Submitted by William Justo Figueiro (williamjf) on 2015-07-06T23:27:47Z No. of bitstreams: 1 09a.pdf: 771261 bytes, checksum: c8d36f36fd0e9b6396c5bad8e1ea3681 (MD5) / Made available in DSpace on 2015-07-06T23:27:47Z (GMT). No. of bitstreams: 1 09a.pdf: 771261 bytes, checksum: c8d36f36fd0e9b6396c5bad8e1ea3681 (MD5) Previous issue date: 2014 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / UNISINOS - Universidade do Vale do Rio dos Sinos / Microrganismos vivendo no interior dos tecidos das plantas sem causar efeitos prejudiciais visíveis de sua presença são chamados endofíticos. As bactérias endofíticas podem promover o crescimento das plantas de várias maneiras: através da secreção de reguladores de crescimento vegetal, pela solubilização de fosfato, entre outras. Na busca de práticas agrícolas ambientalmente sustentáveis, tem sido dada considerável atenção à fixação biológica de nitrogênio atmosférico. Assim, o objetivo do presente estudo foi avaliar, através de métodos dependentes de cultivo, a ocorrência de bactérias endofíticas em colmos e folhas de duas amostras das variedades “RG 121” e “RG 900” de arroz irrigado, num sistema de cultivo convencional. Foram coletadas plantas de arroz de duas variedades na Estação Experimental do Arroz, do Instituto Rio Grandense do Arroz (EEA-IRGA), Cachoeirinha/RS, no ano agrícola 2012/2013. As plantas coletadas em campo foram submetidas à desinfecção superficial, seguida do isolamento e cultivo das bactérias endofíticas em meios de cultura semi-sólidos, livres de nitrogênio: NFb (Azospirillum brasilense/A. lipoferum), JNFb (Herbaspirillum seropedicae/H. rubrisubalbicans) e LGI-P (Gluconacetobacter diazotrophicus). Ao final desses procedimentos, foram obtidos 190 isolados bacterianos, os quais agruparam-se, de acordo com suas características fenotípicas, em 29 morfotipos. A partir disso, o padrão de distribuição dos morfotipos entre oito condições foi avaliado por Análise de Correspondência Destendenciada (DCA). As oito condições foram: C1 = amostra (planta) 1, variedade “RG 121”, colmos; C2 = amostra 1, variedade “RG 121”, folhas; C3 = amostra 1, variedade “RG 900”, colmos; C4 = amostra 1, variedade “RG 900”, folhas; C5 = amostra (planta) 2, variedade “RG 121”, colmos; C6 = amostra 2, variedade “RG 121”, folhas; C7 = amostra 2, variedade “RG 900”, colmos; C8 = amostra 2, variedade “RG 900”, folhas. Os dois primeiros eixos da DCA explicaram 28,5 % da variância total do conjunto de dados - 23,9 % de explicação somente pelo eixo 1. Ainda, foi realizada uma Análise de Componentes Principais (PCA) na tentativa de estabelecer quais morfotipos melhor explicam as diferenças observadas entre as condições. Deste modo, os eixos 1 e 2 da PCA tiveram uma proporção de explicação acumulada correspondente a 45,61 %, sendo o eixo 1 responsável por 25,91 % destes. Por fim, em função do número de isolados obtidos por grupo fenotípico, pôde-se calcular o índice de diversidade de Shannon-Weaver (H’), que relaciona a riqueza e uniformidade de espécies (morfotipos) para cada área (condição) em estudo. Estes foram testados par-a-par pelos métodos de bootstrapping e permutação quanto a possíveis diferenças significativas. De modo que valores de H’ estatisticamente diferentes apresentados por algumas das combinações de condições puderam ser relacionados à amostra (planta), à estrutura vegetal (colmos e folhas) e à variedade, isolada ou conjuntamente. O crescimento bacteriano em meios de cultura livres de nitrogênio, em condições microaerofílicas, infere a ocorrência da capacidade de fixação biológica de nitrogênio. / Microorganisms living within the tissues of plants without causing visible detrimental effects of their presence are known as endophytes. The endophytic bacteria can promote plant growth in various ways: by secreting plant growth regulators, by phosphate solubilization, among others. In pursuit of environmentally sustainable agricultural practices, considerable attention has been given to the biological nitrogen fixation. Thus, the aim of this study was to evaluate, by culture-dependent methods, the occurrence of endophytic bacteria in stems and leaves of two samples of varieties “RG 121” e “RG 900” of irrigated rice, on a conventional cropping system. Rice plants