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Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:30Z No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:42Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Approved for entry into archive by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2015-06-30T13:13:53Z (GMT) No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) / Made available in DSpace on 2015-06-30T13:13:53Z (GMT). No. of bitstreams: 1 Dissertação_Livia.pdf: 2415864 bytes, checksum: 9fef5c6a16beacb29a63be5283790fd6 (MD5) Previous issue date: 2013 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde / Os produtos farmacêuticos que requerem a característica de esterilidade devem ser submetidos ao Ensaio de Esterilidade que deve ser realizado em áreas limpas, a fim de evitar resultados falso-positivos. A legislação brasileira recomenda a identificação de microrganismos provenientes dos Ensaios e do ambiente onde estes foram realizados. A dificuldade da identificação de vários gêneros bacterianos por metodologias fenotípicas têm sido relatada em vários estudos e mostram a necessidade da utilização de metodologias moleculares para esta finalidade. Neste estudo foi realizada a caracterização fenotípica (API e VITEK BioMerieux) e genotípica (análise da sequência do gene 16S rRNA) de 58 estirpes de cocos Gram positivos não fermentadores da glicose, provenientes de produtos farmacêuticos estéreis e ambiente controlado. O resultado da caracterização fenotípica realizada utilizando o sistema VITEK demonstrou que 100% das identificações foram equivocadas quanto ao gênero e espécie bacteriana. O sistema API identificou corretamente 69% das estirpes quanto ao gênero bacteriano quando comparado com a análise da sequência do gene 16S rRNA. Vinte e cinco estirpes foram submetidas ao sistema VITEK 2 e 68% dessas foram identificados corretamente quanto ao gênero bacteriano. A análise da sequência do gene 16S rRNA mostrou-se eficiente na determinação do gênero e mostrou a diversidade bacteriana deste grupo de organismos. Entre os cocos Gram positivos não fermentadores da glicose analisados foram identificados os gêneros Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter e Brachybacterium. Essa análise também mostrou que 8,6% das estirpes avaliadas podem representar espécies ainda não descritas. Esta metodologia possibilita a diferenciação de quase todas as espécies do gênero encontrado com mais frequência, o Micrococcus, exceto o Micrococcus yunnanesis e Micrococcus luteus. Essas espécies também não puderam ser diferenciadas pela análise da sequência de segmentos de genes conservados (rpoB, gyrB, groEL and recA). Os equívocos das identificações fenotípicas alertam para a necessidade da implementação de metodologias moleculares para concluir a identificação correta de estirpes bacterianas provenientes de testes de esterilidade e ambientes controlados. / Sterile pharmaceutical products must be submitted to sterility testing to be carried out in clean rooms, in order to avoid false positive results. Brazilian law recommends the identification of microorganisms from sterility tests and the environment where these tests were performed. It has been reported in several studies difficulty in identifying various genera using phenotypic methods. This suggests the need of molecular methods which are more suitable for this purpose. In this study we performed phenotypic (API and VITEK systems (BioMerieux)) and genotypic (sequence analysis of 16S rRNA) characterization of 58 strains of Gram positive cocci non-fermenting glucose, from pharmaceuticals sterile and controlled environment. The results of phenotypic characterization performed using the VITEK system showed that 100% of the identifications of bacterial genus and species were misleading. The API system correctly identified the bacterial genus of 69% of the strains compared with the sequence analysis of 16S rRNA. Twenty-five strains were identified with the Vitek 2 system and 68% of the strains were identified with the correct bacterial genus. Sequence analysis of 16S rRNA gene was effective in determining the bacterial genus and also showed bacterial diversity of this group of organisms. Among the glucose non-fermenting Gram-positive cocci analyzed, the identified genera were: Micrococcus, Kocuria, Demetria, Macrococcus, Arthrobacter, Dietzia, Janibacter and, Brachybacterium. This analysis also showed that 8.6% of the strains tested may represent species not yet described. This methodology allowed the differentiation of almost all species of the genus Micrococcus, except Micrococcus yunnanesis and Micrococcus luteus. These species were also not differentiated by sequence analysis of fragments of housekeeping genes (rpoB, gyrB, groEL and recA). The mistake phenotypic identifications highlight the need of the implementation of molecular methods to achieve the correct identification of bacterial strains from sterility testing and controlled environments.

