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Terdjman, Muriel. "Etude de la contamination microbiologique de l'air comprimé à usage médical obtenu à partir d'une centrale de production." Paris 5, 1995. http://www.theses.fr/1995PA05P202.
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Duquette-Lozeau, Karine. "Qualité microbiologique de l'air et de la litière de fumier recyclé en production laitière." Master's thesis, Université Laval, 2019. http://hdl.handle.net/20.500.11794/37632.
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La litière de fumier recyclé (LFR) (séparation du fumier et fraction solide remise sous les animaux) est de plus en plus utilisée dans l’industrie laitière au Québec. Pourtant, les risques reliés à son utilisation sur la santé humaine sont méconnus. La présente étude tente donc d’identifier la meilleure méthode de compostage de la LFR quant à la qualité de l’air dans les fermes laitières. Quatre méthodes de compostage du solide ont été testées : SW) statique; TW) retourné quotidiennement; DC24) statique après 24 h dans un composteur rotatif; et DC72) statique après 72 h dans un composteur rotatif. Des échantillons d’air ont été prélevés aux jours 0, 5, 10 à l’aide d’un échantillonneur liquide et d’un autre sur filtre. Les poussières ont été mesurées par compteur optique de particules. Les microorganismes ont été analysés par culture (bactéries et moisissures mésophiles, moisissures thermotolérantes) ou par qPCR pour les bactéries totales et Penicillium/Aspergillus, ainsi que plusieurs agents pathogènes et un gène de résistance aux carbapénèmes. Au jour 0, les poussières et les moisissures mésophiles sont inférieures pour SW, TW et DC24. Les bactéries totales sont plus faibles pour SW et TW et Penicilium/Aspergillus pour DC24. Au jour 5, les poussières sont inférieures pour DC24 et DC72, alors que les moisissures mésophiles, bactéries totales et Penicillium/Aspergillus sont en plus faibles concentrations pour SW et TW. Au jour 10, les poussières et Penicillium/Aspergillus sont plus faibles pour SW et TW, les bactéries totales pour DC72 et les résultats ne diffèrent pas pour les moisissures mésophiles. Pour les trois journées d’échantillonnage, SW a des concentrations inférieures à DC72 en bactéries mésophiles. Aucun des résultats de moisissures thermotolérantes ni d’endotoxines ne diffère et aucun des agents pathogènes ni le gène de résistance n’a été détecté par qPCR. Les traitements SW et TW semblent représenter les meilleurs choix quant à la qualité de l’air.Recycled manure solids (RMS) (solid-liquid separation f fresh manure where the solid fraction is used as bedding) gain rising interest in Quebec’s dairy industry. However, RMS use’s associated risks on human and animal health are unknown. This study tried to identify the best composting method regarding to air quality in dairy barns. Four composting methods were tested: SW) static, TW) daily turned, DC24) static after 24 h in a drum composter and DC72) static after 72 h in a drum composter. Air sampling were done with a liquid sampler and a filter sampler at days 0, 5 and 10. Dust concentrations were measured by an optical particle counter. Microorganisms were analysed by culture (mesophilic bacteria and fungi, thermotolerant fungi) or by qPCR for total bacteria (16s rDNA) and Penicillium/Aspergillus (ITS1), as well for several pathogenic agents and a carbapeneme resistance gene (KPC). At day 0 and 5, SW, TW and DC24 lead to the lowest concentrations for dust and mesophilic fungi. Total bacteria were lower for SW and TW, while Penicillium/Aspergillus were lower for DC24. At day 5, DC24 and DC72 lead to the lowest concentrations for dust, while SW and TW lead to lower concentrations for mesophilic fungi, total bacteria and Penicillium/Aspergillus. At day 10, dust and Penicillium/Aspergillus were lower for SW and TW, while total bacteria were lower for DC72 and no mesophilic fungi did not differ. For the three sampling days, SW lead to lower concentration of mesophilic bacteria than DC72. No thermotolerant fungi or endotoxins results differ and no pathogenic agent or the carbapenem resistance gene were detected by qPCR. Thus, SW and TW seem to be the methods to privilege regarding air quality in dairy barns.
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Hidalgo, Hélène. "Qualité microbiologique de l'air et systèmes de climatisation : étude de la flore bactérienne et fongique et influence des caractéristiques de l'installation." Université Joseph Fourier (Grenoble), 1993. http://www.theses.fr/1993GRE18006.
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Lemieux, Joanie. "Validation d'échantillonneurs d'air et biais sur la diversité." Master's thesis, Université Laval, 2019. http://hdl.handle.net/20.500.11794/36720.
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Différents types d’échantillonneurs d’air existent pour récolter les bioaérosols. Ils ont tous leurs avantages et leurs inconvénients, mais de ces types, un en particulier est susceptible d’introduire des biais dans le traitement et l’analyse des résultats. Les échantillonneurs de type liquide voient une portion de leur liquide d’échantillonnage s’évaporer pendant leur fonctionnement, ce qui favoriserait la perte de bioaérosols par ré-aérosolisation ou leur concentration dans le liquide. Peu de connaissances sont acquises sur la ré-aérosolisation, la concentration et leurs effets sur les résultats. Cette étude avait pour but de documenter comment l’évaporation du liquide de collecte impacte l’échantillonnage de l’air. Des expérimentations in vitro, où les récipients de collection de deux échantillonneurs liquides (Coriolisμ® et BioSampler®) étaient inoculés avec des consortiums bactériens connus, ont permis de conclure que la ré-aérosolisation et la concentration des bactéries sont des phénomènes complexes. En effet, ils sont difficilement prédictibles et semblent influencés par l’évaporation, le genre bactérien, l’hydrophobicité de la membrane bactérienne, l’interaction avec les autres bactéries, la composition du liquide de collection, le débit et le mécanisme de capture de l’échantillonneur. De plus, des expérimentations en milieu naturel ont permis de comparer la diversité récoltée par les échantillonneurs de type liquide et de type filtre, par méthodes de séquençage à haut débit. Une des particularités de cette étude était qu’une cassette contenant un filtre était branchée à la sortie d’air du BioSampler® pour récolter les bactéries ré-aérosolisées pendant l’échantillonnage. Plusieurs genres bactériens sont totalement ré-aérosolisés du récipient de collection du BioSampler®. Plus de la moitié des genres bactériens récoltés par le Coriolisμ® diffèrent de ceux du BioSampler®, et inversement. Les échantillonneurs filtres ont tous deux récolté une diversité bactérienne très similaire. Ces résultats constituent un apport important au domaine scientifique puisqu’ils prouvent les biais potentiels induits par les échantillonneurs de type liquide.Different types of air samplers are available to harvest bioaerosols. They all have their advantages and disadvantages, but of these types, one in particular is likely to introduce bias in the treatment and analysis of the results. Liquid-based samplers see a portion of their collection fluid evaporate during operation, which would favor either the loss of bioaerosols by re-aerosolization or their concentration in the fluid. Very little knowledge is known about re-aerosolization, concentration and their effects on results. The main purpose of this study was to document how the evaporation of the collection fluid impacts air sampling. In vitro experiments, in which collection vessels from two liquid samplers (Coriolisμ® and BioSampler®) were inoculated with known bacterial consortia, concluded that reaerosolization and concentration are complex phenomena. Indeed, they are difficult to predict and seem influenced by evaporation, the bacterial genus, the hydrophobicity of the bacterial membrane, the interaction with other bacteria, the composition of the collection fluid, the flow and capture mechanism of the sampler. In addition, experiments in a natural environment have made it possible to compare the diversity harvested by the liquid-based and filter-based samplers by high throughput sequencing methods. One of the peculiarities of this study was that a cassette containing a filter was connected to the BioSampler® air outlet to collect the re-aerosolized bacteria during sampling. The results are unequivocal, several bacterial genera are totally re-aerosolized from the BioSampler® collection vessel. More than half of the bacterial genera harvested by the Coriolisμ® differ from those of the BioSampler®, and vice versa. The filter samplers both harvested a similar bacterial diversity. These results are an important contribution to the scientific field since they prove the biases induced by liquid type samplers.
