,3-bisphosphoglycerate independent. The enzyme from Saccharomyces cerevisiae has been extensively studied: the high resolution crystal structure of this tetrameric enzyme, subunit Mr 27,000, is known (Winn et al., 1981), the amino acid sequence has been determined (Fothergill and Harkins, 1982) and the gene encoding the enzyme has been isolated and sequenced (White and Fothergill-Gilmore, 1988). Phosphoglycerate mutase from the fission yeast, Schizosaccharomyces pombe, has been purified and partially characterised (Price et al., 1985; Johnson and Price, 1987). It is monomeric, of Mr 23,000, and the sequences of a number of peptides produced by digestion of this enzyme have been determined (Fothergill and Dunbar. unpublished). Alignment of these sequenced peptides with the sequence of S.cerevisiae phosphoglycerate mutase shows 40% identity and the conservation of a number of residues which are known to be essential to the activity of the S. cerevisia enzyme e. g. His-8, Arg-7, Ser-11. Thr-20 and Arg-59. Attempts were made to isolate and sequence the gene encoding S. pombe phosphoglycerate mutase. The S.cerevisiae phosphoglycerate mutase gene failed to detect gene sequence homologies in the S.pombe genome. An oligonucleotide, designed against part of the S.pombe phosphoglycerate mutase sequence (a stretch which was not homologous to the S. cerevisiae sequence) also failed to detect sequence homologies in the S.pombe genome. Thus under the conditions used, neither the S. cerevisiae gene nor the degenerate oligonucleotide appeared to be a suitable molecular probe to screen the S.pombe cDNA expression library in λgt11 (which was synthesised by V. Simanis). A polyclonal antibody against S.pombe phosphoglycerate mutase was prepared and used to screen the S. pombe cDNA expression library. A number of small identical clones were isolated and sequenced. the cDNA inserts encoded 69 residues and part of this sequence was similar to part of the sequence of phosphoglycerate mutase from other sources. Part of the sequence was also similar to a stretch of fructose-2,6-bisphosphatase sequence (fructose-2,6-bisphosphatase appears to be divergently related to phosphoglycerate mutase, Pilkis et al., 1987). A purification scheme for phosphoglycerate mutase from the prokaryote, Streptomyces coelicolor, has been devised. The N-terminal sequence of this enzyme was determined and confirmed that the gene isolated and sequenced by Peter White, encoded phosphoglycerate mutase from S. coelicolor. The enzyme was shown to be a tetramer with a subunit Mr of 29,000. S. coelicolor phosphoglycerate mutase was also shown to be partially 2,3-bisphosphoglycerate dependent.

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A técnica de solarização vem sendo utilizada em pequenas propriedades como uma alternativa de substituição de defensivos agrícolas no controle de fitopatógenos, insetos, plantas daninhas e nematóides de solo. Desta forma, instalou-se um experimento em condições de campo, numa área da Fazenda Experimental Lageado, campus da UNESP no município de Botucatu – SP (latitude 22°51’S e longitude 48°26’W) para se avaliar o impacto desta técnica sobre a comunidade microbiana de um solo caracterizado como Latossolo Vermelho Distrófico, textura média. Inicialmente, incorporou-se uma fonte de matéria orgânica ao solo (couve Brassica oleraceae var. acephala L. fresca e triturada) na quantidade de 4kg.m-2. Posteriormente, umedeceu-se o mesmo e cobriu-se com filme plástico transparente de polietileno aditivado com 150mm de espessura. Fez-se vedação lateral de cada parcela, para se evitar a dispersão de gases e aumentar-se o efeito térmico natural. O experimento obedeceu a delineamento fatorial 2x2x4 (solo solarizado e não solarizado x com e sem incorporação de couve x épocas de coleta). Os tratamentos foram: a)adição de couve sem solarização; b)solarização e adição couve; c)testemunha, sem adição de couve e sem solarização; d)solarização sem adição de couve, com três repetições cada tratamento... .
The soil solarization technique has been used in small properties as an alternative to substitute chemical defensives for phytopathogens, insects, damage causing plants and soil nematode control. A field condition experiment was carried out in an area of Faculdade de Ciências Agronômicas - Botucatu - SP - Brazil (latitude 22°51’S and longitude 48° 26’ W) in order to evaluate the technique impact on the microbial community of soil characterized as Distrofic Red Latosoil, medium texture. Initially, a source of organic material was incorporated to the soil (kale- Brassica oleraceae var acephala L. fresh and ground) in the amount of 4 kg.m-2. After that, it was moisturized a covered with transparent additivated polyethylene plastic film 150mm tick. Lateral sealing of each alloment was made, in order to avoid gas dispersal and to increase natural thermal effect. The experiment followed a 2x2x4 factorial outline (solarized and non solarized soil x with and without kale incorporaton x four times of harvest). The treatments were: a) addition of kale incorporation; b) solarization and addition of kale; c) witness, without addition of kale and without solarization; d) solarization without addition of kale; with three repetitions of each treatment. Samples composed of soil from each allotment were collected from 0-10cm deep, with the first collecting performed seven days after the experiment implantation in the field, and the further ones as intervals of 14 days, from January to March 2001, being afterwards taken to the area of Departamento de Produção Vegetal, (Defesa Fitossanitária) for microbiological analysis... (Complete abstract, click electronic address below).

Orientador: Anita Jocelyne Marsaioli
Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Quimica
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Resumo: A utilização de enzimas para a transformação de compostos orgânicos é cada vez mais utilizada como alternativa à síntese clássica. As enzimas são utilizadas como biocatalisadores para as sínteses in vitro de compostos assimétricos uma vez que elas são intrinsicamente quirais e apresentam alta eficiência catalítica. Diante da biodiversidade de microrganismos existentes na natureza e da necessidade de descobrir novos biocatalisadores para as sínteses de blocos de construções quirais e de produtos químicos de alto valor agregado, esta tese teve como objetivos a avaliação enzimática de células microbianas íntegras, o isolamento e a identificação de compostos enantiomericamente puros obtidos através da ação enzimática de oxidorredutases. Inicialmente, a avaliação da presença de Baeyer-Villiger monooxigenases em 12 espécies de fungos através de biocatálise convencional levou a produção de (R)-(+)-5-metil-e-caprolactona (1b), produzida pelos fungos Aspergillus oryzae CCT 0975 e Geotricum candidum CCT 1205 em excelentes conversões (ambas 98%) e excessos enantioméricos (96% e 91%), respectivamente. Na etapa seguinte foi desenvolvida uma metodologia alternativa a biocatálise convencional, denominada "multibiorreações", objetivando uma triagem mais rápida e eficiente. A metodologia foi aplicada na detecção de atividade de monooxigenase permitindo um aumento no conhecimento do perfil de seletividade dos substratos analisados. A ação enzimática das células íntegras de Trichosporum cutaneum CCT 1903 resultou na redução de metilcicloexanonas orto- e para-substituídas (1 e 6) e na oxidação da cis-jasmona (8). Posteriormente realizou-se o isolamento, identificação, determinação dos excessos enantioméricos, determinação das configurações relativas e absolutas dos produtos obtidos a partir da oxidação da cis-jasmona (8): (7S,8R)-epoxijasmona, 12 (92% e.e.), 7,8-diidróxijasmona, 13 (53% e.e.) e (4S)-hidroxijasmona (86% e.e.). Nesta etapa, a determinação do perfil de seletividade de T. cutaneum CCT 1903 foi avaliado frente a 14 substratos contendo ligações duplas olefínicas (24-37). A atividade de monooxigenase foi verificada sobre os seguintes monoterpenos monocíclicos: (R)-(-)-carvona (25), a- e b- iononas (26 e 27) e (R)-(+)-limoneno (32). As biotransformações destes compostos de fragrâncias levaram às sínteses de: (1S,2R,4R)-neoisodiidrocarveol (41), (6R)-isoprenil- (3R)-metil-2-oxo-oxepanona (42), ácido-(3R)-isopropenil-6-oxo-heptanóico (43), 2,3-epóxi-(5R)-isopropenil-2-metilcicloexanol (44), 4-oxo-7,8-diidro-b-ionona (50), a-homociclogeraniol (51), limoneno-1,2-diol (54) e (+)-(4R)-p-1-menteno-8,9-diol (55), os quais foram identificados espectroscopicamente (RMN de H e C, H e H gCOSY, H e C HSQC, H e C gHMBC). Finalmente, foi realizado um estudo das atividades enzimáticas para os fungos CCT 5632, Rhyzopus oryzae CCT 1022 e a levedura AMA 7, utilizando a metodologia de multibiorreações e reações de biotransformações convencionais (substratos 8, 25-27 e 32). Uma atividade oxidorredutase foi detectada em AMA7 e R. oryzae CCT 1022. A levedura AMA7 produziu a: 7,8-epoxijasmona (12), 7,8-diidroxijasmona (13), 4- hidroxijasmona (14) e a diidrocarvona (45), enquanto que R. oryzae CCT 1022 produziu o composto 14 e o neoisodiidrocarveol (41). O fungo 5632 também apresentou atividade monooxigenase verificada a partir da formação da 4-oxo-7,8-diidro-b-ionona (50)
Abstract: The utilization of enzymes for organic compound transformations is an alternative to classical syntheses. Enzymes are used as biocatalysts for the syntheses in vitro of asymmetric compounds because they are intrinsically chiral and result in high catalytic efficiency. In front of the biodiversity of existing microorganisms in Nature and of the necessity to discover new biocatalysts for the syntheses of blocks of chiral constructions and of chemical products with high added value, this thesis aimed at enzymatic evaluation of oxidoreductase from microbial whole cells and their application of the production of enantiomerically pure compounds. First of all, Baeyer-Villiger monooxygenase (BVMO) activity was monitored using traditional biocatalytic methods. Bioprospection in 14 fungi resulted in the detection of cyclohexanone BVMO in Aspergillus oryzae CCT 0975 and Geotrichium candidum CCT 1205. The lactone (R)-(+)-1b was obtained in high enantiomeric excesses (96% and 91%, respectively) and conversion (98%). Searching for rapid screening method, multibioreaction methodology was implemented and applied to the detection of monooxigenase activity, which increased n times the amount of evaluated microorganisms per unit of time, where n is the number of added substrates. Trichosporum cutaneum CCT 1903 produced outstanding results, reducing the ortho- and para-substituted (1 and 6) methyl-cyclohexanones and oxidizing cis-jasmone (8). After isolation, identification and determination of the enantiomeric excess, the relative and absolute configuration of the cis-jasmone bioproducts were: (7S,8R)-epoxyjasmone, 12 (e.e. 92%), 7,8-dihydroxyjasmone, 13 (e.e. 53%) and (4S)-hydroxyjasmone, 14 (e.e. 86%). The enantioselectivity and substrate specificity of alkene monooxygenase in T. cutaneum CCT 1903 was further investigated using 14 substrates (24-37), applying the multibioreaction approach. Monooxygenase activity was detected in (R)-(-)-carvone, a- and b-ionones and (R)-(+)-limonene. Batch reactions of these fragrance compounds produced: (1S,2R,4R)- neoisodihydrocarveol (41), (6R)-isoprenyl-(3R)-methyl-2-oxo-oxepanone (42), (3R)- isopropenyl-6-oxoheptanoic acid (43), 2,3-epoxy-(5R)-isopropenyl-2-methylcyclohexenol (44), 4-oxo-7,8-dihydro-b-ionone (50), a-homo-cyclogeraniol (51), (R)-(+)-limonene-1,2-diol (54) and uroterpenol (55) as pure samples for spectroscopic identification (H and C NMR, H and H gCOSY, H and C HSQC, H and C gHMBC). Oxidoreductase activity was monitored using multibioreaction methodology and traditional biocatalytic methods with the fungi CCT 5632, Rhyzopus oryzae CCT 1022 and the yeast AMA 7 (substrates 8, 25-27 and 32). Thus AMA7 produced epoxyjasmone (12), 7,8- dihydroxyjasmone (13), hydroxyjasmone (14) and dihydrocarvone (45), while that R. oryzae CCT 1022 produced 14 and neoisodihydrocarveol (41). The fungus 5632 also presented monooxygenase activity confirmed through formation from 4-oxo-7,8-dihydro-b- ionone (50)
Doutorado
Quimica Organica
Doutor em Ciências