Darbe nagrinėjama programinio kodo palyginimo, siekiant rasti funkcinius dublikatus, problema. Tikslas - sukurti bei pasiūlyti universalų trivialiems pakeitimams atsparų programinio kodo palyginimo metodą. Darbe aprašomi du pasirinkti medžių palyginimo algoritmai: tikslus medžių redagavimo atstumo ir apytikslis pq-gram, pasižymintis geresniu našumu. Šie algoritmai praktiškai pritaikyti Java programinio kodo palyginimui: parašytas AST parseris bei programa, lyginanti XML dokumentus arba Java klases. Darbe įvertintos galimos AST transformacijos bei eksperimentiškai parinktos programinio kodo palyginimui tinkamiausios p ir q parametrų reikšmės pq-gram algoritmui. Pasiūlytas pq-gram algoritmo patobulinimas, pq-gram indeksuose saugant maišos funkcijų grąžintus įrašų atvaizdus. / This paper investigates a problem of source code comparison with a goal of finding functional code duplicates. The goal of this paper is to create and present a universal source code comparison method, which would be resistant to trivial changes. Two algorithms were chosen to achieve this goal: exact tree edit distance and approximate pq-gram, which has better performance. Both algorithms were practically applied for Java code comparison: an AST parser and a program, which compares XML documents or Java classes, were created. This paper also evaluates AST transformations as well as presents experimental results of p and q values best suited for code comparison. Args4j library is used to evaluate the quality of source code comparison results.

Bacterial endosymbionts occur in diverse fungi, including members of many lineages of Ascomycota that inhabit living plants. These endosymbiotic bacteria (endohyphal bacteria, EHB) often can be removed from living fungi by antibiotic treatment, providing an opportunity to assess their effects on functional traits of their fungal hosts. We examined the effects of an endohyphal bacterium (Chitinophaga sp., Bacteroidetes) on substrate use by its host, a seed-associated strain of the fungus Fusarium keratoplasticum, by comparing growth between naturally infected and cured fungal strains across 95 carbon sources with a Biolog((R)) phenotypic microarray. Across the majority of substrates (62%), the strain harboring the bacterium significantly outperformed the cured strain as measured by respiration and hyphal density. These substrates included many that are important for plant-and seed fungus interactions, such as D-trehalose, myoinositol, and sucrose, highlighting the potential influence of EHB on the breadth and efficiency of substrate use by an important Fusariurn species. Cases in which the cured strain outperformed the strain harboring the bacterium were observed in only 5% of substrates. We propose that additive or synergistic substrate use by the fungus bacterium pair enhances fungal growth in this association. More generally, alteration of the breadth or efficiency of substrate use by dispensable EHB may change fungal niches in short timeframes, potentially shaping fungal ecology and the outcomes of fungal-host interactions.

A unique undosciribed gramnegative rod is extensively characterized in this study. The cells of this unusual water isolate measure 1.2 X 6.5 microns, The most distinguishing characteristic of the bacterium is a polar tuft of 35-40 flagella that aggregate to function as a single organelle which is visible under phase contrast. Aging cells deposit poly- -hydroxybutyric acid granules which are bound by an inclusion membrane made up of four distinct layers. It also possesses an unusual exterior membrane outside the cell wall which contains large fibrils of protein running at a slight angle to the longitudinal axis of the cell. The guanosine-cytosine ratio was found to be 62.2$. The organism's taxonomic position was further investigated by immunological, morphological, and biochemical methods. It was found to be most closely akin to members of the genus Pseudo onas, although somewhat divergent from other species classified in this genus. After careful evaluation of the findings obtained during this study, the new bacterium was subsequently named Pseudomonas multiflagella.