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Nieguitsila, Adélaïde. "Evaluation de l'aérocontamination fongique dans les environnements intérieurs." Thesis, Paris Est, 2008. http://www.theses.fr/2008PEST0076/document.
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Le contrôle de l'aérocontamination fongique est devenu un objectif majeur pour préserver la santé humaine et animale. L'évaluation de l'aérocontamination fongique fait classiquement appel aux techniques de mise en culture ou de dosage de composants fongiques présents dans l'air. Ces techniques présentent des inconvénients. C'est la raison pour laquelle, nous nous sommes fixés comme objectif de mettre au point des méthodes d'analyse alternatives utilisant des outils de biologie moléculaire. Dans un premier temps, nous avons comparé plusieurs techniques de prélèvements d'air dans des environnements intérieurs présentant une contamination plus ou moins élevée. Nous avons, par la suite, optimisé les conditions d'extraction de l'ADN fongique à partir de prélèvements d'air. L'ADN extrait a été amplifié par PCR à semi-nichée ? avec des amorces universelles permettant d'amplifier une partie de l'ARNr 18S de champignons. Par la suite, nous avons utilisé la TTGE (Temporal Temperature Gradient Electrophoresis) et la D-HPLC (Denaturing High Performance Liquid Chromatography) pour séparer les amplificats. Chaque produit de PCR a été identifié par séquençage direct après purification. La comparaison des espèces identifiées par ces techniques avec celles qui sont obtenues par la méthode classique (culture) apporte de meilleurs renseignements sur la qualité fongique d'un même prélèvement d'air. L'application de ces techniques dans des environnements à diff'rents niveaux de contamination a permis de déduire que l'étude de l'évaluation de l'aérocontamination fongique se fait par l'association de la culture et des méthodes moléculairesFungal spores represent a significant part of the biological contaminants that could be detected in air. Exposure to fungi has been associated with several types of human or animal health problems (mycosis, allergy, mycotoxicosis). To evaluate the relationship between airborne fungi potential and adverse health effect, the fungal types and their relative frequencies in air need to be investigated. Traditional methods for fungal identification (culture and microscopy analysis) are laborious, time-consuming and require expertise. To replace cultivation, several techniques have been proposed. This study showed that molecular techniques (PCR-TTGE or Temporal Temperature Gradient Electrophoresis and PCR-DHPLC or Denaturing High Performance Liquid Chromatography) allowed the separation of amplificons corresponding to distinct fungal species that may be encountered in air. Both methods were proved to be appropriate for analysis of complex fungal communities. The detection and the molecular identification techniques were adapted for the evaluation of indoor airborne fungal contamination. The cultivation method and culture-independent techniques were further compared for the analysis of fungal aerosols from different sites
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Nait, Chabane Yassine. "Caractérisation de biofilms à l'interface air-liquide formés par Acinetobacter baumanii." Rouen, 2013. http://www.theses.fr/2013ROUES022.
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Acinetobacter baumannii est une bactérie opportuniste responsable à l’heure actuelle, de véritables épidémies d’infections nosocomiales à travers le monde. Cette émergence pourrait être due à deux facteurs majeurs remettant en cause son éradication : d’une part, l’apparition de souches multirésitantes aux antibiotiques. D’autre part, la résistance d’A. Baumannii aux produits antiseptiques, associée à une survie prolongée en milieu hospitalier sur les surfaces sèches et sur le matériel médical. Cette persistance pourrait trouver son origine dans la formation de biofilms par cet agent bactérien, en particulier à l'interface air-liquide (pellicules). Précisons que cette caractéristique n’est retrouvée que pour les espèces du genre Acinetobacter présentes à l’hôpital. Dans cette étude nous nous sommes intéressés à ce type de biofilm particulier. Dans un premier temps, nous avons classé les pellicules de 26 isolats cliniques d’A. Baumannii en 3 morphogroupes: i) morphotype oeufs (23%), ii) morphotype boules (50%), iii) morphotype canaux (27%). Les observations en microscopie à l’angle de Brewster et l’évaluation de l’hydrophobicité relative de ces souches ont montré qu’A. Baumannii colonise directement l’interface air liquide et ne requière pas de support solide. L’analyse des glucides totaux de la matrice a montré 3 principaux composants glucose, GlcNac et Kdo. La composition de la matrice des groupes I et III est proche notamment en glucose comme composant principal, alors que au sein du groupe II, c’est le GlcNac qui est majoritaire. Par ailleurs, nous avons identifié trois pilines associées à la matrice: i) CsuaA/B, la sous-unité la plus abondante dans les 3 morphogroupes ce qui confirme l'implication du système pili Csu de type I dans le développement pellicule, ii) la protéine ABYAL1779 appartenant un système chaperonne usher (CU), qui présente 45% d’homologie avec la piline principal F17-A des pili F17chez Escherichia coli entérotoxinogènes, enfin iii) la piline ABYAL2525 d'un deuxième opéron CU bien conservé dans les espèces du complexe ACB. Dans un second temps, nous avons réalisé une analyse protéomique par approche bidimensionnelle afin de déterminer les protéines membranaires fortement impliquées dans la formation de la pellicule. Nous avons comparé le protéome de la souche A. Baumannii A077 (groupe I) cultivée à l’état planctonique et en mode pellicule. Sur 52 spots identifiés, nous avons classé les protéines surexprimées à l'état pellicule en 4 groupes fonctionnels majeurs et constituent de potentiels facteurs de virulence : 3 systèmes d’acquisition et de transport de fer, 3 porines spécifiques, des protéines impliquées dans le métabolisme et le transport de lipides, enfin des protéines appartenant à 3 systèmes de pili dont deux ont été déjà identifié dans la première partie. Enfin, dans le but de lutter contre l’établissement de ces pellicules, nous nous sommes intéressés plus particulièrement aux cibles thérapeutiques que peuvent représenter les pili. Dans cette optique, nous avons testé l'effet d'une molécule pilicide, la virstatine sur la formation de biofilm chez A. Baumannii en mode statique en inhibition et en dispersion puis en mode dynamique en système bioflux. La production de biofilm est diminuée de 38% à 100μM chez A. Baumannii ATCC17978. Nous avons aussi montré que la virstatine agit sur plus de 70% d’isolats cliniques et qu’elle reste active sur la formation de biofilms en mode dynamique. Les résultats de cette étude nous ont permis de mieux comprendre les mécanismes mis en place par A. Baumannii pour former la pellicule. Nous pouvons, par ailleurs, suggérer une relation
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Ledoux-Henebel, Corinne. "Surveillance de la qualité microbiologique de l'air dans les lieux publics et l'hôpital." Paris 5, 1991. http://www.theses.fr/1991PA05P014.