from two varieties were collected in the Estação Experimental do Arroz, from Instituto Riograndense do Arroz (EEA-IRGA), Cachoeirinha/RS, in the agricultural year 2012/2013. Plants collected in the field were submitted to surface disinfection, followed by isolation and cultivation of endophytic bacteria in semisolid, nitrogen-free, culture media: NFb (Azospirillum brasilense/A. lipoferum), JNFb (Herbaspirillum seropedicae/H. rubrisubalbicans) and LGI-P (Gluconacetobacter diazotrophicus). At the end of these procedures, 190 bacterial isolates were obtained, which were grouped, according to their phenotypic characteristics, in 29 morphotypes. From this, the pattern of distribution of morphotypes in eight conditions was assessed by Detrended Correspondence Analysis (DCA). The eight conditions were: C1 = sample (plant) 1, variety "RG 121", stems; C2 = sample 1, variety "RG 121", leaves; C3 = sample 1, variety "RG 900", stems; C4 = sample 1, variety "RG 900", leaves; C5 = sample (plant) 2, variety "RG 121", stems; C6 = sample 2, variety "RG 121", leaves; C7 = sample 2, variety "RG 900", stems; C8 = sample 2, variety "RG 900", leaves. The first two axes of DCA explained 28.5 % of the total variance of the data set - 23.9 % of explanation only by axis 1. In addition, a Principal Components Analysis (PCA) was performed in attempt to establish which morphotypes best explain the observed differences between the conditions. Thus, the axes 1 and 2 of the PCA had a proportion of accumulated explanation corresponding to 45.61 %, being the axis 1 responsible for 25.91 % of them. Finally, according to the number of isolates obtained by phenotypic group, the Shannon-Weaver diversity index (H’) were calculated, which relates the richness and evenness of species (morphotypes) for each area (condition) in study. These were tested pair-to-pair by bootstrapping and permutation methods for possible significant differences. Thus, statistically different values of H' presented by some of the combinations of conditions could be related to the sample (plant), plant structure (stems and leaves) and variety, individually or together. The bacterial growth in nitrogen-free culture media, under microaerophilic conditions, implies the occurrence of biological nitrogen fixation capacity.

O gênero Burkholderia é composto por bactérias de elevada diversidade fisiológica e genética, sendo alvo para uma série de aplicações biotecnológicas. A linhagem Burkholderia seminalis TC3.4.2R3 foi capaz de controlar fungos e bactérias patogênicas, bem como produzir antibióticos. A análise de perfil fisiológico da planta hospedeira indica que não há a elicitação de respostas de defesa no controle dos sintomas da podridão mole por B. seminalis. Foi observado que o sucesso do controle depende do contato direto do agente de biocontrole e o patógeno B. gladioli, e que B. seminalis possui atividade antimicrobiana in vitro contra com a formação de um halo de inibição e redução de 40% da densidade de B. gladioli em co-cultura líquida com B. seminalis. A análise do mutante M01 defectivo no controle da podridão mole, que apresenta a região intergênica da patatina-ferritina nocauteada pelo transposon, não mostrou diferenças significativas comparado ao isolado selvagem para os mesmos ensaios. / The genus Burkholderia is composed of bacteria with high physiological and genetic diversity, and it is being goal for a number of biotechnological applications. The Burkholderia seminalis TC3.4.2R3 strain was able to control fungal and bacterial pathogens as well as produce antibiotics. The physiological profile analysis of the host plant indicates that there is no defense responses elicitation induced by B. seminalis involved in controlling symptoms of soft rot. It was observed that the success of control depends on the full contact of the biocontrol agent and the pathogen B. gladioli. B. seminalis has an antimicrobial activity in vitro showing a inhibition halo and reducing the density of B. gladioli in co-culture with B. seminalis by 40%. The M01 mutant, wich is defective in control of soft rot and showing a insertion of transposon in a intergenic region between patatin-ferritin, presented no significant differences compared to the wild strain for the same assays.