Orientador: Elisa Helena Giglio Ponsano / Resumo: O objetivo deste estudo foi investigar a composição química e as atividades antimicrobiana e antioxidante dos extratos etanólicos de Curcuma longa e Bixa orellana, na busca por substituintes aos aditivos sintéticos utilizados na indústria de alimentos. Pela espectrometria de massa (GC-MS) foram identificados bisdemetoxicurcumina, demetoxicurcumina e curcumina no extrato de C. longa e prunina e naringenina no extrato de B. orellana. C. longa apresentou atividade antimicrobiana frente a Clostridium sporogenes e Staphylococcus aureus, com concentração bactericida mínima (CBM) de 25 mg/mL e 156 µg/mL, respectivamente. O extrato de B. orellana apresentou CBM de 50 mg/mL para C. sporogenes e 625 µg/mL para S. aureus. Nenhum dos extratos apresentou atividade bactericida para Escherichia coli e Salmonella Typhimurium. A atividade antioxidante dos extratos foi evidenciada pelos métodos Poder Antioxidante por Redução Férrica (FRAP) e Capacidade de Absorção do Radical Oxigênio (ORAC). O extrato de B. orellana apresentou maior atividade antioxidante pelos métodos FRAP e ORAC (277,70 e 455,17 mM trolox equivalente/g, respectivamente) do que o extrato de C. longa (129,74 e 217,98 mM trolox equivalente/g, respectivamente). Os efeitos biológicos dos extratos etanólicos de C. longa e B. orellana revelados no presente estudo apontaram seu potencial para a utilização na indústria de alimentos como uma alternativa aos aditivos sintéticos. / Abstract: The objective of this study was to investigate the chemical composition and the antimicrobial and antioxidant activities of Curcuma longa and Bixa orellana ethanolic extracts, in the search for alternatives to the synthetic additives used in the food industry. Mass spectrometry (GC-MS), identified bisdemethoxycurcumin, demethoxycurcumin and curcumin in the extract of C. longa and prunin and naringenin in the extract of B. orellana. C. long showed antimicrobial activity against Clostridium sporogenes and Staphylococcus aureus, with a minimum bactericidal concentration (MBC) of 25 mg/mL and 156 μg/mL, respectively. MBC of B. orellana extract was 50 mg/mL for C. sporogenes and 625 μg/mL for S. aureus. None of the extracts showed bactericidal activity against Escherichia coli and Salmonella Typhimurium. The antioxidant activity of the extracts was evidenced by the methods Iron Reduction Antioxidant Power (FRAP) and Oxygen Radical Absorption Capacity (ORAC). B. orellana extract had higher antioxidant activity by FRAP and ORAC (277.70 and 455.17 mM trolox equivalent/g, respectively) than C. longa extract (129.74 and 217.98 mM trolox equivalent/g, respectively). The biological effects of C. longa and B. orellana ethanolic extracts revealed in this study indicated their potential as an alternative to synthetic additives used in the food industry. / Mestre

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-05-16T17:06:24Z No. of bitstreams: 1 DIS_ANDRE_DA_LUZ_PEREIRA_COMPLETO.pdf: 705105 bytes, checksum: f0e5dcb2aeafdd39832d8260d516c466 (MD5) / Made available in DSpace on 2017-05-16T17:06:24Z (GMT). No. of bitstreams: 1 DIS_ANDRE_DA_LUZ_PEREIRA_COMPLETO.pdf: 705105 bytes, checksum: f0e5dcb2aeafdd39832d8260d516c466 (MD5) Previous issue date: 2017-01-20 / The aim of this work is to compare syntactic approaches to wh-questions, which is based on the generative perspective developed in linguistic studies from the 1950s onwards (CHOMSKY, 1957, 1965, 1972, 1977a, 1981, 1986, 1995). The central point of the comparison is relevance of the notion of movement for the syntactic studies of wh-questions. The mainstream of generative grammar uses the notion of movement to explain syntactic phenomena, and the Simpler Syntax Hypothesis believes that this notion is unnecessary, since