Cyanides enter the environment through both natural and man-made sources. Natural sources include cyanogenesis by bacteria, fungi and plants. A number of cyanide catabolising microorganisms have also been reported in literature. This is the first reported instance of cyanide catabolism in Trichoderma harzianum. Four strains of T. harzianum, one of T. pseudokoningii were evaluated. An investigation was made into the occurrence and distribution of the cyanide catabolising enzymes. Three enzymes, cyanide hydratase, beta-cyanoalanine synthase and rhodanese, were studied. All the strains showed a high capacity to degrade cyanide via both the cyanide hydratase and rhodanese pathways, beta-cyanoalanine synthase activity, however, was not detected in any of the selected strains. In the studies on the kinetic characterization of the rhodanese enzyme, a broad pH optimum of 8.5 - 10.5 was obtained for all the strains and a broad temperature optimum of 35 - 55 °C was also observed. The KmCN and Vmax values ranged from 7-16 mM and from 0.069 - 0.093 betamoles. Min-1. mg protein-1, respectively, between the selected strains of Trichoderma. Strong evidence of cyanide biodegradation and co-metabolism emerged from studies with flask cultures where glucose was provided as a co-substrate. The rate of degradation of 2000 ppm CIST was enhanced almost three times in the presence of glucose. Plant microcosm studies carried out using pea and wheat seeds too gave further corroboration of the cyanide degrading and plant growth promotion capabilities of Trichoderma. Microcosms set-up with cyanide at 50 or 100 ppm CN, in the presence of Trichoderma, showed germination of both pea and wheat seeds. There was no seed germination in any of the controls in the absence of Trichoderma inoculation.

The presence of pharmaceutical residues in water poses a serious treat to human health. The fate of many of these pharmaceuticals in the environment has not been thoroughly investigated. Sulfamethoxazole, sulfamethizole, trimethoprim and carbamazepine are among these pharmaceuticals with significant bioactivity that are considered to be persistent pollutants. The biodegradation of these compounds has been studied in this project in order to assess the fate of these pharmaceuticals in the environment. An easily degradable carbon source was added in these biodegradation experiments to optimize co-metabolism as a removal mechanism. Five microorganisms were used to determine if the selected drugs were biodegradable and, also, to identify the metabolites arising from their biodegradation. It was demonstrated that biodegradation occurred for sulfamethoxazole, sulfamethizole and carbamazepine. Trimethoprim showed a high resistance to biodegradation. It appeared that the microorganism Rhodococcus rhodochrous showed a particular ability to degrade the pharmaceuticals. The presence of metabolites was confirmed by HPLC and mass spectra analyses.
La présence de résidus de produits pharmaceutiques dans l'eau représente une sérieuse menace pour l'environnement et la santé humaine. Le devenir de ces produits pharmaceutiques dans l'environnement n'a pas été adéquatement étudié. Le sulfaméthoxazole, le sulfaméthizole, le triméthoprime ainsi que le carbamazepine sont parmi ces composés pharmaceutiques qui ont une importante bioactivité et sont considérés comme polluants persistants. Dans ce projet, la biodégradation des ces produits à été étudiée afin d'évaluer le devenir de ceux-ci dans l'environnement. Une source de carbone facilement biodégradable a été utilisé lors des expériences afin de stimuler le mécanisme d`élimination par co-métabolisme. Cinq microorganismes ont été utilisés afin d'évaluer la biodégradabilité des produits pharmaceutiques sélectionnés et aussi identifier les métabolites résultant de leur biodégradation. Il a été démontré que la biodégradation est survenue pour le sulfaméthoxazole, le sulfaméthizole ainsi que le carbamazépine. Le triméthoprime à quant lui démontré une forte résistance à la biodégradation. Le microorganisme Rhodococcus rhodochrous a démontré une habileté particulière à dégrader les produits pharmaceutiques. La présence de métabolites a également été confirmée par analyse HPLC et spectrometrie de masse.

Thesis (S.B.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2006.
Includes bibliographical references (leaf 25).
Imaging aquatic microorganisms in 3D space is of interest to biologists and ocean scientists seeking to understand the behavior of these organisms in their natural environments. In this research, digital holographic imaging (DHI), with a 4f system providing transverse magnification of 9.1, is used to study such microorganisms. To test the imaging technique, DHI was used to locate and track 10 micrometer Dunaliella freely swimming in a 30 milliliter tank of artificial ocean water. Multiple holograms were recorded onto one frame with laser pulsing to identify short algae trajectories. An automatic algae locating program was designed, but the signal to noise ratio was too low, and therefore the program could only locate algae reliably with manual confirmation. With refinement to the experimental setup, the signal to noise ratio could be increased, and this imaging technique could be used to analyze many systems of aquatic microorganisms interacting in a 3D space.
by Michael Trevor Wolf.
S.B.

Mestrado em Biologia Aplicada - Microbiologia Clínica e Ambiental
Antimicrobial photodynamic therapy (aPDT) is becoming a promising alternative to inactivate microbial pathogens. This therapy combines three nontoxic components, a photosensitizer (PS), light and oxygen, that when combined leads to the formation of highly cytotoxic reactive oxygen species, mainly singlet oxygen (1O2). This specie can oxidize many types of biological molecules, such as proteins, nucleic acids and lipids. The combination of positively charged groups and carbohydrate moieties with porphyrin derivatives results in an increased cell recognition and water solubility, which improves cell membrane penetration and accumulation in sub-cellular compartments. The aim of this work was to synthesize new meso-substituted glycosyl porphyrins derivatives and evaluated the efficacy of these compounds as PS in the photoinactivation of two environmental gram positive bacteria, Brevibacterium sp. and Micrococcus sp., and one gram negative bacteria, bioluminescent Escherichia coli. Brevibacterium sp. and Micrococcus sp were chosen for these studies because they were, respectively, representative of very sensitive and very resistant types to UV-B irradiation experiments. It was also evaluated the effect of 1O2 at the lipid and protein oxidation level, generated during the aPDT assay, on the two gram positive bacteria. The derivatives of meso-tetrapyridyl porphyrin were cationized by methyl iodide or by carbohydrate moieties. All synthesized compounds were characterized by proton and fluor nuclear magnetic resonance and by mass spectrometry. Two of the compounds synthesized 5,10,15,20-tetrakis(Nmethylpyridinium- 4-yl)porphyrin tetra-iodide (PS 1) and 5-[N-(Isopropylidene-6- deoxy-galactopyranos-6-yl)pyridinium-4-yl]-10,15,20-tris(N-methylpyridinium-4- yl)porphyrin tetra-iodide (PS 2) were used as PS in the aPDT assays. For the aPDT assays pure bacterial suspensions were irradiated after pre-incubation in the dark, at concentrations of 0.5, 1 and 5 μmol dm-3 of PS in the case of gram positive bacteria, and 5 μmol dm-3 in the case of gram negative bacteria. The kinetics of irradiation was evaluated by the quantification of colony forming units in aliquots collected during 15 minutes of irradiation, under 150 mW cm-2. Light and dark controls were included in all experiments. Photophysical testes (photostability and 1O2 genereation studies) were also performed. Lipid oxidation was assessed by Tiobarbituric acid (TBA) assay and results were expressed in terms of Malondialdehyde (MDA) (nmol dm-3). Protein oxidation was evaluated by 2,4-dinitrophenylhydrazine (DNPH) assay and results were expressed in terms of Protein carbonyl concentration (nmol cm-3). aPDT assays revealed that PS 2 was more effective (3.0 log of reduction) than the PS 1 (2.0 log) with 5 μmol dm-3 against E. coli. In the case of gram positive bacteria, both PS showed the same photoinactivation effect, presenting complete inactivation after 2 minutes of irradiation. However, with lower concentration PS 1 showed to be more effective than PS 2 in both gram positive bacteria. Photophysical studies showed that both PS are photostable and good 1O2 producers. ! Lipid peroxidation assays displayed different results for both environmental bacteria. In Micrococcus no lipid oxidation was observed with PS 1 while with PS 2 was observed around 31% of lipid oxidation (0.083 nmol dm-3). In Brevibacterium PS 1 caused 28% (0.063 nmol dm-3) and PS 2 50% (0.093 nmol dm-3) of lipid peroxidation. According to the protein oxidation results, Micrococcus showed around 2.1 nmol mL-1 and 6.2 nmol mL-1 of protein carbonyls with PS 1 and 2, respectively. In the case of Brevibacterium 5.0 nmol mL-1 and 4.8 nmol mL-1 were observed with PS 1 and 2, respectively. Both porphyrins showed good photoinactivation results on gram negative and gram positive bacteria. Susceptibility of Brevibacterium sp. and Micrococcus sp. to aPDT were different to those showed in UV-B irradiation by these same bacteria. Lipid oxidation assays allowed to conclude that, in Brevibacterium, both PS act in plasma membrane while in Micrococcus this only happens with the PS with a carbohydrate moiety. Protein oxidation led to the conclusion that protein damage may have occurred due to lipid oxidation or direct interaction of 1O2 with proteins.
A terapia fotodinâmica antimicrobiana (aPDT) está-se a tornar uma alternativa promissora para inactivar microrganismos patogénicos. Esta terapia consiste na combinação de três elementos não tóxicos, fotosensibilizadores (PS), luz e oxigénio que, quando combinados conduzem à formação de espécies reactivas de oxigénio altamente citotóxicas, nomeadamente o oxigénio singuleto. Estas espécies podem oxidar muitos tipos de moléculas biológicas, como o caso de proteínas, ácidos nucleicos e lípidos. A combinação de grupos com cargas positivas e hidratos de carbono com derivados porfirínicos resulta num aumento de reconhecimento celular e solubilidade em água, melhorando a penetração na membrana celular e acumulação em compartimentos sub-celulares. O objectivo deste trabalho foi sintetizar novos derivados porfirínicos mesoglicosil substituídos e avaliar a eficácia destes compostos como PS na fotoinactivação de duas bactérias gram positivas ambientais, Brevibacterium sp. e Micrococcus sp. e uma bactéria gram negativas, Escherichia coli bioluminescente. Brevibacterium sp. e Micrococcus sp. foram escolhidas para este estudo por serem, respectivamente, representantes de tipos muito sensíveis e resistentes a experiências de irradiação UV-B. Também foi avaliado o efeito do 1O2 a nível de oxidação de lipídios e proteínas, sobre as duas bactérias gram positivas. Os derivados porfirínicos meso-tetrapiridil foram cationizados com iodeto de metilo e unidades glicosídicas. Todos os compostos sintetizados foram caracterizados por ressonância magnética nuclear dos protões e fluor e por espectrometria de massa. Dois desses compostos sintetizados, 5,10,15,20- tetrakis(N-methilpiridinium-4-il)porfirina tetra-iodeto (PS 1) e a 5-[N- (isopropilidene-6-deoxi-galactopiranos-6-il)piridinium-4-il]-10,15,20-tris(Nmetilpiridinium- 4-il)porfirina tetra-iodeto (PS 2), foram usados como PS nos estudos de aPDT. Nos estudos de aPDT foram irradiadas, após uma préincubação no escuro, suspensões bacterianas puras com 0.5, 1 e 5 μmol dm-3 de PS no caso das bactérias gram positivas, e 5 μmol dm-3 no caso da E. coli. A cinética de irradiação foi avaliada através da quantificação de unidades formadoras de colónias (UFC) colhidas durante os 15 minutos de irradiação, sob 150 mW cm-2. Foram incluídos controlos claros e escuros em todas as experiências. Foram também realizados estudos fotofísicos (Fotoestabilidade e Geração de 1O2). A oxidação lipídica foi avaliada através do ensaio com ácido tiobarbitúrico (TBA) e os resultados expressos em termos de malondialdeído (MDA) (nmol dm-3). A oxidação de proteínas foi avaliada através do 2,4- dinitrofenilidrazina (DNPH) e os resultados expressos em termos de teor de proteína carbonilada (nmol cm-3). Os ensaios de aPDT revelaram que o PS 2 foi mais eficaz (3,0 log de recução) do que o PS 1 (2,0 log) com 5 !mol dm-3 contra E. coli. No caso das bactérias gram positivas, ambos os PS demonstraram o mesmo efeito de fotoinactivação, apresentando uma inativação completa após 2 minutos de irradiação. No entanto, nas menores concentrações o PS 1 mostrou ser mais eficaz do que o PS 2 em ambas as bactérias gram positivas. Os estudos fotofísicos demonstraram que ambos os PS são fotoestáveis e bons produtores de 1O2. Os ensaios de peroxidação lipídica apresentaram resultados diferentes para ambas as bactérias ambientais. No Micrococcus não foi observada oxidação lipídica com PS 1, enquanto com o PS 2 foi observado cerca de 31% de oxidação lipídica (0,083 nmol dm-3). No Brevibacterium o PS 1 causou 28% (0,063 nmol dm-3) e o PS 2 50% (0,093 nmol dm-3) de peroxidação lipídica. De acordo com os resultados de oxidação de proteínas, o Micrococcus apresentou cerca de 2,1 nmol mL-1 e 6,2 nmol mL- 1 de carbonilação de proteínas com o PS 1 e 2, respectivamente. No caso de Brevibacterium foram observados 5,0 nmol mL-1 e 4,8 nmol mL-1 com o PS 1 e 2, respectivamente. Ambas as porfirinas mostraram bons resultados tanto na fotoinactivação bactérias gram negativa como nas gram positivas. A suscetibilidade do Brevibacterium sp. e Micrococcus sp. na aPDT foi diferente dos resultados observados na radiação UV-B, por estas mesmas bactérias. Os ensaios de oxidação lipídica permitiu concluir que, no caso Brevibacterium ambos os PS actuam na membrana plasmática, enquanto que no Micrococcus estes danos só acontecem com o PS 2. A oxidação de proteínas levou à conclusão que os danos a nível das proteínas podem ter ocorrido devido à oxidação lipídica, ou interação directa entre o 1O2 e as proteínas.