Foram avaliados 25 reservatórios de água dos equipos odontológicos da Clínica de Dentística/Endodontia da FOB/USP com relação à presença de micro-organismos e a ação do ultrassom (US) na remoção do biofilme. Amostras de 10ml de água foram obtidas e alíquotas de 25l in natura e diluída até 10-4 foram semeadas pela técnica da gota nos meios: R2A Agar (R2A), Plate Count Agar (PCA), Peptona Diluída (PD) e Sabouraud Dextrose Agar com cloranfenicol a 1% (SDA), incubadas a 24º C por 72 horas. A água dos reservatórios foi descartada e 500 ml de água destilada esterilizada foi adicionada, sendo submetidos à ação do ultrassom (US) por 15 minutos, seguidos do mesmo procedimento descrito anteriormente. As colônias de bactérias foram quantificadas e os fungos foram identificados por micro-cultivo. A média da detecção de UFC/ml antes e após o US foi de 173.787 e 15.841 para o R2A, 104.873 e 3.034 para o PCA e de 245.824 e 8.231 para o PD. A média de fungos foi de 52,4 antes e 19,2 UFC/ml após ação do US. Fungos foram detectados em 20 reservatórios antes e em 12 deles após uso do US. O Penicillium sp apresentou prevalência de 36% nos reservatórios de água avaliados. Os resultados obtidos permitem concluir que o US foi eficiente em desestruturar o biofilme, embora não o elimine por completo, apresentando maior efetividade na desestruturação de bactérias. / A total of 25 waterline unit reservoirs of the odontological sets from the Dentistry/Endodontic Clinic of FOB/USP were assessed, in relation to the presence of microorganisms and the ultrasound action (US) on the biofilm removal. Waterline samples of 10ml were obtained from aliquots of 25l in natura and diluted until 10-4, then, they were spread using the dripping technique on the means: R2A Agar (R2A), Plate Count Agar (PCA), diluted Peptone (DP) and Sabouraud Dextrose Agar with cloranfenicol at 1% (SDA), being incubated at 24º C for 72 hours. The waterline units of the reservoirs were discarded and 500 ml of sterilized distilled water was added, submitted to ultrasound action (US) for 15 minutes, following the same procedure described afore. The bacteria colonies were quantified and the fungi were identified through micro-culture. The average of detection of UFC/ml before and after US was 173.787 and 15.841 for R2A, 104.873 and 3.034 for PCA and of 245.824 and 8.231 for PD. The fungi average was 52,4 before and 19,2 UFC/ml after the action of US. Fungi were detected in 20 reservoirs before and 12 after using US. Penicillium sp showed a prevalence of 36% in the waterline reservoirs assessed. The results obtained, led to the conclusion that US was efficient to break the structure of the biofilm, although it did not eliminate it completely, showing more effectiveness to break the bacteria structure.