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Ambroise, Denis. "Influence de la variabilité de la mesure des bactéries de l'air sur l'évaluation du risque infectieux : exemple de la légionellose." Nancy 1, 2003. http://www.theses.fr/2003NAN10008.
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En prenant l'exemple de la légionellose, ce travail pose les bases d'une démarche d'évaluation quantitative du risque infectieux bactérien par inhalation. Deux scenarii ont été étudiés : l'exposition à l'occasion d'une douche et lors d'une activité à l'extérieur à proximité d'une tour aéroréfrigérante. Des facteurs de sensibilité des sujets à la légionellose tels que le sexe, l'âge et le tabagisme ont été pris en compte. Une relation dose-effet, établie à partir d'une étude réalisée chez l'animal, a été retenue pour la caractérisation des dangers. La quantification du risque a été menée par une approche probabiliste utilisant la méthode de simulation de Monte Carlo. Les résultats obtenus montrent qu'un risque annuel de 10-5 correspond à des concentrations en "Legionella pneumophila" dans l'air de l'ordre de 2 UFC. M-3 dans le cas des douches et 0,02 UFC. M-3 dans celui des tours. En dessous de 100 UFC. M-3, la prise en compte de la variabilité de la mesure de l'exposition diminue d'autant plus l'estimation du risque que la concentration en agents dans l'air est faibleThe aim of our study was to develop a quantitative microbiological risk assessment involving the respiratory pathway. We chose the example of Legionnaires'disease, on the basis of two types of exposure : when taking a shower and being outside in the vicinity of a cooling tower. We collected suitable data in international scientific literature, which allowed us to integrate individual susceptibility factors such as sex, age or smoking habits in our calculations. We established a dose-response relationship by fitting the results of an animal exposure experiment to different models used in microbiological foodborne or waterborne risk assesment. We used a probabilistic approach based on Monte Carlo simulations for risk characterization. "Legionella" concentrations of 2 CFU. M-3 in the air near showers and 0,02 CFU. M-3 near cooling towers amount to an annual risk estimate of 10-5. Taking the exposure measurement variability into account does not change our risk estimation for concentrations above 100 CFU. M-3, but it decreases for lowest ones, which probably are more frequently encountered in the case of "Legionella" exposure
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Gendron, Louis. "The use of fluorescent bacteriophages to study viroaerosol characteristics." Thesis, Université Laval, 2014. http://www.theses.ulaval.ca/2014/30227/30227.pdf.
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Pour bien comprendre et contrôler les aérosols contenant des virus (viroaérosol), un modèle de laboratoire approprié est requis. Pour cette étude, trois bactériophages : P008 couplé au SYBR Gold, PP01 exprimant la GFP et ʎ exprimant la EYFP ont été comparés entre eux et à des microsphères fluorescentes non-biologiques pour leur potentiel en tant que modèle de laboratoire en aérovirologie. Les modèles viraux ont été aérosolisés à partir d’un tampon de phage en utilisant un nébuliseur de TSI (modèle 9301) connecté à une chambre d’aérosols. La taille aérodynamique des aérosols ainsi que leur distribution ont été déterminées à l’aide d’un spectromètre de particules aérodynamique (APS, TSI modèle 3321). Les échantillons de viroaérosols ont été capturés à l’aide d’un impacteur Andersen à six étages contenant soit du tampon de phage à l’intérieur des plaques de chaque étage ou un milieu solide (agar à 1.5%). Les techniques des plages de lyse, du qPCR et la microscopie à fluorescence ont été utilisées pour quantifier les virus récupérés sur les étages de l’impacteur. La microscopie à fluorescence a aussi été utilisée pour quantifier et analyser les modèles viraux sur des particules seules et sur milieu solide. L’ADN viral, des plages de lyse ainsi que des particules fluorescentes ont été observées sur les étages 3 à 6 de l’échantillonneur ce qui corrélait avec les données obtenues par l’APS. La microscopie à fluorescence a permis de visualiser les modèles viraux sur ou à l’intérieur des particules d’aérosols. Ces résultats confirment que les virus peuvent être présents dans l’atmosphère sous forme d’aérosol dont la dimension est bien plus grande que celle de leur propre taille, et que les virus en aérosol peuvent être quantifiés et observés en utilisant la microscopie à fluorescence. L’ensemble de ces résultats suggèrent qu’un bactériophage fluorescent serait un excellent modèle de laboratoire pour étudier le comportement des virus dans l’air.In order to understand and control virus aerosols (viroaerosols), an appropriate laboratory model is required. In this study, fluorescent bacteriophages P008 coupled to SYBR Gold, PP01 expressing GFP and ʎ expressing EYFP were compared to non-biological fluorescent microspheres for their potential as viral models in aerovirology. The test viruses were aerosolized in phage buffer using TSI’s 9301 model atomizer attached to a commercially available aerosol chamber. The aerodynamic particle size distribution of the viroaerosols was determined with an aerodynamic particle sizer (APS, TSI’s 3321 model). Samples were collected with a Sixstage Andersen impactor loaded with Petri dishes containing either phage buffer or a solid 1.5% agar medium. Plaque assays, qPCR and fluorescence microscopy were used to quantify the virus load on each stage of the impactor. Fluorescence microscopy was also used to quantify and analyze single aerosol particles in liquid or solid media. Viral DNA, infectious particles and fluorescent particles were detected on stages 3 to 6 of the sampler and correlated with the aerodynamic particle distribution. Fluorescence microscopy permitted visualization of viruses on or encapsulated inside aerosol particles and on a solid medium. These results confirm that viruses may be present in the atmosphere as aerosols, which are much larger than their own particle size, and that viruses could be visualized and quantified in aerosols using fluorescence microscopy. These findings suggest that a fluorescence-expressing bacteriophage would be an excellent laboratory model for the study of viruses in aerosols.
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Signour, Thomas. "Extraction de signatures de bactéries par microspectroscopie Raman et chimiométrie : application à l’étude de la composition biologique des aérosols dans l’environnement." Electronic Thesis or Diss., Lille 1, 2017. http://www.theses.fr/2017LIL10152.