1 pp.
Replaced by AZ1486h
Testing for all specific types of harmful microorganisms is expensive and essentially impossible. However, coliform bacteria are good indicators of the microbiological quality of drinking water. This publication provides information on how to test for coliform bacteria, and the way it is used to decontaminate the well.

3 pp.
1. Drinking Water Wells; 2. Private Water Well Components; 3. Do Deeper Wells Mean Better Water; 4. Maintaining Your Private Well Water System; 5. Private Well Protection; 6. Well Water Testing and Understanding the Results; 7. Obtaining a Water Sample for Bacterial Analysis; 8. Microorganisms in Private Water Wells; 9. Lead in Private Water Wells; 10. Nitrate in Private Water Wells; 11.Arsenic in Private Water Wells; 12. Matching Drinking Water Quality Problems to Treatment Methods; 13. Commonly Available Home Water Treatment Systems; 14. Hard Water: To Soften or Not to Soften; 15. Shock Chlorination of Private Water Wells
This fact sheet is one in a series of fifteen for private water well owners. The one- to four-page fact sheets will be assembled into a two-pocket folder entitled Private Well Owners Guide. The titles will also be a part of the Changing Rural Landscapes project whose goal is to educate exurban, small acreage residents. The authors have made every effort to align the fact sheets with the proposed Arizona Cooperative Extension booklet An Arizona Well Owners Guide to Water Sources, Quality, Testing, Treatment, and Well Maintenance by Artiola and Uhlman. The private well owner project was funded by both the University of Arizonas Water Sustainability Program-Technology and Research Initiative Fund and the USDA-CSREES Region 9 Water Quality Program.

Els efectes del canvi climàtic incideixen directament en les poblacions de microorganismes fotòtrofs dels tapissos microbians, produint alteracions en altres paràmetres ambientals com és l’increment de la temperatura, que pot provocar sequeres i fins i tot la desertització d’aquests ecosistemes, així com altres efectes en l’osmolaritat de les cèl·lules, degut a l’augment de la salinitat. Els microorganismes esmentats són molt abundants en els tapissos microbians, principalment els cianobacteris i les microalgues, que a més de ser els principals estabilitzadors d’aquests ecosistemes, al mateix temps es troben exposats a diferents condicions d’estrès. La majoria dels estudis que es realitzen per valorar l’impacte que tenen sobre els microorganismes condicions ambientals tan variables utilitzen cultius axènics que provenen de microorganismes aïllats de l’ambient natural o bé de col·leccions de cultius; res més lluny de la realitat, ja que en els ambients naturals els microorganismes fotòtrofs estableixen associacions estables amb microorganismes heteròtrofs i, a vegades, amb altres fotòtrofs. A més, hi ha pocs estudis que analitzin, en aquestes condicions i a nivell individual, els efectes dels paràmetres ambientals o de la pol·lució per metalls en aquestes associacions. La manca de metodologies que poden aplicar-se per esbrinar aquests efectes en un microorganisme en concret, quan està associat amb un altre de manera selectiva, in vivo, de manera ràpida i sense l’ús de cap tipus de tinció, és, per tant, un repte a la hora d’analitzar les possibilitats que tenen aquests microorganismes enfront a canvis tan dràstics. En aquest treball, s’ha intentat solucionar aquesta problemàtica mitjançant l’optimització de diferents tècniques que tenen com a base el microscopi làser confocal, considerant la principal característica dels cianobacteris i les microalgues, que és la d’emetre fluorescència natural. La clorofil·la a és el pigment majoritari d’aquests microorganismes i s’ha utilitzat amb anterioritat com a bioindicador, especialment en estudis realitzats en metalls per altres membre del grup. Així mateix, una problemàtica important és esbrinar el paper que juguen aquests microorganismes en la resistència davant canvis sobtats de les condicions naturals o antropogèniques. En aquest sentit, també s’ha accentuat l’interès en les anomenades cèl·lules dorments i en l’estudi de cèl·lules viables i no viables. Per aquest motiu, en aquest treball s’ha posat a punt una nova metodologia que utilitza el microscopi làser confocal i dos làsers específics i que ha permès determinar el percentatge d’aquestes cèl·lules en mostres exposades a diferents condicions d’estrès. Els microscopis electrònics de rastreig i transmissió, ambdós acoblats a un detector d’energia dispersiva de raigs X, juntament amb el microscopi de transmissió de raig X del sincrotró ALBA, s’han aplicat en mostres sotmeses a varis factors d’estrès per avaluar els canvis morfològics en cèl·lules senceres i en seccions ultrafines, així com en estudis de captació de metalls extra- i intracel·lularment. Els objectius del present treball s’han centrat en l’aplicació combinada de totes aquestes metodologies en dos consorcis de microorganismes: Scenedesmus sp. DE2009 i Geitlerinema sp. DE2011. L’efecte de la llum i la salinitat (com a paràmetres ambientals), així com l’impacte del plom, coure i crom (com a contaminants), s’ha estudiat àmpliament en cèl·lules individuals d’ambdós microorganismes. Finalment, aquesta tesi està estructurada en diferents capítols. El Capítol 3 correspon amb els articles publicats (un d’ells en revisió); així doncs, els resultats obtinguts de les investigacions realitzades s’exposen en les Seccions 3.1, 3.2 i 3.3 i es discuteixen globalment en el Capítol 4.
Los efectos del cambio climático inciden directamente en las poblaciones de microorganismos fototróficos de los tapices microbianos, produciendo alteraciones en otros parámetros ambientales como es el incremento de la temperatura, que puede provocar sequías y hasta la desertización de estos ecosistemas, así como otros efectos en la osmolaridad de las células, debido al aumento de la salinidad. Dichos microorganismos son muy abundantes en los tapices microbianos, principalmente las cianobacterias y las microalgas, que además de ser los principales estabilizadores de estos ecosistemas, a veces se encuentran expuestos a diferentes condiciones de estrés. La mayoría de los estudios, que se realizan para valorar los efectos que tienen sobre los microorganismos condiciones ambientales tan variables, utilizan cultivos axénicos que provienen de microorganismos aislados del ambiente natural o bien de colecciones de cultivos; nada más lejos de la realidad, ya que en los ambientes naturales los microorganismos fototróficos establecen asociaciones estables con microorganismos heterotróficos y, a veces, con otros fototróficos. Además, hay pocos estudios que analicen, en estas condiciones y a nivel individual, los efectos de los parámetros ambientales o de la contaminación por metales en estas asociaciones. La falta de metodologías que pueden aplicarse para averiguar estos efectos en un microorganismo en concreto, cuando está asociado con otro de manera selectiva, in vivo, de manera rápida y sin el uso de ningún tipo de tinción, es, por tanto, un reto a la hora de analizar las posibilidades que tienen estos microorganismos frente a cambios tan drásticos. En este trabajo, se ha intentado solucionar dicha problemática mediante la optimización de distintas técnicas que tienen como base el microscopio láser confocal, considerando la característica principal de las cianobacterias y las microalgas, que es la de emitir fluorescencia natural. La clorofila a es el pigmento mayoritario de estos microorganismos y se ha utilizado con anterioridad como bioindicador, especialmente en estudios realizados en metales por otros miembros del grupo. Asimismo, una problemática importante es valorar el papel que juegan estos microorganismos en la resistencia ante cambios repentinos de las condiciones naturales o antropogénicas. En este sentido, también se ha incrementado el interés por las denominadas células durmientes y en el estudio de células viables y no viables. Por este motivo, en este trabajo se ha puesto a punto una nueva metodología que utiliza el microscopio láser confocal y dos láseres específicos y que ha permitido determinar el porcentaje de estas células en muestras expuestas a diferentes condiciones de estrés. Los microscopios electrónicos de rastreo y transmisión, ambos acoplados a un detector de energía dispersiva de rayos X, junto con el microscopio de transmisión de rayos X del sincrotrón ALBA, se han aplicado en muestras expuestas a varios factores de estrés para evaluar los cambios morfológicos en células enteras y en secciones ultrafinas, así como para los estudios de captación de metales extra- e intracelularmente. Los objetivos de este trabajo se han centrado en la aplicación combinada de todas estas metodologías en dos consorcios de microorganismos: Scenedesmus sp. DE2009 y Geitlerinema sp. DE2011. El efecto de la luz y la salinidad (como parámetros ambientales), así como el impacto de metales como plomo, cobre y cromo (como contaminantes), se ha estudiado ampliamente en células individuales de ambos microorganismos. Finalmente, esta tesis está estructurada en distintos capítulos. El Capítulo 3 corresponde con los artículos publicados (uno de ellos en revisión); así pues, los resultados obtenidos de las investigaciones realizadas se exponen en las Secciones 3.1, 3.2 y 3.3 y se discuten globalmente en el Capítulo 4.
The effects of climate change directly affect the populations of phototrophic microorganisms in microbial mats, causing alterations in other environmental parameters such as the increase in temperature. This in turn causes drought and sometimes the desertification of these ecosystems, as well as affecting the osmolarity of cells due to the increase in salinity. The microorganisms referred to are highly abundant in microbial mats; principally, they are cyanobacteria and microalgae, which, apart from being the main stabilisers of these ecosystems, are exposed to distinct stress conditions at the same time. Most studies carried out to assess the impact on microorganisms of such variable environmental conditions use axenic cultures that come from microorganisms isolated from the natural environment or else from culture collections. Nothing could be further from reality, since—in natural environments—phototrophic microorganisms establish stable associations with heterotrophic bacteria and, at times, with other phototrophs. In addition, very few studies analyse (in these conditions and at individual level) the effects of environmental parameters or of metal pollution in these associations. The lack of methodologies that can be applied to ascertain these effects in a specific microorganism, when this is selectively associated with another, in vivo, swiftly and without using any type of staining, is therefore a challenge in analysing the possibilities of these microorganisms when facing such drastic changes. In the current work, an attempt has been made to solve this problem by means of the optimisation of distinct techniques, based on confocal laser microscopy and centring on the main characteristic of cyanobacteria and microalgae, which is the emission of natural fluorescence. Chlorophyll a is the majority pigment in these microorganisms and has previously been used as a bioindicator, in studies carried out with metals by other members of the group. Determining the role played by these microorganisms in the resistance to sudden changes in natural or anthropogenic conditions is a further, and important, issue. In addition, in this respect, the interest in dormant cells and in the study of viable and non-viable cells has increased. For this reason, a new methodology has been developed in this work; using a confocal laser microscope and two specific lasers, this has allowed us to ascertain the percentage of these cells in samples exposed to distinct stress conditions. The electronic scanning and transmission microscopes, both coupled to an X-ray dispersive energy detector, jointly with the X-ray transmission microscope from the ALBA synchrotron, have been used in samples prepared to evaluate morphological changes due to stress factors both in complete cells and in ultrafine sections, as well as in studies of extra- and intracellular metal extraction. The objectives of this work centre on the combined application of all these methodologies on two consortia of microorganisms: Scenedesmus sp. DE2009 and Geitlerinema sp. DE2011. The effect of light and salinity (as environmental parameters), in addition to the impact of lead, copper and chromium (as pollutants) was studied extensively in individual cells for both microorganisms. Finally, this thesis is organised into distinct chapters. The Chapter 3 correspond to the articles that have already been published (one of them currently under review); thus, the results obtained from the research carried out are therefore presented in Sections 3.1, 3.2 and 3.3 and are discussed globally in Chapter 4.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Este trabalho constitui-se de uma pesquisa multidisciplinar envolvendo física, química, e meio ambiente. Nesta pesquisa foi feito um estudo visando avaliar a absorção de radiação infravermelha da produção de CO2 em solo incorporado com substrato (sementes de aveia amarela, capim-seda, milho, milheto e outros invasores mais inoculação microbiana), com a finalidade de quantificar as emissões deste tipo de solo e as diferenças destas emissões de CO2 com relação à esse mesmo tipo de solo sem a incorporação deste substrato. O material foi coletado na Fundação Mokiti Okada localizada no Município de Ipeúna, interior do Estado de São Paulo SP. O solo retirado foi caracterizado como latossolo vermelho-escuro, argiloso, bem compactado, pouco poroso e com grande capacidade de saturação de água . Foram coletados dois tipos de solos: o solo incorporado com substrato e o não compactado com substrato. Ambos, foram devidamente triturados, peneirados com diâmetro de 2,5 mm e secos na sombra por um período de 12 dias e acondicionados em 12 vasos de cerâmica. Seis desses vasos continham solos não tratados com substrato e seis solos tratados com o substrato acima mencionado. Em cada vaso foi colocado uma quantia de 5.000 mL de solo, onde os mesmos foram umedecidos um dia antes da coleta dos dados. Os vasos receberam 1.200 mL de água, valor este gerado em função da capacidade de campo. No interior de cada vaso foi fixado balde de plástico que através do processo de oxidação da matéria orgânica foi produzido CO2 onde o mesmo foi medido pela câmera respirométrica LI-820 a qual possui leitor ótico infravermelho capaz de medir o fluxo de CO2. Os dados coletados demonstraram que os seis vasos tratados com substratos produziram CO2 em uma proporção muito maior do que os vasos com solos não tratados.
Abstrct: This work is constituted of a research multidisciplinar involving physics, chemistry, and environment. In this research it was made a study seeking to evaluate the absorption of infrared radiation of the production of CO2 in incorporate soil with substratum (seeds of oats yellow, grass-silk, corn, milheto and other invaders more microbial inoculation), with the purpose of quantifying the emissions of this soil type and the differences of these emissions of CO2 regarding to that same soil type without the incorporation of this substratum. The material was collected in the Fundação Mokiti located Okada in the Municipal district of Ipeúna, interior of the State of São Paulo - SP. The solitary soil was characterized as latossolo red-darkness, loamy, well compacted, little porous and with great capacity of saturation of water. Two types of soils were collected: the incorporate soil with substratum and the no compacted with substratum. Both, they were triturated properly, drizzled with diameter of 2,5 mm and dry in the shadow for a period of 12 days and conditioned in 12 ceramic vases. Six of those vases contained soils no treated with substratum and six soils treated with the substratum above mentioned. In each vase an amount of 5.000 soil mL was put, where the same ones were moistened one day before the collection of the data. The vases received 1.200 mL of water, value this generated in function of the field capacity. Inside each vase it was fastened bucket of plastic that through the process of oxidation of the organic matter CO2 was produced where the same was measured by the camera respirométrica READ-820 which possesses infrared optic reader capable to measure the flow of CO2. The collected data demonstrated that the six vases treated with substrata produced CO2 in a much larger proportion than the vases with soils no treated.