Submitted by (edna.saturno@ufrpe.br) on 2017-03-22T13:36:02Z No. of bitstreams: 1 Andrezza Araujo de Franca.pdf: 529792 bytes, checksum: 4398977026fd32c6a9f3e713e306c916 (MD5) / Made available in DSpace on 2017-03-22T13:36:02Z (GMT). No. of bitstreams: 1 Andrezza Araujo de Franca.pdf: 529792 bytes, checksum: 4398977026fd32c6a9f3e713e306c916 (MD5) Previous issue date: 2007-02-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Aiming to relate the structural components of cell wall with its degradability, the chemical composition, the secondary compounds, the in situ degradability, anatomy and tissue degradability of hay of “maniçoba” (wild cassava) were evaluated. The stem showed cells with varied degrees of lignification, highlighting the presence of gelatinous fibers, pith parenchyma lignified and tick cell walls inside the xylem. The leaves were highlighted by the presence of a girder structure, which can influence the degradability; it is characterized by the great quantity of mesophyll, constituted by cells with thin walls, contributing for degradability of dry matter. Idioblasts with druses of oxalate were observed around the vascular tissues, in the midrib. It works like defense mechanisms of plant against herbivores and can affect the availability of minerals for animals. The “maniçoba” hay, in spite of its advanced maturity stage (early fruit development), present adequate chemical composition and low concentration of HCN and tannins. The limits mains to degradability are wall cell thickness and lignifications, particularly in tissue of stem. Additionally, several aspects here reported induce to the continuity of researches in several focuses and aim the improvement to use this specie as forage. / Objetivando relacionar os componentes estruturais da parede celular com sua degradabilidade, avaliou-se a composição química, teor de compostos secundários, degradabilidade in situ, anatomia e degradabilidade dos tecidos do feno de maniçoba. O caule apresentou células com variados graus de lignificação, destacando-se a presença de fibras gelatinosas, parênquima medular lignificado e espessas paredes celulares no xilema. As folhas se destacam pela presença da estrutura girder, a qual pode influenciar a degradabilidade se caracterizam pela grande quantidade de mesofilo, constituído por células com paredes delgadas, contribuindo para degradabilidade de matéria seca. Idioblastos contendo drusas de oxalato foram encontrados nos tecidos vasculares, na nervura principal da folha. Eles funcionam como mecanismos de defesa do vegetal contra herbívoros e podem afetar a disponibilidade de minerais para o animal. O feno de maniçoba, apesar de obtido de planta em avançado estágio de maturidade (início da frutificação), possui adequada composição química e baixos teores de HCN e taninos. Os principais limitantes à degradabilidade são o espessamento e lignificação das paredes celulares, especialmente nos tecidos do caule. Adicionalmente, os diversos aspectos aqui relatados induzem à continuidade de pesquisas em diversos focos e visam o melhoramento e a utilização desta espécie como forrageira.

Os pacientes com Síndrome de Down (SD) possuem grande incidência de doença periodontal (DP), caracterizada por um curso precoce e com maior severidade. O estudo de metaboloma pode contribuir para o entendimento deste curso da doença, identificando possíveis metabólitos como biomarcadores nestes indivíduos. Para entender o perfil metabolômico dos indivíduos com síndrome de Down e a sua relação com a doença periodontal, realizamos a identificação de metabólitos salivares de adolescentes e adultos jovens, entre 12 e 21 anos, ambos os gêneros. Foram coletados dados sobre o estado geral de saúde e realizados exames clínicos bucais, como índice de higiene oral simplificado, sangramento e profundidade de sondagem. Para a análise do metaboloma foi coletada amostra de saliva não estimulada, analisadas por meio de cromatografia gasosa acoplada á espectrometria de massas. Saliva e fluido crevicular gengival também foram coletados para identificação microbiana através do MALDI-TOF. Os dados encontrados foram submetidos a análise estátisca por meio da Análise dos Componentes Principais (PCA) e quantificação relativa dos metabólitos foi avaliada por testes não paramétricos, Mann-Whitney e Kruskal-Wallis. Foi possível observar através dos modelos de PCA separação dos indivíduos com SD e controles, independente da doença periodontal. A quantificação relativa revelou maiores níveis de glicina, lprolina, l-leucina, l-serina, ácido palmítico, ácido pentanóico, ácido tetradecanóico, tirosina e l-fenilalanina nos grupos SD quando comparados aos controles. Controles com DP também apresentaram níveis elevados de glicina, l-alanina, l-serina e manopiranose quando comparados com controles saudáveis. A microbiota de indivíduos com SD apresentous diferenças siginificantes em relação aos individuos controles, principalmente para Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia quando avaliado a saliva e A. Actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola no fluido crevicular gengival. Em conclusão, o perfil metabolômico impresso nos indivíduos com SD difere significativamente dos indivíduos controles, independente da doença periodontal. Entretanto, os metabólitos que diferenciam indivíduos controles com e sem DP, apresentam-se elevados em todos indivíduos com SD, promovendo novos \"insights\" para o perfil metabólico relacionado a DP na SD. / Down Syndrome (DS) patients have a high incidence of periodontal disease (PD), characterized by an early course and greater severity. The metabolome study may contribute