Orientador: Reinaldo Marchetto / Banca: Saulo Santesso Garrido / Banca: Clóvis Ryuichi Nakaie / Resumo: O sistema toxina-antitoxina ParE-ParD é um sistema bacteriano de morte póssegregacional codificado numa ampla gama de hospedeiros. ParE é uma proteína pequena, de aproximadamente 12 kDa codificada pelo gene parE. ParD é uma antitoxina de cerca de 9 kDa, codificada pelo gene parD, capaz de complexar com ParE e neutralizá-la. Estudos têm mostrado o envolvimento da enzima DNA girase no processo de morte celular pela ParE, entretanto, não se tem disponível na literatura nenhuma evidência de inibição desta proteína sobre a atividade da topoisomerase IV. Embora encontrada numa ampla diversidade de microorganismos, ParE não possui função celular totalmente elucidada e seu mecanismo de citotoxicidade permanece ainda desconhecido. Com base na estrutura primária da proteína ParE de E. coli e nos escassos dados disponíveis para esta toxina, peptídeos miméticos foram racionalmente projetados visando compreender o mecanismo de inibição de ParE sobre a atividade da DNA girase, bem como, avaliar a ação destes miméticos na atividade da Topoisomerase IV e da Topo II humana. Estas sequências peptídicas foram sintetizadas pela metodologia de fase sólida, purificadas e analisadas por cromatografia líquida de alta eficiência e caracterizadas por espectrometria de massas. A capacidade de inibição destes peptídeos miméticos sobre a atividade das topoisomerases foi testada por ensaios de eletroforese em gel. Os peptídeos que apresentaram melhores resultados de inibição sobre a atividade das topoisomerases foram o ParERM3 e o ParEC3, projetados para conter os resíduos de aminoácidos L61-R100 e L69-R100, respectivamente, presentes na porção C-terminal da estrutura da proteína ParE de Escherichia coli. A sequência "LNIES" (L101 a S105), quando presente nos peptídeos miméticos atenuou a toxicidade dos mesmos frente às topoisomerases, provavelmente por interferir na interação peptídeo-topoisomerase. Embora não existam... / Abstract: The toxin-antitoxin system ParE-ParD is a bacterial system of post-segregational death encoded in a wide range of hosts. ParE is a small protein of approximately 12 kDa encoded by parE gene. ParD is an antitoxin about 9 kDa, encoded by the parD gene, able of complexing with ParE and neutralize it. Studies have shown the involvement of the enzyme DNA gyrase in the cell death process by ParE, however, is not available in the literature any evidence of inhibition of this protein on the activity of topoisomerase IV. Although found in a wide variety of micro-organisms, the ParE function has not been fully elucidated and its cytotoxic mechanism remains unknown. Based on the primary structure of the E. coli ParE and the limited data available for this toxin, mimetic peptides were rationally designed aiming at understanding the mechanism of inhibition of ParE on the activity of DNA gyrase, as well as on the topoisomerase IV and human topoisomerase II activities. These peptides sequences were synthesized by solid phase methodology, purified and analyzed by high performance liquid chromatography and characterized by mass spectrometry. The ability of these mimetics to inhibit the activity of topoisomerases was tested by electrophoresis on agarose gel. The peptides that showed better results on the inhibition activity of topoisomerases were ParERM3 and ParEC3 , designed to contain the L61-R100 and L69-R100 amino acids sequence, respectively, found of the C-terminal portion of the ParE structure of Escherichia coli. The "LNIES" sequence (L101 to S105), when present in the mimetic peptides, attenuated the toxicity of the peptides on topoisomerases activity, probably by interfering in the topoisomerase - peptide interactions. Despite the absence of data in the literature regarding the toxicity of ParE protein on the topo IV and human topo II activities, the peptides ParERM3 and ParEC3 showed better inhibitory activity on these enzymes when compared... / Mestre