Orientadores: Renata de Oliveira Mattos-Graner, Edgard Graner
Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Streptococcus mutans é o principal patógeno da cárie dentária. Antígenos (Ags) secretados ou associados à superfície celular de S. mutans participam deste processo e incluem a proteína ligante de glucano B (GbpB), a qual pode influenciar na susceptibilidade à infecção por S. mutans. Estudos recentes indicam que a expressão de antígenos de superfície reconhecida por fagócitos é controlada por sistemas de dois componentes (SDC) em espécies relacionadas a S. mutans. O objetivo deste projeto foi investigar o efeito de variações na produção de GbpB e a influência de dois SDC, Cov (de control of virulence) e Vic (de virulence control), no padrão de internalização de S.mutans por macrófagos de camundongos. Foram avaliadas 10 cepas clínicas distintas previamente caracterizadas quanto ao padrão de síntese de GbpB e quatro mutantes knock-out de dois SDC: dois mutantes covR- e dois mutantes vicH-. Para isto, macrófagos murinos da linhagem Balb/c foram cultivados em meio RPMI e expostos a diferentes cepas de S. mutans. Os níveis de internalização bacteriana por macrófagos foram determinados através da contagem de macrófagos com bactérias internalizadas, com auxílio de microscópio óptico. Os mecanismos de internalização foram caracterizados em ensaios semelhantes com macrófagos previamente tratados com inibidores específicos dos processos de fagocitose, macropinocitose e endocitose mediada por clatrina e em análises de microscopia eletrônica de transmissão. Não houve diferença estatisticamente significante nos níveis de internalização das cepas que variavam quanto à produção de GbpB. Os mutantes vicH- foram significativamente menos internalizados quando comparados às cepas selvagens e aos mutantes covR. As análises de MET e ensaios com inibidores de processos específicos de internalização indicam que, na ausência de opsoninas, S. mutans pode ser internalizado por diferentes mecanismos incluindo-se fagocitose, macropinocitose e endocitose mediada por clatrina. Esses dados poderão auxiliar na compreensão dos mecanismos de captação e processamento de S. mutans por células apresentadoras de antígenos e dos fatores que influenciam no padrão de resposta imune adaptativa a estes microrganismos
Abstract: Streptococcus mutans are the main pathogens of dental caries. Antigens (Ags) secreted or associated with the bacterial cell surface may influence in the susceptibility to infection by S. mutans. These include the virulence protein Glucan-binding protein B (GbpB). Recent studies indicate that the expression of surface antigens recognized by phagocytes is controlled by systems of two components (TCS, Two Component System) in several streptococci species. The objective of this project was to investigate the effect of variations in the production of GbpB and the influence of the two-component systems (TCS), Cov and Vic, in the pattern of internalization of S. mutans by macrophages from mice under the absence of opsonins. Thus, levels of bacterial internalization by macrophages were evaluated in 10 different clinical strains of S. mutans previously characterized regarding the patterns of GbpB production, and in four knockout mutants of the TCS, two covR- mutants and two vicH- mutants. To this purpose, murine macrophages of the lineage Balb/c were cultured in RPMI medium and exposed to different strains of S. mutans. The levels of macrophages with internalized bacteria were determined with the help of an optical microscope. Patterns of bacteria internalization were analyzed in similar assays using macrophages previously treated with inhibitors of specific mechanisms of internalization (phagocytosis, macropinocytosis and endocytosis mediated by clathrin) and by electron transmission microscopy (ETM) analyses. There was no statistically significant difference in the efficiency of internalization between the strains that differed regarding production of GbpB. The mutants vicH- was significantly less internalized when compared with the respective wild type strains and mutants covR-. Analyses with specific inhibitors and ETM indicated that S. mutans can be internalized by phagocytosis, macropinocytosis and endocytosis mediated by clathrin. These data may help to understand the mechanisms by which S. mutans is internalized and processed by antigen-presenting cells and the factors affecting patterns of adaptative immune response to these bacteria
Mestrado
Microbiologia e Imunologia
Mestre em Biologia Buco-Dental