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Depuis plusieurs années, l’étude et le contrôle de la qualité de l’air sont au cœur de toutes les préoccupations. En 2012, la DGA (Direction Générale de l’Armement) met en place le programme ASTRID (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense) accompagnant les travaux de recherche duale civile et militaire. Cette thèse s’inscrit dans cette démarche et propose d’étudier la faisabilité du concept de détection et d’identification rapides des microorganismes présents dans un échantillon d’air par microspectroscopie Raman, avec une résolution au niveau de l’espèce. Pour cela, nous construisons un modèle chimiométrique de classification des microorganismes représentatifs de la biodiversité naturelle en acquérant, sans a priori, d’une part les spectres Raman de ces microorganismes après biocollecte et étalement sur la lame d’un microspectromètre Raman, et d’autre part les séquences génomiques codant les ARN 16S de ces mêmes microorganismes.Les travaux de recherche présentés dans cette thèse présentent donc les différentes études mises en œuvre lors du développement d’un nouveau protocole permettant l’analyse des bactéries issues d’aérosols naturels environnementaux. Nous démontrons la nécessité d’optimiser l’acquisition des spectres Raman sur les bactéries et le traitement statistique des données spectrales permettant le développement de modèles de classification présentant des taux de reconnaissance élevésFor several years, the study and the control of the quality of the air are at the heart of all the concerns. In 2012, the DGA (Direction Générale de l’Armement) employs the ASTRID program (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense), to accompany the dual civil and military research work. This thesis is part of this approach and proposes the feasibility study, by Raman microspectroscopy, of the concept of rapid detection and identification of microorganisms present in an air sample, with a resolution at the species level. For this, we construct a chemometric model for the classification of micro-organisms representative of the natural biodiversity. Such a model is built by acquiring, without a priori i) the Raman spectra of these microorganisms after biocollection; and ii) the genomic sequences encoding the 16S RNAs of these same microorganisms. The research presented in this thesis therefore presents the different studies carried out during the development of a new protocol allowing the analysis of bacteria from natural environmental aerosols. We demonstrate the need to optimize the acquisition of Raman spectra on bacteria and the statistical processing of spectral data that allows the development of classification models with high recognition rates
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Delanoe, Antoine. "Etude CLIMATOX : contribution à la caractérisation des bioaérosols d'habitats dégradés par les moisissures et à l'évaluation de leurs effets sur la santé." Thesis, Normandie, 2018. http://www.theses.fr/2018NORMC279/document.
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La qualité de l’air dans les habitats est un problème majeur en Europe, où les personnes passent une grande partie de leur temps en milieu intérieur. Selon l’OMS, de nombreux habitats sont touchés par l’humidité entraînant le développement de moisissures qui ont des conséquences sanitaires et économiques.Dans ce travail, une approche globale, associant étude de terrain et expérimentations en laboratoire, a été employée pour décrire l’exposition aux bioaérosols dans des habitations dégradées par les moisissures, étudier leurs effets sur la santé des résidents et proposer une démarche diagnostique applicable aux habitats dégradés. Nous avons ainsi mis en évidence la présence récurrente de certaines espèces fongiques, notamment Aspergillus versicolor, Penicillium chrysogenum et P. crustosum, qui pourraient être utilisées comme indicateurs microbiologiques de contamination fongique de l’air intérieur. Par ailleurs les analyses statistiques ont permis de montrer des liens entre les concentrations de certaines espèces de micromycètes dans l’air et les catégories de surface contaminées proposées par l’ANSES. Des relations ont également pu être établies entre l’exposition à ces moisissures et certaines manifestations respiratoires et cutanées mentionnées chez les résidents. L’évaluation du potentiel cytotoxique des bioaérosols collectés dans les habitats dégradés a, quant à elle, permis de mettre en évidence un lien entre les réponses observées sur deux lignées cellulaires testées (pulmonaire A549 et cutanée HaCaT).D’un point de vue méthodologique, la qPCR couplée à la cytométrie en flux semble être une méthode rapide qui peut être corrélée à l’approche culturale et permet ainsi de suivre l’exposition à Aspergillus versicolor dans les habitats dégradés. Huit espèces fongiques récurrentes dans les bioaérosols ont également été sélectionnées pour une étude en enceinte climatique montrant l’impact de la température et de l’humidité relative sur la croissance et la toxinogenèse fongiqueAir quality in houses is a major concern in Europe, as people spend most of their time indoors. According to the WHO, numerous houses are exposed to dampness that can lead to mold growth, causing health and economic damages.In this work, a global approach including both field study and laboratory experimentations was used to characterize the human exposure to bioaerosols in mold-damaged houses, to study their health effects on inhabitants and to propose a diagnostic process that could be applied to mold-damaged houses. We showed the recurrence of several fungal species, specifically Aspergillus versicolor, Penicillium chrysogenum and P. crustosum, which could be used as microbial indicators of airborne fungal contamination. In addition, our statistical analyses showed relations between the concentrations of recurrent molds in air and the levels of surface contamination by molds proposed by ANSES. Statistical link has also been found between mold exposure and respiratory or cutaneous symptoms described by the inhabitants. A cytotoxic potential evaluation of bioaerosols collected in these mold-damaged houses allowed to highlight a relation between the responses observed for the two cell lines (pulmonary A549 and cutaneous HaCaT).From a methodological point of view, qPCR coupled with flow cytometry appears to be a fast and reliable method that can be correlated to cultural approach, allowing to monitor the human exposure to Aspergillus versicolor in mold-damaged buildings. Eight recurrent fungal species identified in bioaerosols were also selected for a study in a climatic chamber that showed the effects of temperature and relative humidity on fungal growth and toxinogenic potential
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Parat, Sylvie. "Étude des relations entre climatisation, micro-organismes aéroportés et santé : une approche médicale, métrologique et technique." Université Joseph Fourier (Grenoble), 1997. http://www.theses.fr/1997GRE19010.
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Létourneau, Valérie. "Impact des systèmes de gestion des lisiers sur la contamination en microorganismes des porcheries et des producteurs de porcs." Thesis, Université Laval, 2010. http://www.theses.ulaval.ca/2010/27567/27567.pdf.
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Signour, Thomas. "Extraction de signatures de bactéries par microspectroscopie Raman et chimiométrie : application à l’étude de la composition biologique des aérosols dans l’environnement." Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10152/document.
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Depuis plusieurs années, l’étude et le contrôle de la qualité de l’air sont au cœur de toutes les préoccupations. En 2012, la DGA (Direction Générale de l’Armement) met en place le programme ASTRID (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense) accompagnant les travaux de recherche duale civile et militaire. Cette thèse s’inscrit dans cette démarche et propose d’étudier la faisabilité du concept de détection et d’identification rapides des microorganismes présents dans un échantillon d’air par microspectroscopie Raman, avec une résolution au niveau de l’espèce. Pour cela, nous construisons un modèle chimiométrique de classification des microorganismes représentatifs de la biodiversité naturelle en acquérant, sans a priori, d’une part les spectres Raman de ces microorganismes après biocollecte et étalement sur la lame d’un microspectromètre Raman, et d’autre part les séquences génomiques codant les ARN 16S de ces mêmes microorganismes.Les travaux de recherche présentés dans cette thèse présentent donc les différentes études mises en œuvre lors du développement d’un nouveau protocole permettant l’analyse des bactéries issues d’aérosols naturels environnementaux. Nous démontrons la nécessité d’optimiser l’acquisition des spectres Raman sur les bactéries et le traitement statistique des données spectrales permettant le développement de modèles de classification présentant des taux de reconnaissance élevésFor several years, the study and the control of the quality of the air are at the heart of all the concerns. In 2012, the DGA (Direction Générale de l’Armement) employs the ASTRID program (Accompagnement Spécifique des Travaux de Recherches et d’Innovation Défense), to accompany the dual civil and military research work. This thesis is part of this approach and proposes the feasibility study, by Raman microspectroscopy, of the concept of rapid detection and identification of microorganisms present in an air sample, with a resolution at the species level. For this, we construct a chemometric model for the classification of micro-organisms representative of the natural biodiversity. Such a model is built by acquiring, without a priori i) the Raman spectra of these microorganisms after biocollection; and ii) the genomic sequences encoding the 16S RNAs of these same microorganisms. The research presented in this thesis therefore presents the different studies carried out during the development of a new protocol allowing the analysis of bacteria from natural environmental aerosols. We demonstrate the need to optimize the acquisition of Raman spectra on bacteria and the statistical processing of spectral data that allows the development of classification models with high recognition rates
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Merai, Mouna. "Caractérisation expérimentale et modélisation des transferts thermique/hydrique et de la croissance microbienne au cours du transport frigorifique de carcasses de porc." Thesis, Paris, Institut agronomique, vétérinaire et forestier de France, 2018. http://www.theses.fr/2018IAVF0014/document.