there would be a simpler explanation (CULICOVER; JACKENDOFF, 2005, 2006). Specifically, the explanations about the construction of wh-question structures will be compared by the Simpler Syntax Hypothesis, which presents the alternative concepts to the concept of movement to describe the phenomenon of wh-questions, such as indirect licensing and discontinuous dependencies. The idea of movement is an important axis in the mainstream generative theory, since it evolves from the theory of traces, presented in the 1970s, but which gains strength and consolidates itself as movement theory especially from the Principles and Parameters (PP) program in the 1980s. Although words such as displacement and movement have already been mentioned in the course of the generative enterprise, they are consolidated in the PP program and remain in the Minimalist Program (PM), the next stage of the generative enterprise begun in the 1990s. Another aspect identified in the development of the research was the parallel architecture of the language structure proposed by the Simpler Syntax, which challenges the central role of syntax in linguistic studies, sharing with semantics and phonology the responsibility for generating structures from Conceptual Structure. To achieve the proposed objectives, a hypothetical-deductive model was used, in which the premises proposed by the theoretical lines under study are considered. The examples and sentences analyzed were not generated in situations of use by speakers. They were extracted from the theoretical sources analyzed or generated by the author. / Tendo por base a perspectiva gerativista, que se desenvolve nos estudos lingu?sticos a partir da d?cada de 1950 (CHOMSKY, 1957, 1965, 1972, 1977a, 1981, 1986, 1995), este trabalho tem por objetivo comparar abordagens sint?ticas sobre interrogativas-QU (em ingl?s, whquestions). O ponto central da compara??o ? relev?ncia da no??o de movimento para os estudos sint?ticos de interrogativas-QU. A Tradi??o da Gram?tica Gerativa se utiliza da no??o de movimento para explicar os fen?menos sint?ticos, e a Hip?tese da Sintaxe mais Simples acredita que esta no??o seja desnecess?ria, j? que haveria uma explica??o mais simples (CULICOVER; JACKENDOFF, 2005, 2006). Especificamente, ser?o comparadas as explica??es sobre a constru??o de estruturas interrogativas-QU pela hip?tese da Sintaxe mais Simples, que apresenta os conceitos alternativos ao conceito de movimento para descrever o fen?meno das interrogativas-QU, como licenciamento indireto e depend?ncias descont?nuas. A ideia de movimento ? um eixo importante na tradi??o da teoria gerativista, pois evolui a partir da teoria dos vest?gios, apresentada na d?cada de 1970, mas que ganha for?a e se consolida como teoria do movimento especialmente a partir do programa de Princ?pios e Par?metros (PP), na d?cada de 1980. Embora palavras como deslocamento e movimento j? tenha sido mencionadas anteriormente no percurso do empreendimento gerativista, elas se consolidam no PPP e se mant?m no Programa Minimalista (PM), etapa seguinte do empreendimento gerativista, iniciado na d?cada de 1990. Outro aspecto identificado no desenvolver da pesquisa foi a arquitetura paralela da estrutura da linguagem proposta pela Sintaxe mais Simples, que contesta o papel central da sintaxe nos estudos lingu?sticos, compartilhando com a sem?ntica e com a fonologia, a responsabilidade por gerar estruturas a partir do conceito de Estrutura Conceitual. Para atingir os objetivos propostos, foi utilizado um modelo hipot?tico-dedutivo, em que s?o consideradas as premissas propostas pelas linhas te?ricas em estudo. Os exemplos e senten?as analisadas n?o foram gerados em situa??es de uso por falantes. Foram extra?dos das fontes te?ricas analisadas ou geradas pelo autor.