Orientador: Valéria Nogueira Dias Paes Secco / Banca: Ana Maria Bormio de Rosis / Banca: Wilson Baliotti Júnior / Resumo: O Sistema Rh é constituído por 48 antígenos eritrocitários, sendo o antígeno Rh D, considerado o mais imunogênico seguido dos antígenos c, E, C, e. A probabilidade de imunização por Rh D é muito mais alta quando comparada com outros antígenos de grupos sanguíneos humano que ocasionalmente produzem anticorpos irregulares, tornando esse sistema de grande importância clínica. O antígeno Rh D é composto por 37 epítopos, formando um mosaico. Qualquer mudança de aminoácidos em uma parte da proteína pode afetar a expressão da seqüência de epítopos ou resultar em novos epítopos. Por isso, os antígenos do grupo sanguíneo Rh D podem apresentar variações de sua expressão na membrana eritrocitária. As hemácias com fenótipo Rh D fraco diferem quantitativamente das células Rh D positivas normais, por serem definidas como tendo todos os epítopos Rh D presentes, porém com uma redução dos níveis de antígeno Rh D. As hemácias Rh D parcial diferem qualitativamente das células Rh D positivas normais, pois carecem de um ou mais epítopos que formam o antígeno Rh D. Em geral, os fenótipos Rh D parcial se expressam devido à presença de genes híbridos, os quais ocorrem predominantemente em indivíduos de origem africana. O número de amostras classificadas como Rh D fraco e Rh D parcial depende das características do reagente utilizado para a fenotipagem Rh D. Anteriormente os reagentes utilizados eram os policlonais, porém atualmente, com a descoberta do anticorpos monoclonais e a dificuldade de obtenção dos policlonais, tem-se utilizado apenas os reagentes monoclonais. Desde então, diferenças entre a reatividade de vários reagentes, especialmente na detecção de Rh D fraco e Rh D parcial, têm criado uma discussão sobre o teste adequado para doadores de sangue, pacientes e receptores. / Abstract: The Rh System is constituted by 48 red cells antigens, where the Rh D antigen is considered the most immunogenic followed by c, E, C, e antigens. The probability of immunization by Rh D is higher when is compared with other human sanguineous groups antigens, that occasionally produce irregular antibodies, what makes this system very important clinically. The antigen Rh D is composed by 37 epitopes, which forms a mosaic. Any amino acid change in a protein part can affect the expression of the epitopes sequence or results in new epitopes. Therefore, the sanguineous group Rh D antigens can present variations in the red cells membrane expression. The red cells with weak D phenotype are quantitatively different from the Rh D normal positive cells, because of the presence of all epitopes in their definition, but with a reduction of Rh D antigen levels. The red cells D partial are qualitatively different from the Rh D normal positive cells, because they need one or more epitopes that form Rh D antigen. In general, the partial D phenotype occurs due to the presence of hybrid genes, which occur predominantly in African individuals. The number of classified samples as weak D and partial D depends on the reagent used for the Rh D phenotyping characteristics. Previously the used reagents were polyclonal, but currently, with the discovery of the monoclonal antibodies and the difficulty of attainment of the polyclonal, it has been used only monoclonal reagents. Since then, differences between the reactivity of many reagents, especially in the detention of weak D and partial D, have been created a discussion about the apropriated tests for blood donators, patients and receivers. / Mestre