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L’objectif de ce travail est de développer une démarche permettant de prédire l’évolution de la charge microbienne à la surface de carcasses de porc lors d’un transport frigorifique selon les conditions opératoires (température et humidité de l’air de soufflage) et des conditions initiales (profil de température en sortie de chambre froide d’abattoir). La croissance microbienne dépendant notamment de la température et de l’activité de l’eau, il est nécessaire d’étudier les transferts de chaleur et de matière de type diffusif au sein des carcasses et de type convectif autour des carcasses. Ces derniers dépendent de la circulation d’air dans le véhicule frigorifique lorsqu’il est chargé de centaines de demi-carcasses ce qui rend la géométrie particulièrement complexe.De ce fait, ce travail fait appel à diverses disciplines : mécanique des fluides, transferts thermiques et microbiologie prévisionnelle. Le couplage de ces trois disciplines permet d’apporter des réponses scientifiques quant à la qualité sanitaire des carcasses de porc.En travaillant sur un dispositif expérimental reproduisant une semi-remorque chargée de carcasses de porc à l’échelle réduite, les écoulements d’air ont pu être caractérisés par vélocimétrie laser Doppler 2D dans deux configurations de distribution d’air (avec et sans conduits). De plus, les coefficients de transfert convectifs locaux ont pu être estimés à la surface de différentes parties des carcasses de porc et à différentes positions dans la semi-remorque à l’échelle réduite. Un schéma simplifié des écoulements d’air a été établi, il permet de localiser les « zones à risque » dans la semi-remorque chargée (faible circulation d’air et faible coefficients de transfert convectif).En se basant sur les résultats de l’étude expérimentale à l’échelle laboratoire et sur ceux récoltés au cours de vrais transports frigorifiques, la variabilité des paramètres caractérisant l’air circulant autour des carcasses a pu être estimée. Ces informations ont servi de conditions aux limites d’un modèle de transfert de chaleur et de matière (eau) au sein de la partie la plus sensible au niveau microbiologique: le jambon. Ce modèle 3D, résolu par la méthode des éléments finis, permet de prédire l’évolution de la température, de la teneur en eau et de la charge microbienne (Pseudomonas) à la surface de la partie maigre du jambon pour différents scénarios de transport frigorifique. Les résultats ont montré que si le transport commence alors que le cœur des carcasses est encore tiède (15°C au lieu de 7°C selon la réglementation actuelle) la croissance des microorganismes à la surface des carcasses de porc n’est globalement pas plus importante entre l’abattage et l’arrivée sur le site de découpe.Enfin, une étude de terrain a permis de valider les données obtenues à l’échelle du laboratoire et de réaliser une étude énergétique. Il apparait que quelle que soit le pourcentage de carcasses tièdes dans la semi-remorque, la capacité frigorifique du système de production de froid est généralement suffisante pour évacuer la chaleur des carcasses.Cette étude a permis de développer des méthodes de caractérisation des écoulements et des transferts dans une géométrie particulièrement complexe. Elle a montré l’intérêt de coupler des modèles de transfert et de microbiologie prévisionnelle. Les expérimentions à l’échelle laboratoire ont été construites en reproduisant au plus près les conditions réelles grâce à l’appui de spécialistes de la filière viande. Ainsi les carcasses modèles ont été réalisées dans des moules obtenus par impression 3D d’après des scanners X de vraies carcasses. Les résultats de cette étude sont directement utilisables par la profession et les pouvoirs publics pour l’adaptation de la réglementation des transports réfrigérés. La démarche développée pourra être adaptée pour des problèmes similaires dans des enceintes ventilées très encombréesThe objective of this work is to develop an approach allowing to predict the evolution of the microbial load on the surface of pork carcasses during a refrigerated transport according to the operating conditions (temperature and humidity of the blowing air) and initial conditions (temperature profile at the outlet of the slaughterhouse cold room). Since microbial growth depends mainly on temperature and water activity, it is necessary to study heat and mass transfer the transfer within and around the carcasses. These phenomena depend on the circulation of air in the refrigerated vehicle loaded with hundreds of half-carcasses which makes the geometry particularly complex.Thus, this work involves various disciplines: fluid mechanics, heat transfer and predictive microbiology. The coupling of these three disciplines makes it possible to provide scientific answers as to the sanitary quality of the pork carcasses.By conducting experiments on a semitrailer loaded with pork carcasses on a reduced scale, the air flows could be characterized by 2D Doppler laser velocimetry in two air distribution configurations (with and without air ducts). In addition, local convective heat transfer coefficients could be estimated at the surface of different parts of pork carcasses and at different positions in the reduced-scale trailer. A simplified model of the airflow has been established, that makes it possible to identify the "risk zones" in the loaded semi-trailer (low air circulation and low convective transfer coefficients).Based on the results of the experimental laboratory scale study and those collected during actual refrigerated transport, the variability of the parameters characterizing the air circulating around the carcasses could be estimated. This information served as boundary conditions for a model of heat and mass (water) transfer within the most sensitive part at the microbiological level: the ham. This 3D model, solved by the finite element method, makes it possible to predict the evolution of the temperature, the water content and the microbial load (Pseudomonas) on the surface of the lean part of the ham for different scenarios. The results showed that if the transport begins while the heart of the carcasses is still warm (15°C instead of 7°C according to current regulation) the growth of microorganisms on the surface of pork carcasses is generally not more between slaughter and arrival at the cutting site.Finally, a field study validated the data obtained at the laboratory scale and carried out an energy study. It appears that whatever the percentage of warm carcasses in the semi-trailer, the cooling capacity of the cooling system is generally sufficient to evacuate the heat of the carcasses.This study has made it possible to develop a method that characterizes airflow and heat transfer methods in a particularly complex geometry. It showed the interest of coupling transfer models and predictive microbiology models. Experiments at the laboratory scale were built by reproducing the real conditions as closely as possible thanks to the support of specialists in the meat sector. Thus the model carcasses were made in molds obtained by 3D printing from X-Ray scanners of real carcasses. The results of this study are directly usable by the profession and the public authorities for the adaptation of the refrigerated transport regulations. The approach developed may be adapted for similar problems in very congested ventilated enclosures
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Haddadin, Jamal. "Études microbiologiques et cinétiques de la lixiviation bactérienne en réacteurs : effet de différents paramètres physico-chimiques, développement d'un procédé en réacteurs air-lift et lit-fluidisé et application à l'extraction de l'antimoine." Vandoeuvre-les-Nancy, INPL, 1995. http://www.theses.fr/1995INPL063N.