The rise of antimicrobial resistance (AMR) has culminated in the most pressing problem in modern medicine. The situation is most acute with regards to the management of multi- drug resistant Gram-negative infections (MDRGNB) with common infections increasingly untreatable due to rapidly dwindling therapeutic options. A solution to the problem of AMR is unlikely to be easily found, but revisiting and re-purposing existing antimicrobials is a viable approach in the medium term. This study investigated the use of unorthodox antimicrobial combination therapies for the treatment of MDRGNB, with particular focus on agents of last resort. A systematic review of clinical studies highlighted the potential for polymyxin (colistin) combination therapies (e.g. colistin-rifampicin, colistin-carbapenems), although this could not be supported in a formal meta-analysis. A systematic approach for screening MDRAB for susceptibility to novel colistin combinations using multiple methods was employed and uncovered a number that were more potent than those previously identfied. The most potent combination that was consistently identified was colistin when combined with fusidic acid, despite this drug having no useful activity against MDRGNB on its own. The combination was further evaluated in static time-kill assays against a range of Gram-negative pathogens with defined resistance mechanisms, including to polymyxins and using invertebrate (Galleria mellonella) and murine models of MDRGNB infection. Colistin and fusidic acid combination therapy was subsequently used to successfully treat a case of ventilator-associated pneumonia due to MDR A. baumannii. This work highlights how older drugs can be re-purposed to tackle the problem of AMR using a precision medicine approach. Further studies to elucidate the mechanism of action of the colistin- fusidic acid combination and a formal clinical trial are warranted to investigate the potential utility of this combination in the treatment of MDRGNB with the expressed goal of bridging the current antimicrobial development gap.

Acidithiobacillus ferrooxidans is a gram-negative bacterium that is able to extract energy from oxidation of Fe²⁺ and reduced sulfur compounds and fix carbon dioxide from atmosphere. The facts that A. ferrooxidans thrives in acidic pH (~2), fixes carbon dioxide from the atmosphere and oxidizes Fe²⁺ for energy make it a good candidate in many industrial applications such as electrofuels and biomining. Electrofuels is a new type of bioprocess, which aims to store electrical energy, such as solar power, in the form of chemical bonds in the liquid fuels. Unlike traditional biofuels made from agricultural feedstocks, electrofuels bypass the inefficient photosynthesis process and thus have potentially higher photon-to-fuel efficiency than traditional biofuels. This thesis covers the development of a novel bioprocess involving A. ferrooxidans to make electrofuels, i.e. isobutyric acid and heptadecane. There are four major steps: characterization of wild-type cells, engineering of medium for improved electrochemical performance, genetic modification of A. ferrooxidans and optimization of operating conditions to enhance biofuel production. Each is addressed in one of the chapters in this thesis. In addition, applications of A. ferrooxidans in biomining processes will be briefly discussed. An economic analysis of various applications including electrofuels and biomining is also presented. Wild-type A. ferrooxidans were first characterized in both batch and continuous cultures. A modified 9-K medium suggested by American Type Culture Collection (ATCC) was used as a starting point which has 72 mM Fe²⁺ at pH 1.8. The Fe²⁺ concentration and pH were varied in the experiments to assess their impacts on growth rate, cell yield (g cells/g Fe²⁺) and maintenance (energy used to keep cell viability). Citrate was added to the growth medium to dissolve precipitates which can be problematic in a continuous operation. It was found out that cells exhibited higher cell yield (g cells/g Fe²⁺) and lower maintenance with higher pH and addition of citrate. This indicates that cells grow in a more energy-efficient manner at such conditions since cells spend less energy in maintenance and more energy in biomass formation. Next the growth medium containing 72 mM Fe³⁺ and 70 mM citrate at pH 2.2 was explored during the electrochemical reduction of Fe³⁺. It turned out that electrochemical reduction of Fe³+ could not be carried out effectively due to a low electrolyte conductivity and low energy density of the medium. Citrate was also found to negative affect electrochemical performance due to a strong complexation with Fe³⁺. The conductivity was improved by adding 500 mM Mg²⁺ to the medium. Vanadium was used as an alternative redox mediator that has a much better solubility than Fe³⁺ to improve the energy density. Genetic modification was achieved by introducing genes from two foreign pathways i.e. valine synthesis and fatty acid synthesis into A. ferrooxidans to enable cells to produce either isobutyric acid (IBA) or heptadecane. Transformed cells were characterized based on the findings in wild-type cells. Isobutyric acid production was found to increase with increasing pH and Fe²⁺ concentration and addition of citrate. Further optimization of the growth medium was done by increasing Fe²⁺ to 288 mM, holding pH at 2.2 and using gluconate as the iron chelator instead of citrate. An economic analysis was performed on the electrofuel process and applications of genetically modified A. ferrooxidans in copper biomining processes. At electricity prices of $0.05/kWh, further improvement in biological efficiency needs to be achieved before the electrofuel process may become economically viable. The use of genetically modified cells in copper biomining process could open new opportunities to co-produce valuable chemicals and copper from the reduced material associated with the copper ores. The chemicals co-produced during copper processing could be sold for additional revenue or used on-site.