to the understanding of the disease course, identifying possible metabolites as biomarkers in these individuals. To understand the metabolomic profile of the DS and their relationship with PD, we conducted the identification of salivary metabolites of adolescents and young adults between 12 and 21 years, both genders. Data were collected on general health and was performed oral clinical examination, as the IHOS, bleeding index and probing depth. For metabolome analysis was collected unstimulated saliva sample, analyzed by gas chromatography coupled to mass spectrometry. Saliva and gingival crevicular fluid were also collected for microbial identification by MALDI-TOF. Data were submitted to analysis-statistic by PCA and relative quantification of metabolites was evaluated by Mann-Whitney and Kruskal-Wallis tests. It can be observed through the PCA models separation of DS groups and controls groups, regardless of periodontal disease. Relative quantification showed higher levels of glycine, L-proline, L-leucine, L-serine, palmitic acid, pentanoic acid, tetradecanoic acid, tyrosine and L-phenylalanine in the SD groups when compared to controls groups. Controls with PD also showed high levels of glycine, L-alanine, L-serine and mannopyranose compared with healthy controls. The microbiota of individuals with DS groups show significant differences compared to control groups, especially for Rothia dentocariosa, Staphylococcus epidermidis, Tannerella forsythia when evaluated saliva and A. actinomycetemcomitans, Micrococcus luteus, Rothia aeria, Treponema denticola in gingival crevicular fluid. In conclusion, the printed metabolomic profile in individuals with Down syndrome differs significantly from control subjects, regardless of periodontal disease. However, the metabolites that distinguish controls group with and without PD, show up high in all DS individuals, promoting new \"insights\" to the metabolic profile related to PD in DS.

Drinking water is produced from raw water and is either from groundwater or surface water. This thesis aims to find out if the cleaning process of raw water is sufficiently effective. This is important because consumers are otherwise at risk of waterborne infection caused by pathogens. There are three groups of pathogens; bacteria, virus and parasite. These have different characteristics which mean that they require different water treatment to be separated. In addition to normal operation, a number of scenarios were examined. This is to investigate how water treatment would do if they became a reality. The thesis has examined Borg´s waterworks operated by Norrköping Vatten AB. It was defined to cover the distance from water source to the consumer. In the work, the model Quantitative Microbial Risk Assessment (QMRA) was used to perform risk analysis by simulating the normal operation and different scenarios of the water purification process. Thus, knowledge can be obtained about the effectiveness of separation by bacteria, viruses and parasites. However, the QMRA-model is considered to contain some flaws and for that reason the Norwegian model called Good Disinfection Practice (GDP) was also used. GDP is a theoretical model which is based on formulas and tables. The model takes into account the raw water quality and also provides deductions for various measures that the water plant possesses to ensure a good supply of water. The results obtained with both models were similar and showed that the water treatment is sufficient for the bacteria, but not viruses and parasites. Both models were considered to be reliable but viruses and parasites are very difficult to analyze, which has resulted in uncertain literature values and hence in the results. The result also showed that neither viruses nor parasites exceeded the limit by so much that more hygienic barriers to the reduction of them are necessary. The conclusion which may be drawn from the fact that no parasites have been detected in the raw water is that the water treatment still might be sufficient. To determine the effects that an exclusion of various barriers may give, the normal operation was simulated and a purification step at a time was excluded. The result showed that the purification steps which are most important to maintain the treatment process are chemical precipitation followed by rapid filtration, slow filtration and disinfection with chlorine. If any of these cleaning steps were to fail, this introduces a large increase in the risk of waterborne disease. The results showed that the chemical precipitation step gave the greatest separation effect on the virus but also on the parasites. However, the slow filtration gave the largest separation of the parasites. Free chlorine had the greatest effect on bacteria. The investigated scenarios were assumed to be wastewater discharges, sewage discharges in relation to flood the nearby pastures, and sewage overflows due to heavy rainfall. The results of the simulated scenarios were the same when it was only bacteria that in all cases produced a result within the limits of the daily infection probability. Both viruses and parasites exceeded both values. However, there were few studies on these and thus literature values needed to be implemented in the QMRA-model. Hence, the uncertainty of the results was great. The QMRA-model also contained deficiencies in the simulation of the discharge of effluents, where the amount of virus was about 1000-10000 times too much. If this problem as well as more specific data for the investigated area, and more Swedish studies were available, a more credible simulation of the scenarios could be implemented.