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La lixiviation bactérienne est l'application biotechnologique la plus importante dans l'industrie métallurgique, celle-ci est utilisée pour la récupération des métaux par lessivage, utilisant les capacités que possèdent certains micro-organismes à solubiliser les métaux. Ce travail est divisé en quatre sections: premièrement, nous avons identifié la composition microbiologique de la culture mixte en notre possession. Cette culture mixte est composée de trois populations bactériennes. Deuxièmement, nous avons étudié l'influence de certains paramètres physico-chimiques tels que le pH, la température, la concentration en CO2, le taux de solides et l'ajout de Fe3+ au milieu réactionnel sur la cinétique de la biolixiviation, en culture discontinue. Ces études ont notamment conduit à la détermination d'un pH (1. 75), d'une température (32-37°C) et d'une concentration en CO2 (0,03 et 2% v/v) optimaux pour ce procédé. Troisièmement, nous avons comparé différents systèmes réactionnels (air-lift, lit fluidisé et mécaniquement agité) pour aider au choix d'une future technologie alternative. Notre dernière contribution a porté sur l'étude de la capacité de la culture mixte à oxyder des déchets industriels contenant de l'antimoine, en culture discontinue. Nous avons étudié le rôle important de l'interaction galvanique entre la pyrite et les résidus antimoniés sur la récupération de l'antimoine
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Dutil, Steve. "Caractérisation des bioaerosols dentaires : un regard sur l'eau des unités dentaires." Doctoral thesis, Université Laval, 2008. http://hdl.handle.net/20.500.11794/20120.
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La tubulure des unités dentaires (UD) démontre la présence d'un biofilm, lequel assure la croissance et le maintien d'une abondante population microbienne planctonique incluant plusieurs pathogènes et pathogènes opportunistes, tels que des légionelles et des mycobactéries non-tuberculeuses (NTM). Les instruments dynamiques connectés aux UD génèrent des bioaérosols. Ainsi, outre la bouche des patients, l'eau de l'UD peut être une source considérable de bioaérosols. De plus, l'analyse par culture sous-estime grandement la biomasse microbienne. Les principaux objectifs de cette thèse sont de : (i) mesurer la génération et la persistance des bioaérosols lors de traitements dentaires; (ii) mesurer l'exposition du personnel et des patients à ces bioaérosols; (iii) adapter et utiliser des méthodes d'analyse des microorganismes non reliées à la culture afin de mieux caractériser l'environnement de travail qu'est le cabinet dentaire et d'augmenter ainsi de manière importante les connaissances reliées aux risques d'infection ou de sensibilisation du personnel et des patients aux bioaérosols. Dans nos conditions expérimentales, il a été établi que les traitements de nettoyage dentaire génèrent des milliers de bioaérosols cultivables provenant de la bouche des patients et possiblement de l'eau des UD. De plus, il a été démontré que le personnel et les patients sont exposés à des concentrations de bioaérosols pouvant atteindre 1,9 E+05 bactéries/m3 . Par ailleurs, le faible diamètre (0,73 um) des aérosols générés, suggère un risque d'exposition. Toutefois, bien que l'eau des UD soit contaminée par des légionelles et des NTM, l'aérosolisation non-significative de ces bactéries propose un faible risque d'exposition pour ces pathogènes. L'utilisation de méthodes d'analyse non reliées à la culture a permis de mieux caractériser l'environnement dentaire : l'hybridation in situ en fluorescence avec un enrichissement préalable par culture a permis de détecter rapidement la présence du pathogène respiratoire Legionella spp. dans l'eau des UD; la microscopie à fluorescence a permis de déterminer la charge bactérienne totale des échantillons environnementaux (bioaérosols et l'eau des UD). Toutefois, la cytométrie en flux ne semble pas une approche adéquate pour la quantification bactérienne. En conclusion, l'utilisation de méthodes d'analyse non reliées à la culture a démontré que le personnel dentaire et les patients sont exposés à des bioaérosols générés lors de traitements dentaires.
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Joblin, Yaël. "Elaboration d’un microsystème d’analyse de l’air destiné à la détection rapide d’un développement fongique dans les espaces clos." Thesis, Paris Est, 2011. http://www.theses.fr/2011PEST1027/document.
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Les champignons sont des biocontaminants courants des environnements intérieurs. De nombreuses études ont démontré leur rôle dans la dégradation des supports que ces microorganismes colonisent tels que les matériaux de construction, les ouvrages ou les œuvres d'art. De plus, ces biocontaminants sont susceptibles d'induire des allergies, des infections, des toxi-infections ou encore des irritations. Depuis 2005, une technique de détection de la croissance fongique, basée sur la recherche de traceurs chimiques spécifiques dans l'air, et un indice de contamination fongique (ICF), ont été développés et validés au cours de différentes campagnes de mesures dans l'habitat, les bureaux, les écoles, les crèches… L'objectivation d'une croissance fongique dans un environnement s'appuie sur des prélèvements par adsorption, une analyse chromatographique au laboratoire (GC/MS) et le calcul de l'ICF. Dans le cadre de la surveillance de la qualité microbiologique de l'air des environnements intérieurs, cette thèse a pour ambition de prolonger ces travaux en développant notamment un système de microcapteurs chimiques adapté à la mesure in situ. Cette recherche repose à la fois sur la méthode de détection fongique développée au CSTB et sur l'expertise scientifique et technique de l'ESIEE en matière de miniaturisation d'instruments de mesure, grâce à l'apport des microtechnologies. Le premier axe de cette étude a consisté à identifier expérimentalement les COV du métabolisme fongique spécifiques d'un développement sur des matériaux du patrimoine. Ces molécules ont permis de consolider l'ICF et de définir deux indices spécifiques à la problématique des sites patrimoniaux, validés dans des châteaux, musées, bibliothèques, grottes ornées… Le second axe porte, d'une part, sur la conception la réalisation et la caractérisation des briques élémentaires du microsystème d'analyse, à savoir un module de préconcentration (adsorption TENAX), un module de séparation (microGC) et un module de détection (capteurs polymères) et d'autre part sur les intégrations et pilotage de ces briquesFungi are common microbial contaminants of indoor environments. Many studies have demonstrated their role in the partial or total degradation of materials they colonize such as building materials, or works of art. Moreover, those microbial contaminants are likely to lead to allergies, infections, poisoning or irritation. Since 2005, a new technique based on researching specific chemical tracers in the air, was developed and validated during different measurement campaigns. This approach is now applied to various indoor environments (houses, offices, schools, child care centers…) and allows the detection of recent and/or hidden contamination. The purpose of this work is to study and characterize a rapid and continuous air analysing microsystem for detection of fungal contamination in closed spaces. This study falls within the field of monitoring air microbiological quality in indoor environments. In addition to the time saved by the absence of any laboratory analysis, this system must provide a permanent monitoring of environments frequented by people, such as museums, schools, hospitals... This research is based both on the fungal detection method developed by CSTB and on scientific and technical expertise of ESIEE : specialised in design and manufacturing of miniaturized analysis systems obtained using microtechnology. The first step of this study was to define the compounds' nature to be detected for different cases of contamination along with the sampling strategy for the system. The second step focuses on the microstructures design and fabrication to be used in microanalytical system based on gas chromatography and the development of a miniaturised analysis system. So the first part of the study consisted in defining specific fungal contamination tracers for heritage conservation sites. This list allowed to reinforce a fungal contamination index for indoor environments and to define two specific indexes designed for heritage conservation sites. The validation of these different indexes allowed checking their compliance with those types of environments (castles, museums, libraries, decorated caves...) by detecting all cases of contamination, along with the control remediation of former contaminated environments. The second part of the study enabled the design and validation of three main modules constitutive of the microanalytical system based on gas chromatography. A miniaturised analysis system based on three modules has been developed
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Babini, Valentina. "Formaggi tradizionali della regione Marche: caratterizzazione compositiva, microbiologica e sensoriale." Doctoral thesis, Università Politecnica delle Marche, 2011. http://hdl.handle.net/11566/242002.