Made available in DSpace on 2014-06-11T19:23:28Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-02-27Bitstream added on 2014-06-13T20:30:13Z : No. of bitstreams: 1 murari_cs_me_sjrp_parcial.pdf: 225376 bytes, checksum: f1c467b09ceb77e424e43229434d3924 (MD5) Bitstreams deleted on 2015-03-16T11:30:19Z: murari_cs_me_sjrp_parcial.pdf,Bitstream added on 2015-03-16T11:31:02Z : No. of bitstreams: 1 000713892.pdf: 1800564 bytes, checksum: ec4b7820479ba135ca9dd0c43a1b89ff (MD5) / O soro de leite é um subproduto de elevado valor nutricional, composto principalmente por lactose e proteínas. Representa uma fonte rica de nutrientes, podendo ser utilizado em diversos processos fermentativos, porém quando descartado de maneira incorreta pode gerar sérios problemas ao meio ambiente devido a sua elevada carga orgânica. Desta maneira, o objetivo do trabalho foi avaliar a produção de etanol e redução da matéria orgânica por Kluyveromyces marxianus 229 em meios compostos por soro de leite. Os meios foram fermentados por 48 horas em condições aeróbias e anaeróbias em diferentes concentrações de soro de leite (100%, 80%, 50% e 25%) e inoculo inicial (5%,10%,15% e 20%). Em intervalos de 6 horas alíquotas dos meios fermentativos foram retiradas para realizar análise de etanol, carboidratos, proteínas, e demanda química de oxigênio (DQO). A maior produção de etanol ocorreu nos meios compostos por maiores concentração de nutrientes (MSL1), e com 10% de inoculo inicial, chegando a produzir 18,70 g/L de etanol em 10 horas de fermentação e 17,20 g/L de etanol em 18 horas de fermentação em condições aeróbias e anaeróbias respectivamente. Porém, em termos de rendimento etanólico teórico, os meios que apresentaram 50% de soro de leite (MSL3), foram os que obtiveram melhores resultados, chegando a um rendimento de 71,80% em aerobiose e 68,11% em anaerobiose . Em relação à produção e produtividade celular, os meios MSL1 com 15% de inoculo foram os que obtiveram resultados mais satisfatório, com 6,45 g/L e 0,53 g/L.h respectivamente em aerobiose e 5,44 g/L e 0,22 g/L.h respectivamente em anaerobiose. A levedura K. marxianus 229 apresentou melhores resultados quanto a redução da DQO nos meios de menor concentração de nutrientes (MSL4), chegando a reduzir até 94,26% e 83,28%... / Whey, a by-product with high nutritional value, mainly composed by lactose and proteins, represents a rich source of nutrients that can be used in different fermentation processes. However, when it´s incorrectly disposed, can cause, however when it‘s incorrectly disposed it can cause serious problems to the environment due its high organic load. Thus, the aim of this study was to evaluate the ethanol production and the organic matter reduction by Kluyveromyces marxianus 229 in a media composed of whey. The media was fermented for 48 hours under aerobic and anaerobic conditions at different concentrations of whey (100%, 80%, 50% and 25%) and initial inoculums (5%, 10%, 15% and 20%). Aliquots of fermentation media were taken every 6 hours to perform analysis of ethanol, carbohydrates, proteins and chemical oxygen demand (COD). Ethanol production was higher in media containing higher nutrients concentration (MSL1) and 10% of initial inoculums, reaching 18.70g/L of ethanol after 10 hours and 17.20g/L after 18 hours of fermentation under aerobic and anaerobic conditions, respectively; though in terms of theoretical ethanol yield, the media containing 50% of whey (MSL3) obtained better results, reaching a yield of 71.80% under aerobic condition and 68.11% under anaerobic condition. Regarding the cellular production and productivity, the media MSL1 containing 15% of inoculum obtained more satisfactory results, presenting 6.45g/L and 0.53 g/L.h respectively under aerobic condition and 5.44g/L and 0.22 g/L.h respectively under anaerobic condition. The yeast K. marxianus 229 showed better results for the COD reduction in the media with lower nutrients concentration (MSL4), reducing to 94.26% and 83.28% after 48 hours of aerobic and anaerobic fermentation respectively. These results... (Complete abstract click electronic access below)

Orientador: Laura Satiko Okada Nakaghi / Resumo: A androgênese é um mecanismo de reprodução uniparental no qual a progênie possui material genético exclusivamente paterno. Essa forma de manipulação cromossômica pode ser utilizada como estratégia de reconstituição de espécies ameaçadas. O presente trabalho foi desenvolvido com o objetivo de estabelecer protocolos de indução à androgênese dispérmica na espécie Astyanax altiparanae, padronizando um pacote tecnológico de manipulação da espécie. Inicialmente, foi investigado se o tempo de estocagem dos oócitos (tempo 0 - controle, com 60 minutos pós extrusão (mpe), 120 mpe, 180 mpe e 240 mpe) afetava sua viabilidade em ser fertilizado e a sua ploidia. Os resultados apontam que uma estocagem superior a 60 minutos pós extrusão pode afetar a ploidia e causar reflexos sobre a viabilidade e sobrevivência, com 4,44% das amostras analisadas sendo triploides. Considerando essas informações, foi posteriormente estabelecido um meio de cultura para os oócitos após a extrusão (Ringer, Hanks e dPBS) e a faixa ideal de irradiação UV para inativar o material genético materno (30’, 60’, 90’ e 120’ segundos). A solução ideal para estocagem dos oócitos pós extrusão foi a de dPBS, por até 120 minutos, permitindo um largo intervalo para manipulação. Nesse tratamento, a taxa de eclosão foi de 92,08 ± 1,75%. A faixa de irradiação mais eficaz em inativar o material genético materno foi a de 90 mW/cm2, com 100% das amostras analisadas haploides. Com o estabelecimento deste protocolo, tornou-se possível... (Resumo completo, clicar acesso eletrônico abaixo) / Doutor