During a volcanic eruption, the extrusion of lava onto surfaces destroys biological activity creating virgin land surfaces. Through time this new land will be subject to soil formation and colonisation under relatively similar climatic conditions and parent materials. Soils formed from volcanic deposits present a unique opportunity to study microbial community development. Soils at different developmental stages and differing in vegetation cover were selected from four locations on the slopes of Mount Etna, Sicily. Three main research objectives were determined in order to test the hypothesis that the microbial communities from soils at later stages of development would have a greater biomass, be more diverse, be more efficient at utilising carbon sources and recover from an environmental disturbance at a greater rate. A field experiment was conducted to ascertain the long term in situ catabolic abilities of the microbial communities in each soil and to establish the effects of litter mixing on decomposition rate. Litter bags containing either Genista aetnensis (Etnean Broom), Pinus nigra (Corsican Pine) or a mixture of the two were buried at each of the sites and their decomposition monitored over a 2.5 year period. PLFA diversity, community composition and function was assessed for each of the soils. The soils were also subject to a disturbance and the recovery of key community parameters was monitored over a six month period in order to establish each soil community’s resistance and resilience to disturbance. A laboratory experiment was conducted in order to investigate functional diversity and decomposition functions of each soil community using a range of simple and complex substrates. The relationship between PLFA diversity and functional diversity was also investigated. No correlation was found between soil C and N contents, microbial biomass or soil respiration and soil developmental stage and there was no detectable difference in litter bag mass loss between the soil types. No non- additive effects were noted in mixed litters. The more developed soil had a greater PLFA diversity and PLFA biomass however the more developed soil was not more resistant or resilient to disturbance. Developed soils showed greater catabolic diversity compared with less developed soils broadly correlating with PLFA diversity. Despite increased PLFA diversity and functional diversity in the more developed soils, residue decomposition in situ was unaffected. Reduced PLFA diversity and community complexity did not result in reduced function. Soils at different developmental stages had similar catabolic responses and were able to degrade simple and complex substrates to a similar degree. Microbial diversity in soil has the potential to be very high thus resulting in a high rate of functional redundancy i.e. many species within the same community which have the same functional role. It is possible that only a few key functional groups present within the soil community contribute to the main decomposition function within the soil and were able to maintain function during perturbation. Both Etna soils had similar PLFA’s present in similar concentrations and these groups in general were maintained during disturbance. This suggests that total microbial community diversity may not be as important to community function as the presence of key functional groups.

La détection et l’identification de micro-organismes pathogènes est un réel enjeu de santé public tant au niveau alimentaire, que clinique ou d’intérêt national tel qu’illustré en biodéfense. Dans tous ces domaines, il est important d‘avoir des méthodes d’identification et de détection qui soient à la fois rapide, sensible et robuste. Cette thèse a pour objectif de contribuer au développement d’une approche rapide d’identification de micro-organismes sans a priori par spectrométrie de masse en tandem. Cette approche innovante, appelée phylopeptidomique, repose sur l’alliance de la peptidomique, i.e. analyse à large échelle des peptides provenant de la digestion enzymatique d’un échantillon biologique, et de la phylogénie des organismes cellulaires. Après extraction des protéines présentes dans l’échantillon à ausculter, des peptides sont générés et analysés par spectrométrie de masse en tandem. La déconvolution des signaux MS/MS à l’aide du logiciel « µOrg.ID » développé en propre au laboratoire permet d’identifier et quantifier les organismes présents dans l’échantillon en fonction des organismes indexés dans les bases de données. L’étude du protéome de Bacillus atrophaeus, agent simulant de l’anthrax, sous forme sporulée et végétative a permis d’illustrer une méthode d’identification de biomarqueurs protéiques permettant de déterminer le ratio entre les deux formes dans un échantillon. La limite de détection de la phylopeptidomique dans des échantillons purs et des échantillons en mélange équimolaire a été établie sur des bactéries modèles d’intérêts médical et environnemental. La limite de détection de spores de Bacillus atrophaeus en présence de 14 matrices interférentes (alimentaires, environnementales