Orientador: Francisco Jose de Souza Filho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Os objetivos deste trabalho foram: investigar microbiologicamente, por método de cultura e pelo método do Checkerboard DNA-DNA hybridization, canais radiculares antes (C1) e após preparo químico-mecânico (C2); e, analisar radiograficamente, lesões periapicais induzidas em dentes de cães, avaliando a possível relação entre comprimento de dente e tamanho de lesão periapical. Foram utilizados 21 pré-molares inferiores de quatro cães. Os dentes foram acessados, as polpas removidas e permaneceram expostos ao meio oral por 120 dias. Após este período, as imagens das reações periapicais induzidas foram avaliadas pelo software Imagelab 2.4, mensurando área e perímetro. O preparo químico-mecânico foi procedido, sendo os dentes divididos em três diferentes grupos, de acordo com a substância química-auxiliar testada: G1- NaCl 0,09%; G2- Clorexidina 2% gel; e G3- NaOCl 5,25% + EDTA 17%. Em cada uma das amostras foi investigada a microbiota por método de Cultura que propicia crescimento de bactérias anaeróbias estritas; e, a presença de até 40 espécies bacterianas foi investigada pela técnica de Checkerboard DNA-DNA hybridization. As médias das áreas das imagens radiográficas das lesões foram de 0,06 cm2, para os segundos pré-molares, 0,12 cm2 para os terceiros pré-molares e 0,18 cm2 para os quartos pré-molares. Houve associação entre os dentes de maior comprimento com as maiores áreas de lesão periapical. Em C1 foram isoladas 60 bactérias cultiváveis, pertencendo a 32 diferentes espécies, variando de 0 a 9 espécies por canal, sendo 70% anaeróbios facultativos e 18% anaeróbios estritos, com predominio de Gram-positivos (73%). Os gêneros bacterianos mais freqüentemente isolados foram: Streptococcus, Staphylococcus, Neisseria, Propionibacterium, Actinomyces e Prevotella. Pelo Checkerboard foram detectadas 158 bactérias, pertencendo a 29 diferentes espécies, variando de 0 a 19 espécies por canal, sendo 67% anaeróbios estritos, com predomínio de Gram-negativos (67%). Os microrganismos mais frequentemente encontrados foram Campylobacter gracilis, Capnocytophaga sputigena, Leptotrichia bucallis, Prevotela intermedia, Streptococcus gordonii e Gemella morbilorum. Em C2 todos os canais apresentaram ausência de crescimento microbiano pela Cultura, porém, pelo Checkerboard foram detectadas 33 bactérias, pertencentes a 9 diferentes gêneros e 11 diferentes espécies, variando de 1 a 8 por canal, em 10 dos 21 dentes analisados. Gemella morbilorum, Capnocytophaga sputigena, Leptotrichia bucallis e Fusobacterium nucleatum sp. Polymorphum (não detectada em C1), foram detectadas com maior freqüência após o preparo químico mecânico. No G1 houve uma redução 89% de células bacterianas. A utilização de clorexidina gel 2% (G2) e do NaOCl 5,25 (G3) permitiram redução de 97% e 99%, respectivamente, não diferindo entre si, ao nível de significância de 5%. Concluíu-se que, a microbiota de canais radiculares de dentes de cães com lesão periapical induzida, por exposição ao meio oral, após 120 dias, apresenta grande número de espécies; que o preparo químico mecânico promoveu uma grande redução de microrganismos; e que os resultados sugerem existência de relação positiva entre comprimento de dente e tamanho de lesão periapical
Abstract: The objectives of this study were: to investigate, by Culture and by Checkerboard DNA-DNA hybridization, microorganisms from infected root canals before (S1) and after chemomechanical preparation (S2), and analyse radiographically induced periapical lesions in dog's teeth, for a possible relation between tooth length and lesion size. Twenty one lower premolars of four mongrel dogs were used. Their chambers were accessed and their pulps removed and left open to the oral environment for the total period of 120 days. The radiographs were evaluated by Imagelab 2.4 software to measure the area and perimeter images of the induced periapical lesions. Chemomechanical preparation was proceded, the teeth were assigned to three groups, according to the substance tested: G1- NaCl 0.09%; G2- chlorhexidine gel 2%; G3-. NaOCl 5.25% + EDTA 17%. Methodology for handling, culture and incubation for growth of strict anaerobe species was used; and, 40 bacterial species were investigated in each one of the samples by Checkerboard DNA-DNA hybridization. Measurements of the radiographic image data of the lesions were 0.06 cm2 for the second premolars, 0.12 cm2 for the third premolars and 0.18 cm2 for the fourth premolars. There was a positive association between tooth length and size of periapical lesion. In S1, a total of 60 cultivable isolates were recovered from 32 different species, ranging from 0 to 9 per canal. Facultative anaerobe species comprised 70% of the samples and 18% were strict anaerobic species with one microbiota predominantly Gram-positive (73%). The most frequent genera recovered from the canals were: Streptococcus, Staphylococcus, Neisseria, Propionibacterium, Actinomyces and Prevotella. One hundred and fifty eight bacteria were detected from 29 different species, ranging from 0 to 19 per canal. Strict anaerobe species comprised 67% of the samples with one microbiota predominantly Gram-negative (67%). The most frequent species detected by Checkerboard from the canals (S1) were: Campylobacter gracilis, Capnocitophaga sputigena, Leptotrichia bucallis, Prevotela intermedia, Streptococcus gordonii e Gemella morbilorum. In S2, no microbial growth was observed by culture. However, by checkerboard, 33 bacteria were detected, from 9 different genera and 11 differet species, ranging from 1 to 8 per canal, in 10 of the 21 investigated teeth. Gemella morbilorum, Capnocytophaga sputigena, Leptotricchia bucallis e Fusobacterium nucleatum sp. Polymorphun (not detected in S1), were detected with higher frequency after chemomechanical preparation. There was 89% of bacteria reduction in G1. Use of chlorhexidine gel 2% (G2) and NaOCl 5.25% (G3) allow bacteria reduction of 97% and 99%, respectively, with no statistical difference, with 5% significance. In conclusion, the root canal microbiota of dog teeth with induced periapical lesion, by exposure to the oral environment, after 120 days presents a great number of different species; results suggest positive relationship between the tooth length and size of the periapical lesion.
Doutorado
Endodontia
Doutor em Clínica Odontológica

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Os solos de regiões tropicais necessitam da aplicação de altas doses de fertilizantes fosfatados, cuja utilização é limitada pelo custo. Fosfatos naturais podem ser uma alternativa interessante, porém apresentam baixa solubilidade. A utilização de microorganismos solubilizadores pode aumentar a solubilidade desses fosfatos. O objetivo do trabalho foi avaliar os efeitos da inoculação das bactérias Pseudomonas fluorescens e Pseudomonas SB via semente, associadas a fontes de baixa solubilidade de fósforo, no desenvolvimento e componentes de produtividade do milho. Foram implantados dois experimentos de campo, em um Argissolo vermelho amarelo distrófico arênico no município de Pompéia (SP), na safra 2014. No experimento 1 o delineamento experimental utilizado foi o de blocos casualizados, com quatro repetições, num esquema fatorial 3 x 3 +1, envolvendo fontes de fósforo (sem fósforo, fosfato de Arad, apatita), microorganismos (sem inoculação, Pseudomonas fluorescens, Pseudomonas SB) e um tratamento adicional (superfosfato triplo), com solo corrigido com calcário. No experimento 2, o delineamento experimental foi o mesmo, num esquema fatorial 2 x 3, envolvendo fontes de fósforo (apatita, fosfato de Arad) e microorganismos (sem inoculação, Pseudomonas fluorescens, Pseudomonas SB), com o solo sem correção de acidez. As variáveis analisadas foram: teor de fósforo na folha, altura da planta e diâmetro do colmo (no florescimento), comprimento da espiga, diâmetro da espiga, número de fileira de grãos por espiga, número de grãos por fileira, número de grãos por espiga, massa de 100 grãos, produtividade, teor de fósforo no grão e índice de eficiência agronômica dos fosfatos. No experimento 1, a inoculação não promoveu alterações na altura da planta e diâmetro do colmo. Para teor de fósforo na folha a inoculação com Pseudomonas SB promoveu aumento no teor de fósforo foliar quando não foi utilizada nenhuma fonte de fósforo. Para as variáveis de produção, a inoculação com Pseudomonas fluorescens aumentou o diâmetro da espiga e número de fileira de grãos quando associada com apatita e o número de grãos por espiga. A inoculação com Pseudomonas fluorescens aumentou a produtividade em relação a inoculação com Pseudomonas SB, porém não diferiu das plantas que não receberam inoculação. A associação de Pseudomonas fluorescens com apatita e fosfato de Arad aumentou o teor de fósforo no grão. No experimento 2, a inoculação com Pseudomonas SB proporcionou um decréscimo na altura da planta e diâmetro do colmo em relação às plantas inoculadas com Pseudomonas fluorescens e aumento no teor de fósforo no grão em relação às inoculadas com Pseudomonas fluorescens e não inoculadas e adubadas com apatita.
Tropical soils require the application of high doses of phosphate fertilizers, whichusageis limited by cost.Natural phosphates can be an interesting alternative, but exhibit low solubility.The use of solubilizing microorganisms can increase the solubility of these phosphates.The objective of this paper wastoevaluate the effects of Pseudomonas fluorescensandPseudomonas SBbacteriainoculationon seed, associated with sources of low solubilityphosphorus, on the development and productivity components of maize.Two field experiments were conducted in a yellow Paleudalf in the city of Pompeia (SP), in 2014. In experiment 1 the experimental design was a randomized complete block design with four replications, in a factorial 3 x 3 + 1, involving phosphorus sources (without phosphorus, Arad phosphate, apatite), microorganisms (without inoculation,Pseudomonas fluorescens,Pseudomonas SB)and an additional treatment (triple superphosphate) with soil acidity adjusted with lime.In experiment 2, the experimental design was the same, in a factorial 2 x 3, involving phosphorus sources (apatite, Arad phosphate) and microorganisms (without inoculation,Pseudomonas fluorescens,Pseudomonas SB),without soil acidity adjustment.The variables analyzed were: phosphorus content in the leaf, the plant height and stem diameter (at flowering), ear length, ear diameter, row number of grains per ear, number of kernels per row, number of grains per ear, weight of 100 grains, productivity, phosphorus content in grain and phosphate agronomic efficiency.In experiment 1, inoculation did not change the height of the plant and stem diameter.For phosphorus content in the leaf, the inoculation with Pseudomonas SB promoted increase in leaf phosphorus content when it was not used any source of phosphorus.For production variables, inoculation withPseudomonas fluorescensincreased the ear diameter and row number of grains when associated with apatite and the number of grains per ear.Inoculation withPseudomonas fluorescensincreased productivity compared to inoculation withPseudomonas SB,but did not differ from plants that received no inoculation.Pseudomonas fluorescensassociation with apatite and Arad phosphate increased the phosphorus content in the grain.In experiment 2, inoculation withPseudomonas SBprovided a decrease in plant height and stem diameter compared to plants inoculated withPseudomonas fluorescensand increased phosphorus content in the grain compared to plants inoculated with Pseudomonas fluorescens and not inoculated and fertilized with apatite.

O emprego de enzimas lignocelulolíticas secretadas por microrganismos para a produção de etanol de segunda geração tem motivado pesquisas na área da engenharia de processos fermentativos, genômica e secretômica. Entre os microrganismos com potencial para a produção de celulases destacam-se variantes genéticos do fungo filamentoso Penicillium echinulatum caracterizados por produzirem altos títulos enzimáticos. Neste trabalho, estudouse a secretômica da linhagem selvagem 2HH e do mutante S1M29 de P. echinulatum, em cultivo submerso, empregando diferentes fontes de carbono: glicose, glicerol, celulose e bagaço de cana-de-açúcar pré-tratado por explosão a vapor. Análises enzimáticas possibilitaram identificar que P. echinulatum produz celulases, hemicelulases, esterases e, em menor proporção, pectinases e amilases. Outrossim, os maiores títulos enzimáticos para a maioria das enzimas foram verificados na linhagem mutante. Nos meios formulados com bagaço de cana-de-açúcar ou celulose verificou-se a indução das maiores produções enzimáticas para ambas as linhagens. A análise do secretoma por 1D-PAGE seguido de LCMS/ MS das amostras de 96 horas de cultivo permitiu identificar que em ambas as linhagens há predominância de enzimas CAZy, sendo celulases, hemicelulases e enzimas degradadoras de parede celular fúngica as mais predominantes. Celobiohidrolases, endoglicanases, β-glicosidases, xilanases, β-xilosidases e mananases foram identificadas e, em quantidades menores, ligninases, pectinases, amilases, esterases e solenina, entre outras proteínas (adesão, chaperonas, oxidoredutases, proteases, peptidases, lipases, glutaminases e hipotéticas). Os meios elaborados com glicose ou glicerol foram utilizados pelo fungo para a produção de amilases, ligninases e enzimas degradadoras da parede celular fúngica. Destaca-se a secreção 2 a 3 vezes maior de celulases pela linhagem mutante, sendo que o meio de cultivo elaborado com bagaço de cana-de-açúcar proporcionou a secreção de maiores quantidades de celulases para o mutante. Nesta condição, o complexo celulolítico da linhagem S1M29 constitui-se de 55% de celobiohidrolases, 38% de endoglicanases e 1% de β-glicosidases. Estes dados sugerem que durante o melhoramento genético do fungo ocorreram mudanças, embora não direcionais, possivelmente em nível da regulação da expressão gênica, modificações póstraducionais e alterações na capacidade para secretar proteínas extracelulares que tornaram a linhagem mutante S1M29 com potencial para ser empregada na hidrólise de lignocelulósicos.
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The use of lignocellulolytic enzymes secreting by microorganisms for the production of second-generation ethanol has motivated research in the field of fermentation processes engineering, genomics and secretomics. Among the microorganisms with the potential for cellulases production are genetic variants of the filamentous fungus Penicillium echinulatum characterized by produce high enzymatic titers. In this work, it was studied the secretome of the wild type 2HH and mutant S1M29 of P. echinulatum in submerged cultivation on different carbon sources: glucose, glycerol, cellulose and sugar cane bagasse pretreated by steam explosion). Enzymatic analysis allowed verifying that P. echinulatum produces cellulases, hemicellulases, esterases, and minor proportion, pectinases and amylases. Furthermore, the major enzymes titers for most enzymes dosed were verified in the mutant strain. It was verified in the media formulated with sugar cane bagasse or cellulose the induction of the highest enzyme production for both strains. The analysis of secretome by 1DPAGE followed by LC-MS/MS, of samples collected at 96 hours of cultivation, showed that in both strains there is a predominance of CAZy enzymes, being cellulases, hemicellulases and fungal cell wall degrading enzymes the most prevalent. Cellobiohydrolases, endoglucanases, β-glucosidase, xylanase, endoxylanase, β-mannanases and xylosidases were identified and, in smaller amounts, ligninases, pectinases, amylases, esterases and swollenin, among other proteins (adhesion, chaperones, oxidoreductases, proteases, peptidases, lipases, glutaminases and hypothetical). The media elaborated with glucose or glycerol were used for producing of amylases, ligninases and fungal cell wall degrading enzymes. Highlights the secretion of 2-3 times more cellulases by the mutant, being the medium prepared with sugar cane bagasse afforded the secretion of large cellulases quantities for the mutant. In this condition, the cellulolytic complex of S1M29 strain consists of 55% cellobiohydrolases, 38% endoglucanases and 1% β-glucosidase. These data suggest that during the genetic improvement of the fungus changes occurred, although not directional, possibly at the level of regulation of gene expression, post-translational modifications and changes in the ability to secrete extracellular proteins, that have made the mutant S1M29 a potential strain to be employed in hydrolysis of lignocellulose.