Orientador: Julieta Rodini Engracia de Moraes / Coorientador: Jair Rodini Engracia Filho / Banca: Rogério Salvador / Banca: Gilson Pereira de Oliveira / Resumo: Na natureza os bijupirás estão sujeitos à infecção por bactérias, vírus, parasitos e às doenças de origem nutricional. No entanto, dados a respeito dessas doenças em bijupirás em cativeiro são escassos no Brasil e em outros países. As infecções podem causar grandes perdas econômicas, seja pela redução no crescimento, custo de tratamento e morte dos peixes. O presente trabalho teve como objetivo caracterizar os aspectos ictiopatológicos de bijupirás (Rachycentron canadum) criados em tanques-rede no litoral norte do estado de São Paulo (23°48'54"S 45°22'14"O) durante o ano de 2011. As investigações avaliaram a ocorrência de doenças caracterizando sinais clínicos e achados anatomopatológicos de 46 bijupirás coletados nos meses de março, maio, julho e setembro de 2011. Nos resultados do exame bacteriológico foram caracterizados e isolados micro-organismos compatíveis com a bactéria gram-negativa, Photobacterium damselae piscicida. No exame parasitológico foram identificados Neobenedenia melleni e Tuxophorus caligodes. Na análise histopatológica, órgãos como fígado, rins, baço, intestino e cecos pilóricos apresentaram lesões granulomatosas. No fígado e rins prevaleceram lesões como esteatose e degeneração tubular, respectivamente. No baço e nos intestinos foram identificados centros de melanomacrófagos e enterite crônica. As brânquias exibiram áreas de hiperplasia epitelial acompanhada de fusão lamelar, edema subepitelial e proliferação de células mucosas no ápice das lamelas primárias. Neste período houve 97,21% mortalidade atribuída à infecção por Photobacterium damselae piscicida, caracterizada e isolada pelos exames microbiológico e molecular / Abstract: In nature cobias are subject to infection by bacteria, viruses, parasites and diseases of nutritional origin. However, data about these diseases in cobias in captivity are rare in Brazil and other countries. The infections can cause major economic losses, either by reduced growth, cost of treatment and death of fish. The objective of this current study was to characterize ictiopathological aspects of cobias (Rachycentron canadum) farmed in net tanks in north coast of state of São Paulo (23°48'54"S 45°22'14"W) during the year 2011. The research evaluated the occurrence of diseases characterized clinical signs and pathological findings of 46 cobias collected in March, May, July and September 2011. In the results of the bacteriological analysis were characterized and isolated micro-organisms compatible with gram-negative bacterium, Photobacterium damselae piscicida. In parasitological examination was identified Neobenedenia melleni and Tuxophorus caligodes. In the histopathological analysis, organs like liver, kidneys, spleen, intestine and pyloric caecum showed granulomatous lesions. In the liver and kidney prevailed lesions as tubular degeneration and steatosis, respectively. In the spleen and intestines were identified melanomacrophages centers and chronic enteritis. The gills exhibited areas of epithelial hyperplasia accompanied fusion lamellar, subepithelial edema and proliferation of mucosal cells at the apex of the primary lamellae. In this period there was 97,21% mortality attributed to infection by Photobacterium damselae piscicida, isolated and characterized by molecular and microbiological examinations / Mestre