et autres) a permis de mettre en évidence les avantages et limitations de l’approche. Enfin, un mélange artificiel standardisé de 24 organismes a été développé afin d’évaluer les outils de bio-informatique en métaprotéomique. / The detection and identification of pathogenic microorganisms is a real public health issue for the food industry and the clinics or national interest as illustrated in the biodefense field. Thus, it is important to have identification and detection methods that are fast, sensitive and robust. This PhD thesis aims at contributing to the development of a rapid approach to identify microorganisms without any a priori by tandem mass spectrometry. This innovative approach, called phylopeptidomics, is based on the combination of peptidomics, i.e. large scale analysis of peptides derived from the enzymatic digestion of a biological sample, and the phylogeny of cellular organisms. After extraction of the proteins from the sample of interest, peptides are generated and analyzed by tandem mass spectrometry. The deconvolution of MS/MS signals using the "μOrg.ID" software developed in the laboratory enables the identification and quantification of organisms present in the sample according to the organisms indexed in generalist databases. The study of the proteome of Bacillus atrophaeus, a simulant agent of anthrax, in sporulated and vegetative form, has provided an illustration of a new method of identification of protein biomarkers, which allows determining the ratio between both forms. The limit of detection of phylopeptidomics in pure samples and equimolar mixtures was established with model bacteria of medical and environmental interests. The limit of detection of B. atrophaeus spores in the presence of 14 interfering matrices (food, environmental and others) has highlighted the advantages and limitations of the approach. Finally, a standardized artificial mixture of 24 organisms was developed in order to evaluate bioinformatics tools in metaproteomics.

Au sens de la législation, les compléments alimentaires sont des denrées alimentaires et doivent donc subir un traitement de pasteurisation pour détruire les microorganismes potentiellement pathogènes. L’utilisation du CO2 comprimé peut être une alternative basse température aux traitements thermiques classiques. Dans ce travail, la technologie au CO2 est optimisée pour traiter des compléments alimentaires fortement chargés par trois microorganismes.L’impact des conditions expérimentales sur l’inactivation et la préservation des actifs a été mis en évidence via un plan d’expériences de type composite centré. La température et dans une moindre mesure l’interaction pression-durée ont été identifiés comme étant les facteurs les plus influents. Pour les actifs, la teneur en polyphénols totaux est préservée alors que la vitamine C est conservée à plus de 70%. Les résultats ont été analysés au regard de l’effet de la température et de la pression sous azote et sous air, de l’interaction pH/température, de la charge en microorganismes et de la nature de la matrice.La pasteurisation est également examinée sous l’angle du transfert de masse et des temps caractéristiques dans un contacteur gaz-liquide afin d’estimer la cinétique de dissolution du CO2 dans la matrice. L’étude de sensibilité montre que c’est la solubilité du CO2 dans l’eau qui impacte la cinétique de dissolution plus que les paramètres liés au contacteur. Des essais avec deux dispositifs batch et un mini-dispositif continu illustrent ce propos.La durée de vie des produits ainsi pasteurisés et l’apport du traitement CO2 comparé à un traitement thermique ont été aussi étudiés. / According to the law, food dietary supplements belong to food products and must then be pasteurized to kill potentially pathogenic microorganisms. Pressurized carbon dioxide can be a low-temperature alternative to usual thermal treatments. In this study, a high pressure carbon dioxide process is optimized to pasteurize dietary supplements highly contaminated with 3 microorganism species.This work reviews the effect of operating parameters on microbial inactivation and active ingredients thanks to a central composite design. Temperature and, at a lower extent, the pressure-duration interaction were identify as influent. Concerning active ingredients, total phenolic concentration is fully preserved whereas vitamin C retention rate is at least of 70%. Results were analyzed taking into account the synergistic effect of temperature and pH, temperature and atmospheric or N2 pressure, contamination level and the composition of the food matrix.Pasteurization was evaluated from the mass transfer and characteristic times point of view in a gaz-liquid contactor. The sensitivity analysis underlines that CO2 solubility in water is the main factor that affects the dissolution kinetic, beyond contactor constraints. Experiments led with 2 different batch set-ups and a mini‑continuous reactor illustrate that point.Finally this work studies the shelf-life of our CO2-pasteurized products and estimates the added value of such a treatment compared to a thermal one.