Orientador: Marli Teixeira Almeida Minhoni
Resumo: A técnica de solarização vem sendo utilizada em pequenas propriedades como uma alternativa de substituição de defensivos agrícolas no controle de fitopatógenos, insetos, plantas daninhas e nematóides de solo. Desta forma, instalou-se um experimento em condições de campo, numa área da Fazenda Experimental Lageado, campus da UNESP no município de Botucatu - SP (latitude 22°51'S e longitude 48°26'W) para se avaliar o impacto desta técnica sobre a comunidade microbiana de um solo caracterizado como Latossolo Vermelho Distrófico, textura média. Inicialmente, incorporou-se uma fonte de matéria orgânica ao solo (couve Brassica oleraceae var. acephala L. fresca e triturada) na quantidade de 4kg.m-2. Posteriormente, umedeceu-se o mesmo e cobriu-se com filme plástico transparente de polietileno aditivado com 150mm de espessura. Fez-se vedação lateral de cada parcela, para se evitar a dispersão de gases e aumentar-se o efeito térmico natural. O experimento obedeceu a delineamento fatorial 2x2x4 (solo solarizado e não solarizado x com e sem incorporação de couve x épocas de coleta). Os tratamentos foram: a)adição de couve sem solarização; b)solarização e adição couve; c)testemunha, sem adição de couve e sem solarização; d)solarização sem adição de couve, com três repetições cada tratamento... (Resumo completo, clicar acesso eletrônico abaixo).
Abstract: The soil solarization technique has been used in small properties as an alternative to substitute chemical defensives for phytopathogens, insects, damage causing plants and soil nematode control. A field condition experiment was carried out in an area of Faculdade de Ciências Agronômicas - Botucatu - SP - Brazil (latitude 22°51'S and longitude 48° 26' W) in order to evaluate the technique impact on the microbial community of soil characterized as Distrofic Red Latosoil, medium texture. Initially, a source of organic material was incorporated to the soil (kale- Brassica oleraceae var acephala L. fresh and ground) in the amount of 4 kg.m-2. After that, it was moisturized a covered with transparent additivated polyethylene plastic film 150mm tick. Lateral sealing of each alloment was made, in order to avoid gas dispersal and to increase natural thermal effect. The experiment followed a 2x2x4 factorial outline (solarized and non solarized soil x with and without kale incorporaton x four times of harvest). The treatments were: a) addition of kale incorporation; b) solarization and addition of kale; c) witness, without addition of kale and without solarization; d) solarization without addition of kale; with three repetitions of each treatment. Samples composed of soil from each allotment were collected from 0-10cm deep, with the first collecting performed seven days after the experiment implantation in the field, and the further ones as intervals of 14 days, from January to March 2001, being afterwards taken to the area of Departamento de Produção Vegetal, (Defesa Fitossanitária) for microbiological analysis... (Complete abstract, click electronic address below).
Mestre

El cultiu in vitro de teixits vegetals és una tècnica molt utilitzada per a l’obtenció d’importants quantitats de material genèticament idèntic i lliure de malalties. No obstant això, en alguns cultius de fruiters, aquesta tècnica es pot veure limitada per la baixa capacitat d’arrelament i aclimatació d’alguns genotips, així com per las grans pèrdues ocasionades per la presència de contaminacions endòfites. L’ús de microorganismes que milloren el creixement de plantes pot constituir una alternativa interessant gràcies a la seva capacitat de produir hormones vegetals i controlar el creixement de patògens. A més a més, també és important desenvolupar productes basats en aquests microorganismes per a l’escalat de la seva aplicació a camp. En aquesta tesi, els tres microorganismes Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 i Phoma spp. PGP03 es van aïllar de cultius in vitro contaminats de Prunus i Pyrus que mostraven un millor creixement que els no contaminats. En plantes produïdes a partir d’embrions de nectarina rescatats in vitro (Capítol 1), es va demostrar que P. oryzihabitans PGP01 augmentava el desenvolupament radicular que afavoria la seva aclimatació en hivernacle. En portaempelts comercials micropropagats in vitro (Capítol 2), C. ramotenellum PGP02 i Phoma spp. PGP03 milloraven el percentatge d’arrelament in vitro, d’un 56,3% a un 100%, en explants tractats amb IBA del portaempelt difícil d’arrelar Py12. En el mateix capítol, P. oryzihabitans PGP01 també promogué el desenvolupament de les arrels del porta-empelt de Prunus RP-20. Tots aquests resultats podrien estar relacionats amb la producció de IAA per part dels tres microorganismes. Mitjançant l’ús de mutants d’Arabidopsis thaliana, es va suggerir que les modificacions en les arrels induïdes per P. oryzihabitans PGP01 podrien estar mediades per les estrigolactones (SLs) i el glutatió (GSH) (Capítol 3). En un cultiu en medi líquid (Capítol 4), es va observar que els efectes en les arrels produïts per P. oryzihabitans PGP01 podrien estar mediats pel contingut d’auxines en el medi de cultiu. A més a més, utilitzant aquest mateix sistema, en presencia de C. ramotenellum PGP02, es va suggerir un efecte del baix pH en el medi de cultiu sobre el creixement d’endòfits en plantes de RP-20. Aquesta hipòtesi va quedar demostrada al Capítol 5, verificant que un pH àcid, en absència de microorganismes, reduïa la concentració d’endòfits sense afectar la multiplicació in vitro. Finalment, es van provar tres subproductes de la industria de la patata, tomàquet i cereals per a l’elaboració d’ún medi de cultiu barato per la producció de P. oryzihabitans PGP01. El creixement d’aquest bacteri en un medi basat en subproductes de patata es va obtenir un creixement màxim de 4.4x109 UFC mL-1 sense afectar la seva activitat biològica (Capítol 6). Els resultats obtinguts aporten noves aproximacions sobre l’ús de microorganismes beneficiosos com alternatives més sostenibles per a promoure el creixement de plantes in vitro.
El cultivo in vitro de tejidos vegetales es una técnica muy útil para obtener grandes cantidades de material genéticamente idéntico y libre de enfermedades. Sin embargo, esta técnica se ve limitada en algunas plantas frutales por la poca capacidad de enraizamiento y aclimatación de algunos genotipos, o por las pérdidas de material vegetal causadas por la presencia de contaminaciones endófitas. El uso de microorganismos que mejoran el crecimiento de plantas puede ser una alternativa muy interesante debido a su capacidad de producir hormonas vegetales o de controlar el crecimiento de patógenos. Es importante desarrollar productos basados en estos microorganismos para escalar su posible aplicación en campo. Los tres microorganismos Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 y Phoma spp. PGP03 se aislaron de cultivos in vitro contaminados de Prunus y Pyrus que mostraban un mayor crecimiento que los no contaminados. En plántulas obtenidas a partir de embriones de nectarina rescatados in vitro (Capítulo 1), P.oryzihabitans PGP01 indujo mayor desarrollo radicular que favoreció la aclimatación de las plantas en invernadero. En patrones comerciales micropropagados in vitro (Capítulo 2), C. ramotenellum PGP02 y Phoma spp. PGP03 mejoraron el porcentaje de enraizamiento in vitro, de un 56.3 a un 100%, de explantos tratados con IBA del portainjerto Pyrus Py12 difícil de enraizar. En este mismo capítulo, P.oryzihabitans PGP01 también promovió el desarrollo de las raíces del patrón de Prunus RP-20. Todos estos resultados podrían estar relacionados con la producción de IAA por parte de los tres microorganismos. Utilizando mutantes de Arabidopsis thaliana, se sugirió que los efectos en las raíces producidos por P. oryzihabitans PGP01 podrían estar mediados por estrigolactonas (SLs) y glutatión (GSH) (Capítulo 3). En cultivo con medio líquido (Capítulo 4), se observó que los efectos en la raíz producidos por P. oryzihabitans PGP01 podrían estar mediados por el contenido de auxinas en el medio de cultivo. En este mismo sistema de crecimiento en líquido, en presencia de C. ramotenellum PGP02, se sugirió el efecto de un pH bajo en el medio de cultivo sobre el crecimiento de microorganismos endófitos en plantas de RP-20. Esta hipótesis fue finalmente confirmada en el Capítulo 5, demostrando que un pH ácido en ausencia de microorganismos reducía la concentración de endófitos sin afectar la micropropagación in vitro. Finalmente, se probaron tres subproductos de la industria de la patata, tomate y cereales para elaborar un medio barato para la producción de P. oryzihabitans PGP01. El crecimiento de esta bacteria en un medio basado en subproductos de patata proporcionó un crecimiento máximo de 4,4x109 UFC mL-1 sin afectar la actividad biológica del mismo (Capítulo 6). Los resultados presentados en esta tesis proporcionan hallazgos muy novedosos acerca del uso de microoganismos beneficiosos como alternativas más sostenibles para promover el crecimiento de plantas in vitro.
In vitro tissue culture constitutes a very versatile technique to obtain large amounts of true-to-type and disease-free-plant materials. However, in some fruit tree crops, the poor in vitro rooting or acclimatization of some genotypes, or the high losses of plant material associated to endophytic contaminations may limit the effectiveness of the process. The use of microorganisms with plant-growth promoting ability might represent a sustainable alternative to overcome those limitations, knowing their ability to produce plant hormones or control pathogens growth. On the other hand, for the scale-up of the application to field conditions, it is of crucial importance to develop a product based on microorganism showing potential agronomical interest. The three microorganisms Pseudomonas oryzihabitans PGP01, Cladosporium ramotenellum PGP02 and Phoma spp. PGP03 were isolated from Prunus and Pyrus contaminated in vitro cultures showing a greater growth than those non-contaminated. In seedlings obtained from in vitro nectarine rescued embryos (Chapter 1), P. oryzihabitans PGP01 promoted root development, favouring the acclimatization to greenhouse conditions. In in vitro micropropagated commercial rootstocks (Chapter 2), C. ramotenellum PGP02 and Phoma spp. PGP03 increased the in vitro rooting percentage, from 56.3 to 100%, of the hard-to-root Pyrus rootstock Py12 explants treated with 3-indolebutyric acid. An effect of P. oryzihabitans PGP01 on root development of the Prunus rootstock RP-20 was observed. The in vitro ability of the three microorganisms to produce IAA supported these results. Using Arabidopsis thaliana defective mutants, the role of strigolactones (SLs) and glutathione (GSH) in the root events induced by P. oryzihabitans PGP01 was suggested (Chapter 3). In a liquid culture (Chapter 4), it was established a link between auxin levels in the medium and root development in the presence of P. oryzihabitans PGP01. Furthermore and regarding endophytes growth in the culture medium, the role of acidic pH to control their growth in RP-20 cultures was suggested in the presence of C. ramotenellum PGP02, being this assumption finally confirmed in Chapter 5 in the absence of microorganisms. In this chapter, the micropropagation at low pH reduced endophytes population without affecting in vitro micropropagation. Finally, wastes based on potato, tomato and cereals industries were tested for the development of a cheap culture medium for P. oryzihabitans PGP01. The growth of this bacterium in a potato wastes-based medium provided a maximum of 4.4x109 CFU mL-1 without losing the plant growth-promoting activity (Chapter 6). The results obtained in the present thesis provide novel insights regarding the use of beneficial microorganisms as more sustainable alternatives to promote in vitro plant growth.