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

Marine sediments are the main accumulation reservoir of organic recalcitrant pollutants such as polychlorinated biphenyls (PCBs). In the anoxic conditions typical of these sediments, anaerobic bacteria of the phylum Chloroflexi are able to attack these compounds in a process called microbial reductive dechlorination. Such activity and members of this phylum were detected in PCB-impacted sediments of the Venice Lagoon. The aim of this work was to investigate microbial reductive dechlorination and design bioremediation approaches for marine sediments of the area. Three out of six sediment cultures from different sampling areas exhibited dechlorination activities in the same conditions of the site and two phylotypes (VLD-1 and VLD-2) were detected and correlated to this metabolism. Biostimulation was tested on enriched dechlorinating sediment cultures from the same site using five different electron donors, of which lactate was the best biostimulating agent; complementation of microbial and chemical dechlorination catalyzed by biogenic zerovalent Pd nanoparticles was not effective due to sulfide poisoning of the catalyst. A new biosurfactant-producing strain of Shewanella frigidimarina was concomitantly obtained from hydrocarbon-degrading marine cultures and selected because of the low toxicity of its product. All these findings were then exploited to develop bioremediation lab-scale tests in shaken reactors and static microcosms on real sediments and water of the Venice lagoon, testing i) a bioaugmentation approach, with a selected enriched sediment culture from the same area, ii) a biostimulation approach with lactate as electron donor, iii) a bioavailability enhancement with the supplementation of the newly-discovered biosurfactant, and iv) all possible combinations of the afore-mentioned approaches. The best bioremediation approach resulted to be a combination of bioaugmentation and bioremediation and it could be a starting point to design bioremediation process for actual marine sediments of the Venice Lagoon area.

Aiming to reduce the cost of production of polyhydroxyalkanoates (PHAs), it was verified the use of two agro-industrial wastes as alternatives carbon sources for the production of: A- poly(hydroxybutyrate-co-hydroxyvalerate), from olive mill wastewater (2012 campaign; 60% dephenolised). It was possible to used up to 25% v/v in the culture media due to the presence of polyphenolic inhibitors in the matrix. B- polyhydroxybutyrate (PHB) with OMW belonging to 2013 (>70% dephenolised): the culture medium could contain 100% v/v without causing inhibition. An integrated biorefinery scheme was defined and tested sequentially: polyphenols recovery, organic acids PHAs and biogas production. C- PHB from dephenolised and fermented grape pomace. An integrated biorefinery scheme was proposed for the first time for the valorisation of grape pomace. D- medium chain length polyhydroxyalkanoates by performing the anaerobic acidogenic digestion of GP before dephenolisation. From the research work on OMW, a study about total polyphenols determination by colorimetric method was carried out. Indeed, solid phase extraction break-through curves obtained during polyphenols recovery experiment were employed as a tool for the analysis. To increase the volatile fatty acids (VFAs) concentration, for obtaining a feeding solution which allow fed-batch fermentation, it was proposed to concentrate VFAs produced in acidogenic digestion using nanofiltration (NF). A preliminary feasibility study has been carried out, getting rejections in the range 30-90%, using a plant-counter and prepared in the laboratory of VFAS solution (C2 to C6) salts and buffers in distilled water. The optimization of the PHAs downstream process was also studied. It is been developed a procedure for the extraction of PHAs by cell pretreatment with, heat, acids, digestion with NaOH and ethanol-water washing. In this study it was verified that the implementation of a pre-treatment with H2SO4 allows to recover 85% of PHAs products reaching a purity of >95%.

No description available.

This study aims to measure the impact of protein and amino acid fermentation on the composition and metabolic output of gut microbiota. Although dissimilatory pathways have been described for most amino acids, microbial degradation routes within the gut microbiota are relatively unexplored. The objectives were (1) to characterize amino acid breakdown by the colonic microbiota, (2) to determine the fermentation products formed from individual amino acids/protein (3) to examine how amino acid metabolism is impacted by the presence of a fermentable fiber (prebiotic inulin) and finally (4) to evaluate with an in vivo model (trout fish) diet:microbe interactions and the development of gut microbiota during fish farming. Interactions between the healthy human intestinal microbiota of the distal colon and different combinations of nutrients were simulated using in vitro pH-controlled anaerobic batch cultures of human faeces. Combining high-throughput sequencing of 16S rRNA amplicons, with high-throughput 1H NMR, changes in faecal microbiota composition and metabolic output were measured. During exogenous substrate microbial fermentation (e.g. beef, Trp or fish feed) in the large bowel bioactive compounds (harmful or beneficial) are produced. Many factors affect the gut-microbial metabolism including pH, type and quantity of growth substrate (e.g. protein/carbohydrate) and make up of the gut microbiota. Considerable interindividual variation was observed in response to different digested substrates but over all, the beneficial impact of prebiotic fiber fermentation on production of bioactive compounds from amino acids/proteins was confirmed in this study. In trout, although our dietary intervention with essential oils had little impact on the gut microbiota, the study showed for the first time a dramatic shift in the composition and diversity of the gut microbiota in juvenile, compared to adult fish. Thse observations may have relevance in designing dietary strategies to reduce chronic diseases like colon cancer and heard disease and for fish farming respectively.