Made available in DSpace on 2014-12-17T14:10:25Z (GMT). No. of bitstreams: 1 AbinadabeJM_DISSERT.pdf: 2202389 bytes, checksum: c6fb8bcc059666158ed3af8030f88987 (MD5) Previous issue date: 2012-07-19 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Industrial activities, oil spills and its derivatives, as well as the incomplete combustion of fossil fuels have caused a great accumulation of hydrocarbons in the environment. The number of microorganisms on the planet is estimated at 1030 and prokaryotes the most abundant. They colonized diverse environments for thousands of years, including those considered extreme and represent an untapped source of metabolic and genetic diversity with a large biotechnological potential. It is also known that certain microorganisms have the enzymatic capacity to degrade petroleum hydrocarbons and, in many ecosystems, there is an indigenous community capable of performing this function. The metagenomic has revolutionized the microbiology allowing access uncultured microbial communities, being a powerful tool for elucidation of their ecological functions and metabolic profiles, as well as for identification of new biomolecules. Thus, this study applied metagenomic approaches not only for functional selection of genes involved in biodegradation and emulsification processes of the petroleum-derived hydrocarbons, but also to describe the taxonomic and metabolic composition of two metagenomes from aquatic microbiome. We analyzed 123.116 (365 ? 118 bp) and 127.563 sequences (352 ? 120 bp) of marine and estuarine metagenomes, respectively. Eight clones were found, four involved in the petroleum biodegradation and four were able to emulsify kerosene indicating their abilities in biosurfactants synthesis. Therefore, the metagenomic analyses performed were efficient not only in the search of bioproducts of biotechnological interest and in the analysis of the functional and taxonomic profile of the metagenomes studied as well / Atividades industriais, derramamentos de petr?leo e seus derivados, bem como a combust?o incompleta de combust?veis f?sseis t?m causado um grande ac?mulo de hidrocarbonetos no meio ambiente. O n?mero de microrganismos no planeta ? estimado em 1030, sendo os procariotos os mais abundantes. Eles colonizaram diversos ambientes durante milhares de anos, incluindo aqueles considerados extremos e representam uma fonte inexplorada de diversidade gen?tica e metab?lica com um grande potencial biotecnol?gico. Sabe-se que muitos microrganismos possuem vias metab?licas complexas atuando na biodegrada??o de hidrocarbonetos derivados de petr?leo e, em muitos ecossistemas, existe uma comunidade aut?ctone capaz de realizar essa fun??o. A metagen?mica tem revolucionado a Microbiologia permitindo o acesso ?s comunidades microbianas n?o cultiv?veis, sendo uma potente ferramenta para elucida??o de suas fun??es ecol?gicas, dos perfis metab?licos, bem como para identifica??o de novas biomol?culas. Assim, o presente estudo aplicou abordagens metagen?micas n?o apenas para sele??o funcional de genes envolvidos nos processos de biodegrada??o e biossurfacta??o de hidrocarbonetos derivados do petr?leo, mas tamb?m para descri??o da composi??o taxon?mica e metab?lica de dois metagenomas de microbiota aqu?tica. Foram analisadas 123.116 (365 ? 118 pb) e 127.563 sequ?ncias (352 ? 120 pb) dos metagenomas marinho e estuarino, respectivamente. Oito clones foram encontrados, sendo quatro envolvidos na biodegrada??o de petr?leo e quatro capazes de emulsificar querosene, indicando a habilidade de sintetizar biossurfactantes. Portanto, as an?lises metagen?micas realizadas foram eficientes n?o apenas na busca de bioprodutos de interesse biotecnol?gico como tamb?m na an?lise do perfil funcional e taxon?mico dos metagenomas estudados