Organic-walled microfossils are abundant and taxonomically diverse in Cambrian-Ordovician strata; some are important for biostratigraphy and for the correlation of geological successions. New assemblages of Cambrian-Ordovician acritarchs from Kolguev Island, Arctic Russia and Middle Cambrian ichnofossils of endoliths from Peary Land, North Greenland are studied. Twenty-seven acritarch species are described in detail and 10 taxa are left under open nomenclature. The diagnosis of one genus is restricted, and two other are emended. New combinations are proposed for three species and one new species is recognised. The studied acritarch assemblages are taxonomically rich and age-diagnostic and used to recognise Upper Cambrian and Tremadoc strata on Kolguev Island. The sedimentologically continuous successions provide for the first time palaeontological evidence of Cambrian strata in the north-eastern sector of Europe. The exact level of the Cambrian-Ordovician boundary was distinguished together with stratigraphic intervals equivalent to the Peltura and Acerocare zones of the Upper Cambrian of Baltica. The newly established relative age of the lowermost sedimentary succession overlying the Timanian unconformity allows verification of the minimum age of the Timanian deformation and the time-span of the hiatus bound to this unconformity. Endoliths occur in the fossil record from the Early Archean and they played an important role in the formation of stromatolites and the process of bioerosion and biodegradation. Endoliths that have actively bored into brachiopod shells or carbonate grains (euendoliths), and some that inhabited the cavities inside brachiopod shells (cryptoendoliths) are described. Borings within the carbonate grains extended with a dentritic pattern, whereas those within the brachiopod shells were formed by a multifilamentous euendolith which produced characteristic longitudinally ridged galleries. The cryptoendolithic morphologies include indeterminate coccoid masses and at least two filamentous forms. However, considerable variation in the dimensions of the currently phosphatised diagenetic crusts of the cryptoendoliths hinders discrimination.

Metal-reducing microorganisms reduce a variety of metals in metabolic processes coupled to the oxidation of organic compounds. These bacteria play an important role in the biogeochemical cycling of metals and organic matter in anaerobic aquatic and sediment ecosystems. It has been proposed recently that metal-reducing microorganisms also are active in deep subsurface environments such as petroleum reservoirs. Only two metal-reducing bacteria have been isolated from petroleum reservoir fluids, Shewanella putrefaciens and Deferribacter thermophilus. This project studied the occurrence and distribution of metal-reducing microorganisms in petroleum reservoirs. The research focused on the isolation, characterisation and identification of anaerobic bacteria from petroleum reservoirs that were capable of reducing metals and the potential roles of these isolates in the microbial ecology and biogeochemical cycling of petroleum reservoirs. Petroleum reservoirs were selected for this study on the basis of physio-chemical conditions such as temperature, salinity, pH and the presence of organic and inorganic compounds, that were likely to provide a suitable environment for anaerobic bacteria capable of reducing metals. Factors such as the stratigraphic features of the sedimentary basin, age of reservoir and past oil field practices also were considered in choosing the reservoir for study. Seven petroleum reservoirs in the USA and Azerbaijan were chosen for extensive investigations. The physico-chemical conditions in these reservoirs varied substantially. A systematic study of the production water from these petroleum reservoirs revealed a consistent presence of iron- and manganese-reducing microorganisms. It was found that salinity and temperature play a significant and defining role in the occurrence and distribution of these metal-reducing microorganisms. Biotic metal reduction was detected from production waters from all but one of the oil wells sampled. It was significant that the water from this well (Neftcala #1074) was the most saline (78 g/l NaCI). Metal-reducing activity was detected at temperatures up to 70°C. Two pure cultures, strains RED1 for Redwash petroleum reservoir (USA) and NEF1 from the Neftcala petroleum reservoir (Azerbaijan) were isolated and characterized. The strains had diverse physiological and metabolic properties including the ability to oxidize a wide range of carbon compounds and reduce a variety of metals. Their temperature, salinity and pH optima varied markedly. Phylogenetic analyses of the 16S rRNA of strain RED1 showed that the strain represented a new species of a new genus in the domain Bacteria. The bacterium most closely related to strain RED1 is the fermentative Fe(III)-reducer, Pelobacter acetylenicus (similarity value, 92.8%). Strain NEF1 possesses a unique combination of phenotypic traits and a low mol % G+C. From preliminary analyses and comparative biochemistry, NEF1 appears to be a novel metal-reducing bacterium of the Flexistipes group. The bacteria isolated in this study were able to grow at temperatures and salinities consistent with the reservoir from which they were isolated. This indicated that petroleum reservoirs are a new source of physiologically diverse, novel, metal-reducing microorganisms. The bacteria isolated also demonstrated a number of characteristics that would enable them to survive and persist in extreme subsurface conditions and develop a selective ecological advantage in petroleum reservoir environments. Significantly, the metal-reducing bacteria isolated were able to utilize an array of metabolic products produced by bacteria indigenous to petroleum reservoirs. This has resulted in a new proposed model for the ecological succession of bacteria in petroleum reservoirs.

The rising demand for arable land, to meet the competing needs of food and energy for a growing population, will soon become unmanageable. There is therefore a pressing need to increase the efficiency with which these demands are met, or find alternative ways of meeting them. In this work, E. coli transformed with a copy of its own ADP-glucose pyrophosphorylase gene (glgC), under a lac promoter, was found to synthesise large inclusion bodies when grown in media supplemented with lactose and IPTG. Analysis of these inclusion bodies suggests that they are formed of polysaccharide, giving the cell a significantly higher total sugar content than a control grown under the same conditions. The inclusion bodies were found to react strongly with iodine, turning a blackish brown colour normally associated with iodine-starch reactions. Results also indicate that the bacteria are unable to digest these inclusion bodies once they have been formed. These findings suggest the presence of long α-helices, which would both bind with iodine and prohibit enzymatic digestion. It was therefore hypothesised that the extra GlgC enzymes were allowing glucan chains to extend at a faster rate during glycogen synthesis, leading to unbranched regions that were long enough to wind themselves into helices. The subsequent introduction of a copy of the E. coli branching enzyme gene (glgB), under the same lac promoter, was therefore expected to abolish the inclusion body phenotype, by allowing the branching of the polysaccharide to keep speed with the synthesis of its linear chains. This was indeed found to be the case. However, cells transformed with both additional gene copies were also found to accumulate a significantly higher total sugar content again: more than twice that of cells transformed with glgC alone and more than seven times that of a control, when grown under conditions designed to optimise polysaccharide synthesis. These transformants were also observed to grow to higher cell densities that a control, in various growth media. The results of both these transformations could be significant in meeting the demands of our growing society. In particular, the use of cyanobacterial glycogen as a carbon source for biofuels has recently been gaining interest, and the work presented here may well be applicable in this field, providing the possibility to significantly increase yields. Lastly, the effects of Isoamylases and Granule Bound Starch Synthase, taken from two starch producing organisms – Zea mays and Ostreococcus tauri – were investigated in an E. coli host. Results were inconclusive, but suggest many avenues for continuing the work.

Honeybees (Apis mellifera and other species) are considered as the most economically important insect species for humans and the ecosystems, not only as honey producers but also and especially as pollinators of agricultural, horticultural crops and wild plants, contributing at the pollination of 35% of the global food production. Unfortunately, honeybee decline started about 30 years ago, with the arrival from Asia of the bee mite Varroa destructor. Since then, honeybees have been damaged by different kinds of biotic and abiotic stressor factors, cumulating any kind of damages, and posing a serious threat to the agricultural field. Many scientists agree that bee decline is a multifactorial process in which a mechanism seems to be more important in a given period of the year than in another, and different mechanisms may predominate in another period or in other environments. Of those multifactorial processes, leading factors are the new emergent pathogens, such as Nosema ceranae a gut pathogen causing serious threat to bees and the consequent death of the colony; Pesticides and other environmental stress factors are furthering enhancing the high pathogenicity on bees, weakening more and more the delicate beehive superorganism balance. The major science concern about the bees usually regards the study of the bee pathogens and their interaction with an increasingly anthropized environment (e.g.: pollution and sub lethal poisonings). Only few research projects (of high scientific importance) have been carried out using an approach aimed to fix the problems linked with it. Even less are the researches investigating probiotic microorganisms as growth promoter, in order to obtain a better wealth and wellbeing of the bees. In the light of these possibilities the aim of my research is the development of -environmental friendly- microbial technologies aimed to increase the health of the bees.

Aquesta tesi s'ha focalitzat en el desenvolupament de noves eines immunoquímiques per al diagnòstic de malalties infeccioses amb l'objectiu d'incrementar l'eficiència dels actuals mètodes de diagnòstic. Concretament, aquesta tesi s'ha centrat en el desenvolupament de tècniques immunoquímiques per a la detecció de Staphylococcus aureus i Pseudomones aeruginosa en mostres biològiques. L'estratègia ha consistit a seleccionar dianes específiques de cadascun dels microorganismes i, mitjançant el disseny racional d'haptens d'immunització, desenvolupar anticossos policlonals específics. Els anticossos han estat avaluats mitjançant assajos de tipus ELISA (enzyme-linked immunosorbent assay) i posteriorment s'han implementat en immunoassajos ELISA en microplaca o de micromatrius (microarrays) fluorescents per a l'anàlisi de mostres biològiques (bacteris en medi de cultiu) i mostres clíniques d'origen respiratori (esput, rentats broncoalveolars i broncoaspirats). Els immunoassajos obtinguts per ambdós bacteris són capaços de detectar les dianes seleccionades amb nivells de detecció útils per a la seva implementació en l'àmbit de diagnòstic, oferint una alternativa prometedora al diagnòstic de malalties causades per aquests bacteris. Com a resultat de la investigació realitzada, s'han generat dues patents (PCT/ES2014/070161 i P201530780), les quals es troben en fase de negociació amb empreses de l'àmbit del diagnòstic. A més s'ha publicat un article de revisió que descriu l'estat de la qüestió pel que fa al potencial de la nanobiotecnologia en el diagnòstic de microorganismes patogen, un article de recerca original, que ja ha estat publicat en la revista Analytica Chimica Acta i que descriu el treball realitzat pel que fa a la detecció immunoquímica de S. aureus, i un tercer article que es troba sota avaluació pels revisors i editors de la revista Analytical Chemistry i que reporta la recerca realitzada pel que fa al desenvolupament d'eines immunoquímiques per al diagnòstic de malalties causades per P. aeruginosa. Per aquest motiu, la tesi s'ha estructurat en format de compendi de publicacions. La tesi també inclou un annex fruit d'una estada predoctoral al grup del Prof. Kim Janda de The Scripps Research Institute (TSRI, CA, EEUU). L'objectiu de l'estada era l'avaluació com a eines terapèutiques dels anticossos produïts en aquesta tesi contra la P. aeruginosa. En aquest sentit, es pretenia investigar el potencial dels anticossos generats per neutralitzar els efectes citotòxics causats per factors de virulència d'aquest microorganisme sobre línies cel•ulars de macròfags. Tot i l'interès d'aquests estudis, aquest treball no es va poder concloure, i no es descarta reprendre'l més endavant.
This thesis has focused on the development of immunochemical new tools for the diagnosis of infectious diseases with the aim of increasing the efficiency of current diagnostic methods. Specifically, this work has focused on the development of immunochemical techniques for the detection of Staphylococcus aureus and Pseudomonas aeruginosa in biological samples. The strategy consisted in selecting specific targets for each of the microorganisms and, specific polyclonal antibodies were developed through rational design of immunization haptens. The antibodies were evaluated by ELISA tests (enzyme-linked immunosorbent assay) and later implemented in microplate ELISA immunoassays and fluorescent microarrays for the analysis of biological samples (bacteria in culture medium) and clinical samples of respiratory origin (sputum and broncoalveolars lavages and broncoaspirats). The immunoassays obtained for both bacteria are capable of detecting the selected targets with detection levels useful for its implementation in the diagnosis field, offering a promising alternative for diagnosing diseases caused by these bacteria. As a result of the research carried out two patents have been generated (PCT/ES2014/070161 and P201530780), which are under negotiation with companies in the field of diagnosis. It has published a review article that describes the state of the art regarding the potential of nanobiotechnology in the diagnosis of pathogenic microorganisms, an original research paper, which was published in the Analytica Chimica Acta journal describing the work regarding the immunochemistry detection of S. aureus, and a third article that is under evaluation by the reviewers and editors of the Analytical Chemistry journal and reporting research conducted regarding the development of immunochemistry tools to diagnose diseases caused by P. aeruginosa.