Orientador: Cecília Laluce / Coorientador: Karen F. de Oliveira. / Banca: Sandra Regina Pombeiro Sponchiado / Banca: Nilce maria Martinez Rossi / Resumo: A biomassa lignocelulósica contém quantidades significativas de fontes de carbono, sendo assim uma fonte de energia renovável. O presente trabalho teve como objetivo estabelecer meios líquidos e sólidos para o estudo dos efeitos dos inibidores presentes nos hidrolisados do bagaço de cana-de-açúcar e, por fim, estabelecer um coquetel de inibidores que permitisse o crescimento e a fermentação da levedura Saccharomyces cerevisiae no meio líquido estabelecido. Por esta razão, o conjunto de resultados deste trabalho compreende três partes. A primeira parte foi dedicada ao estabelecimento de meios sólidos e líquidos para comparação de leveduras na presença do inibidor mais abundante do hidrolisado do bagaço de cana, o ácido acético. No meio sólido YPD, as diferentes linhagens de leveduras ensaiadas sofreram menos os efeitos inibitórios deste ácido do que o meio sintético solidificado. Em meio líquido, foi necessário adicionar 2% de extrato de levedura para ativar o crescimento da levedura 63M, mas este efeito foi suprimido em presença de ácido acético. As variações em pH inicial (3,5 - 5,5) afetaram mais a produção de etanol do que a biomassa, ao passo que a viabilidade não variou. A segunda parte deste trabalho foi dedicada ao estabelecimento de um meio sintético que permitisse o crescimento e fermentação da linhagem 63M em presença de ácido acético. O aumento de 33% em biomassa na presença de 83 mmol/L de ácido acético foi devido à elevação da glicose (10-18%) e do inóculo (5-10 mg/mL). As concentrações crescentes e variadas de ácido acético em meio sintético foi utilizado para estudar seus efeitos sobre o crescimento (inibição acima de 50 mmol/L), a viabilidade (acima de 250 mmol/L) e a produção de etanol (acima de 83 mmol/L). As concentrações de outros inibidores... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Lignocellulosic biomass contains significant quantities of carbon sources, being a renewable energy source. The aim of the present work was to determine liquid and solid media for the study of the effects of inhibitors present in hydrolysates of sugarcane bagasse, and to establish a cocktail of inhibitors that allow the growth and the fermentation of the yeast Saccharomyces cerevisiae in the liquid medium established. Thus, the results of this work consist of three parts. The first part was dedicated to establishing solid and liquid media in order to compare different yeast strains in the presence of the most abundant inhibitor found in hydrolysates of sugarcane bagasse, the acetic acid. On YPD solid medium, this acid had less inhibitory effect on the different yeast strains tested than on the synthetic solid medium. In liquid medium, it was necessary to add 2% yeast extract to activate the growth of the strain 63M, but this effect was abolished in the presence of acetic acid. Variations in the initial pH (3.5 - 5.5) affected more the ethanol production than the biomass, while the viability was not influenced. The second part of this work was dedicated to establishing a synthetic medium that allows the growth and fermentation of the strain 63M in the presence of acetic acid. The 33% increase in biomass in the presence of 83 mmol/L acetic acid was due to the high glucose concentration (10-18%) and inoculum (5-10 mg/mL) used. The increasing concentrations of acetic acid in the synthetic medium were used to study its effects on growth (inhibition above 50 mmol/L), viability (over 250 mmol/L) and ethanol production (above 83 mmol/L). The concentrations of the other inhibitors had different limits of tolerance by the yeast... (Complete abstract click electronic access below) / Mestre