Orientadores: Brenda Paula Figueiredo de Almeida Gomes, Morgana Eli Vianna
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: Os objetivos deste estudo foram: identificar a microbiota dos canais radiculares (CR) e bolsas periodontais (BP) de dentes com polpas necrosadas, lesões periapicais, sangramento gengival e bolsas periodontais pelos métodos de cultura microbiológica e PCR; verificar a capacidade do preparo químico-mecânico (PQM) e do uso de medicação intracanal por 7 (clorexidina gel 2%) e 14 dias (hidróxido de cálcio e associação entre hidróxido de cálcio e clorexidina gel 2%) em reduzir a contagem de unidades formadoras de colônias (UFC/mL) no canal radicular e na bolsa periodontal associada e investigar possíveis associações entre as espécies bacterianas detectadas e entre microrganismos e sinais e sintomas clínicos. Amostras foram coletadas de CR (E1) e BP (P1) antes e após o PQM (E2, P2) e após o uso de medicação (E3, P3). Foram utilizados meio de transporte, cultura e incubação adequados e os microrganismos identificados por testes bioquímicos. O DNA bacteriano das amostras foi extraído e "primers" específicos para Treponema denticola, Treponema socranskii, Gemella morbillorum (Gm), Tannerella forsythia, Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas endodontalis, Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Prevotella tannerae (Pt), Prevotella nigrescens (Pn), Fusobacterium nucleatum, Filifactor alocis, Parvimonas micra (Pm) foram utilizados para detecção destas espécies por PCR. Os resultados mostraram que as UFC/mL iniciais médias foram 17,33 x 10 6 nos CR e 17,93 x 108 nas BP, após o PQM foram 15,25 x 104 nos CR e 8,53 x 108 nas BP e após o uso das medicações intracanais 7,6 x 104 nos CR e de 3,88 x 108 nas BP. Pela cultura, os microrganismos mais encontrados nos CR foram Pi/Pn/Pt e Fn em 45% de E1, Pi/Pn/Pt (5%), P propionicum (5%), S salivarius (5%), M varians (5%) em E2, A naeslundii e P propionicum em 36,6% de E3. Nas BP, os mais encontrados foram Pg e Gm em 50% dos casos de P1, Pm em 62,5% de P2 e Fn em 73,3% de P3. Por PCR, nos CR e nas BP, em todos os momentos da terapia endodôntica, Pm foi o microrganismo mais frequente. Verificou-se que Pm, Td e Tf foram encontrados em maior número em P2 que em E2 e que Pe e Fn estavam mais presentes em P3 que em E3. Nas primeiras coletas não houve diferenças estatisticamente significantes. Concluiu-se que, os canais radiculares e as bolsas periodontais de dentes com comprometimento pulpar e bolsa periodontal apresentaram-se infectados com uma combinação de espécies de microrganismos formada principalmente por bactérias anaeróbias estritas e facultativas, grampositivas, o PQM foi o grande responsável pela redução dos microrganismos dos CR, correlações estatísticas positivas foram observadas entre a presença de sinais e sintomas de origem endodôntica e periodontal e algumas espécies bacterianas, e também entre as espécies, quaisquer das medicações intracanais testadas não foram capazes de alterar ou reduzir a microbiota da bolsa periodontal associada no período de 7 ou 14 dias, não houve diferenças de eficiência antimicrobiana entre as medicações intracanais utilizadas tanto no canal radicular como na bolsa periodontal no período estudado.
Abstract: The aims of this study were to investigate the microbiota of root canals (RC) and periodontal pockets (PP) from teeth with pulp necrosis, periapical lesion, gingival bleeding and periodontal pockets by culture and PCR; to verify the ability of chemo-mechanical preparation (CMP) and intracanal dressings (2% chorhexidine, calcium hydroxide and association of both medications) on reducing colony forming unit (CFU/mL) counting in the period of 7 or 14 days in the root canal and periodontal pocket; and to investigate possible associations among detected bacterial species and microorganisms and between signs and symptoms. Samples were collected from RC (E1) and PP (P1) before and after CMP (E2 and P2) and after the use of intracanal dressings (E3, P3). Adequate transport medium, culture, incubation and biochemical tests were used. DNA of samples was extracted and primers species-specific to Treponema denticola, Treponema socranskii), Gemella morbillorum (Gm), Tannerella forsythia (Tf), Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas endodontalis (Pe), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Prevotella tannerae (Pt), Prevotella nigrescens (Pn), Fusobacterium nucleatum (Fn), Filifactor alocis (Fa), Parvimonas micra (Pm) were used to detect these species by PCR. The results showed that the average of initial CFU/mL was 17.33 x 10 6 in RC and 17.93 x 108 in PP, after CMP 15.25 x 104 in RC and 8.53 x 108 in PP and after the use of intracanal medications 7.6 x 104 in RC and 3.88 x 108 in PP. By culture, the most frequent microorganisms in RC were:Pi/Pn/Pt and Fn in 45% of the E1, Pi/Pn/Pt (5%), P propionicum (5%), S salivarius (5%), M varians (5%) in E2, A naeslundii and P propionicum in 36.6% of the E3. In PP, Pg and Gm were in 50% of P1 while Pm was the most isolated in P2 (62.5%) and Fn in P3 (73.3%). By PCR, in both sites, in all moments of the endodontic therapy, Pm was the mostly found microorganism. It was verified that Pm, Td and Tf were found in greater number in P2 than in E2; and Pe and Fn were more presented in P2 than in E2. There were no statistic differences in the initial samples. It was concluded that, RC and PP from teeth with endo-perio lesion were infected with a combination of microorganisms species, mainly strict and facultative anaerobe bacteria, grampositives; CMP was responsible for the mainly microbial reduction in root canals and the use of intracanal dressings in the established time did not achieve any antimicrobial effect in adjacent periodontal pockets, positive statistic correlations were noted between detected bacterial species and microorganisms and signs and symptoms, there were no statistic differences in the antimicrobial efficacy among the intracanal dressings in RC and PP in the studied period.
Doutorado
Endodontia
Doutor em Clínica Odontológica

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Todo produto agrícola interfere na situação termodinâmica de solos, afetando as interações entre solo-microorganismos-plantas. Biochar é definido como um material carbonáceo multifuncional que atua como condicionante/ fertilizante e eleva a qualidade de solos. Pode estar presente naturalmente em solos férteis ou produzido a partir da pirólise de resíduos agrícolas gerando produtos de maior valor agregado. A literatura destaca casos de sucesso de sua aplicação no solo e como material adsorvente de contaminantes orgânicos e inorgânicos. No entanto, poucas informações estão disponíveis sobre possíveis efeitos antagônicos do biochar aos microorganismos do solo e alterações diretas no desenvolvimento de plântulas. Visando entender interações do biochar no sistema solo-microorganismos-plantas, este trabalho objetivou identificar os efeitos de diferentes doses de biochar produzidos a partir de bagaço de cana-de-açúcar e palha de sabugo de milho na interação com nutrientes, desenvolvimento de fungos ligninolíticos, bactérias promotoras do crescimento de plantas e de plântulas de milho. Para tanto, as amostras de biochar foram produzidas por meio de pirólise em nove regimes de temperatura para compreender quais mudanças morfológicas e físico-químicas ocorrem na formação do biochar. Com o objetivo de preservar as interações multitróficas do solo, foram explorados os efeitos de diferentes doses de biochar no crescimento radial de micélios três diferentes espécies de fungos ligninolíticos: Bjerkandera adusta, Pleurotos ostreaus e Trametes versicolor. Foi encontrado um efeito dependente da espécie, onde as mesmas amostras de biochar podem causar toxicidade aguda e efeito aditivo no crescimento desses fungos. A espécie T. versicolor mostrou ser a espécie mais adaptada. Da mesma forma, as bactérias das espécies Bacillus aryabhattai (CMAA-1363) e Leocobacter sp (CMAA-1422) foram incubadas em meios de cultivo contendo biochar em diferentes doses. Foram verificadas que ambas as espécies não sobreviveram à incubação direta, liberando no processo exsudados (biopolímero) higroscópicos no meio. Experimentos de germinação de sementes de milho mostraram que apenas as baixas doses de biochar produzidos a partir de bagaço de cana-de-açúcar nas temperaturas de 400 e 600 ºC apresentaram efeitos aditivos ao desenvolvimento das plântulas. Quando os exsudatos bacterianos foram testados como fertilizante líquido, apenas o biochar produzido de palha de milho a 300 ºC graus e em baixa dose apresentou efeitos aditivos no crescimento das plântulas. Assim, este trabalho aponta a necessidade de estudos específicos e multitróficos de materiais destinados ao sistema pedológico.
Any agricultural product that reaches the pedological system interferes in the thermodynamic situation of soil non-equilibrium, affecting the soil-microorganisms-plants set. In recent years, biochar has been identified as a conditioning / fertilizing material that helps raise the soil qualities. Biochar is a multifunctional carbonaceous material already existing naturally in fertile soils. However, the non-natural production of biochar has been considered as an alternative destination to agricultural waste that generates value-added products. The literature highlights successful cases of its application in the soil and as adsorbent material of organic and inorganic contaminants. However, little information is available on possible antagonistic effects of biochar on soil microorganisms and direct changes in seedling development. In order to understand possible points of bifurcations that biochar can generate in the soil-microorganisms-plants system, this work aimed to identify the effects of different doses of biochar produced from sugarcane bagasse and corn cob in the growth and development of ligninolytic fungi, plant growth promoting bacteria and maize seedlings. For this, the biochar samples were produced by means of pyrolysis in nine temperature regimes to understand which morphological and physicochemical changes occur in the formation of biochar. With the objective of preserving the soil multitrophic interactions, it was explored the effects of different doses of biochar on the radial growth of mycelium three different species of ligninolytic fungi: Bjerkandera adusta, Pleurotos ostreaus and Trametes versicolor. A species-dependent effect was found where the same biochar samples can cause acute toxicity and additive effect on the growth of these fungi. The species T. versicolor showed to be the most adapted species. Similarly, bacteria of the species Bacillus aryabhattai (CMAA-1363) and Leocobacter sp (CMAA-1422) were incubated in culture media containing biochar at different doses. It was verified that both species did not survive the direct incubation, releasing hygroscopic exudates (biopolymer) in the medium. Germination experiments of corn seeds showed that only the low doses of biochar produced from sugarcane bagasse at temperatures of 400 and 600ºC had additive effects to the development of the seedlings. When the bacterial exudates were tested as liquid fertilizer, only the biochar produced from corn straw at 300 degrees and at low dose had additive effects on the growth of the seedlings. Thus, this work points out the need for specific and multitrophic studies of materials destined to the pedological system.
FAPESP: 2013/08373-0