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La tradizione casearia dell’Italia centrale comprende una grande varietà di formaggi prodotti con latte locale ed antiche tecniche di caseificazione. Tali formaggi sono parte integrante del patrimonio storico e culturale delle comunità locali, nonché risorse da conservare e valorizzare, anche a fronte del sempre più incalzante processo di globalizzazione. La regione Marche, in particolare, vanta un cospicuo numero di produzioni casearie tipiche, come riportato nell’”Elenco nazionale dei prodotti agro-alimentari tradizionali” (DM 18/07/2000). La valorizzazione di tali produzioni rappresenta un obiettivo primario delle politiche volte a promuovere la salvaguardia delle risorse e dello sviluppo socio-economico dei territori locali. Obiettivo della presente ricerca è stato la caratterizzazione compositiva, microbiologica e sensoriale di produzioni tradizionali di caciotta, pecorino e caprino della regione Marche, anche caratterizzate da tratti peculiari quali l’impiego di caglio d’origine vegetale (estratto acquoso di Cynara cardunculus), l’aggiunta di ingredienti caratteristici (erbe locali, olio d’oliva, buccia di limone grattugiata), la stagionatura in botte o all’interno di fosse tufacee. I dati ottenuti dalle analisi fisico-chimiche, microbiologiche e gas-cromatografiche sono stati elaborati statisticamente al fine di individuare correlazioni con le specifiche tecniche di caseificazione ed identificare marcatori oggettivi di qualità. A tale scopo, sono stati campionati complessivamente 29 formaggi, di cui 8 caprini, 12 pecorini e 9 caciotte, di cui 2 a latte vaccino e 7 a latte misto (bovino ed ovino); di questi, le produzioni a latte crudo (19 campioni) sono state prelevate presso caseifici artigianali della regione Marche, mentre i formaggi a latte pastorizzato (10 campioni) sono stati acquistati presso supermercati della provincia di Ancona. Tutti i campioni sono stati sottoposti a: (i) determinazione del contenuto di NaCl, proteina grezza e grassi totali; (ii) misurazione di pH, attività dell’acqua (aw), numero di perossidi della sostanza grassa; (iii) ricerca di Listeria monocytogenes, Salmonella spp. ed enterotossine stafilococciche; (iv) enumerazione di batteri lattici mesofili e termofili; (v) analisi PCR-DGGE della popolazione batterica; (vi) analisi della componente volatile mediante SPME-GC. L’insieme dei dati composizionali, microbiologici e del profilo aromatico è stato sottoposto ad analisi PCA (Principal Component Analysis) e PLS-DA (Partial Least Square Discriminant Analysis) utilizzando il software SIMCA-Pv 11.5 (UMETRICS). Tutte le produzioni in studio sono risultate conformi ai limiti legislativi per i parametri igienico-sanitari considerati. L’analisi multivariata mediante PCA dell’insieme dei dati ottenuti ha prodotto un modello a 2 componenti che spiega il 31.5% della varianza. Nello score plot t1/t2, le 3 tipologie di formaggio sono risultate ben separate; al contrario, non è stato possibile separare i formaggi sulla base del processo produttivo (da latte crudo o pastorizzato), indipendentemente dalla tipologia considerata (caciotta, pecorino o caprino). Il corrispondente loading plot ha permesso di evidenziare 3 gruppi di variabili; nello specifico, le variabili relative al profilo aromatico sono risultate responsabili della distinzione dei formaggi appartenenti alla tipologia caprino; quelle relative a conte microbiche, proprietà fisico-chimiche e composizionali hanno permesso di separare i formaggi appartenenti alla tipologia caciotta; infine, le variabili relative all’ecologia microbica sono risultate utili per discriminare i formaggi appartenenti alla tipologia pecorino. In particolare, conte più elevate di batteri lattici e valori più alti di aw hanno permesso di differenziare le caciotte dai pecorini e caprini, questi ultimi caratterizzati da concentrazioni più elevate di composti aromatici, mentre i pecorini si sono distinti per la presenza delle specie Lactobacillus coryniformis, Lb. rhamnosus e Lb. plantarum. L’analisi PLS-DA ha prodotto un modello a 2 componenti; più in dettaglio, lo score plot t1/t2 ha confermato la separazione delle 3 tipologie di formaggio. Dall’analisi del corrispondente loading plot, è emerso che tale separazione è principalmente dovuta a 10 descrittori legati alla definizione del profilo aromatico: acetone, etanolo, acido acetico, butirrico, caproico, enantico, caprilico, caprico, undecanoico ed undecenoico. L’approccio polifasico applicato nel presente studio ha permesso, nel complesso, di caratterizzare le produzioni casearie considerate, nonché di identificare marcatori utili per una discriminazione oggettiva delle stesse.The dairy tradition of central Italy territories is characterized by a large variety of cheeses, manufactured with local milk according to ancient cheese-making techniques. These cheeses are an integral part of the historical and cultural heritage of local communities and resources to preserve and enhance, even in the face of increasingly insistent globalization process. The Marche region, in particular, boasts a large number of typical dairy products, as reported in the “Italian list of traditional agro-alimentary products” (DM 18/07/2000). The exploitation of these dairy products is a primary objective of politics to promote the conservation of resources and socio-economic development of local territories. The aim of this study was the compositional, microbiological and sensory characterization of traditional caciotta, caprino and pecorino cheeses produced in the Marche region, also characterized by peculiar traits such as the use of vegetable rennet (aqueous extract of Cynara cardunculus flowers), the addition of particular ingredients (herbs, olive oil, grated lemon peel) and ripening under unconventional conditions (in wooden barrels or pit ageing). All the data collected from physico-chemical, microbiological and chromatographic assays were statistically processed in order to assess the relationships between cheese-making techniques and compositional, microbiological and aromatic traits and identify objective markers of quality. To this aim, 29 cheeses manufactured in the Marche region, according to traditional techniques, were sampled; these included 8 goats’ (caprino), 12 ewes’ (pecorino) 2 cows’ and 9 mixed (ewes’ and cows’) milk (caciotta) cheeses. Raw milk cheeses (19 samples) were supplied by local artisan dairies, while pasteurized milk cheeses (10 samples) were bought from supermarkets. All the cheese samples were subjected to physico-chemical analyses for the determination of pH and aw values, NaCl, lipid and protein contents and peroxide index. Conventional microbiological analyses were carried out for the detection of pathogenic micro-organisms (Listeria monocytogenes and Salmonella spp.) and their metabolites (staphylococcal enterotoxins) as well as for viable counting of mesophilic and thermophilic lactic acid bacteria. The composition of the bacterial community was also evaluated by PCR-DGGE analysis of the bacterial DNA extracted directly from either the cheese samples or the bulk cells harvested from the MRS and M17 dilution plates. The cheese volatile profiles were determined by solid phase micro-extraction coupled with gas-chromatography (SPME-GC). All the data collected were subjected to Principal Component Analysis (PCA) and Partial Least Square (PLS) analysis by using SIMCA-P v 11.5 software (UMETRICS). The absence of bacterial hazard was demonstrated in both pasteurized and raw milk cheeses. Principal Component Analysis of the outcomes from physico-chemical, microbiological and chromatographic assays yielded a two components model, explaining the 31.5% of the variance. In the t1/t2 score plot, the three cheese types (pecorino, caciotta and caprino) were well separated; by contrast, no separation was seen between raw and pasteurized milk cheeses, irrespective of the cheese type considered. The corresponding loading plot revealed that variables clustered in three separated groups. In more detail, variables referring to aromatic profile are responsible for the grouping of caprino cheeses; variables referring to microbial loads, physico-chemical and compositional traits are responsible for the grouping of caciotta cheeses, while those referring to the PCR-DGGE results are responsible for the grouping of pecorino cheeses. Indeed, higher loads of mesophilic and thermophilic lactic acid bacteria and higher aw values differentiated caciotta from pecorino and caprino cheeses. This latter cheese type was characterized by higher amounts of volatile compounds, while pecorino cheeses were distinguished for the presence of distinctive bacterial species, namely Lactobacillus coryniformis, Lb. rhamnosus and Lb. plantarum. Partial Least Square Discriminant Analysis resulted in a 2 components model. The score plot of the first principal component vs the second principal component confirmed that the three cheese types were clearly separated. The analysis of the corresponding loading plot showed that such separation is mainly due to 10 descriptors related to the definition of the flavour profile: acetone, ethanol, acetic, butyric, caproic, enanthic, caprylic, capric, undecanoic, and undecenoic acids. The polyphasic approach used in this study allowed, in general, to characterize the dairy products concerned, and to identify useful markers for their discrimination.
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Wutke, Maria Cristina Bahia 1959. "Desinfecção do ar em ambientes confinados pela ação combinada de dioxido de titanio e luz ultravioleta (TiO2/UV)." [s.n.], 2006. http://repositorio.unicamp.br/jspui/handle/REPOSIP/257837.
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Orientadores: Antonio Roberto Siviero, Jose Roberto GuimarãesDissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Civil, Arquitetura e Urbanismo
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Resumo: O crescimento populacional e o desenvolvimento de novas tecnologias e equipamentos com os quais se procura viabilizar mais qualidade de vida, podem muitas vezes contribuir para o aumento da produção de agentes poluentes e da proliferação de agentes patogênicos. As doenças respiratórias no Brasil têm sido importante causa de preocupação e de internações hospitalares. Diversos processos bioquímicos têm sido pesquisados e aplicados no tratamento e desinfecção do ar. A fotocatálise heterogênea com dióxido de titânio (TiO2/UV) é um dos processos oxidativos avançados, utilizados devido às suas inúmeras vantagens em sua aplicação: mais eficiência a longo prazo, produção de resíduos com menor toxidade e alta potencialidade de aplicação com baixo custo. No presente trabalho confeccionou-se um reator fotocatalítico para coletas e tratamento de amostras do ar ambiente de laboratório de Saneamento da UNICAMP, em Campinas, SP, de 19 de junho a 28 de julho de 2006. Trabalhou-se com um total de 540 placas de Petri, em dois experimentos, com tratamentos distintos entre si, em períodos matutinos, vespertinos e noturnos, sendo utilizadas 135 placas com o meio de cultivo Ágar Infusão Cérebro Coração - BHI para bactérias e outras 135 com o meio de cultivo Saboraud Dextrose para fungos, por experimento. Em cada experimento as amostras foram submetidas a quatro sistemas de desinfecção - S1: coleta de ar do interior do reator sem TiO2/UV, S2: coleta de ar do interior do reator com uma luz UV e sem TiO2 e S3: coleta de ar do ), interior do reator com duas luzes UV e sem TiO2, no experimento 1 e S5: coleta de ar do interior do reator com TiO2 e sem luz UV, S6: coleta de ar do interior do reator com TiO2 e uma luz UV e S7: coleta de ar do interior do reator com TiO2 e duas luzes UV no experimento 2, submetidos a três tempos de coleta de ar ? 1, 2 e 3 minutos (t1, t2 e t3) com 15 repetições (R) por sistema. Para os dados referentes às colônias de bactérias foram utilizados testes de qui-quadrado, para freqüências, e o teste não paramétrico ?de Friedman?, nos níveis de 1% e 5% de significância. Aqueles relativos às colônias de fungos foram analisados por teste paramétrico, com análise de variância e testes de diferenças entre médias, com transformação prévia dos dados em ?N, e comparação de médias pelos testes de t e Tukey, ao nível de 5% de significância. O processo de desinfecção de bactérias do ar ambiente é mais eficiente com a utilização de uma ou duas lâmpadas UV-C no interior do reator, sendo necessários, entretanto, 1 e 3 minutos de exposição quando utilizados no sistema, os processos de fotocatálise heterogênea (com TiO2) e de fotólise (sem TiO2), respectivamente. Em relação à desinfecção de fungos, o tempo de 1 minuto é eficiente tanto sem quanto com a utilização de TiO2 no interior do reator, constatando-se ação germicida com uma ou duas lâmpadas UV-C. O processo de fotocatálise heterogênea (TiO2/UV) é um método efetivo na desinfecção adequada de microrganismos - fungos e bactérias, presentes no ar de ambiente confinado laboratorial, por tempo de exposição de 1 minuto com uma ou duas lâmpadas UV-C
Abstract: The populational growth and development of new technologies and equipments to improve quality of life can many times contribute to an increase of polluent agents and the proliferation of pathogenic ones. Respiratory-tract diseases in Brazil have been an important cause of concern and hospitable internations. Several biochemical processes are under research and applied for air treatment and disinfection. Heterogeneous photocatalysis with titanium dioxide (TiO2/UV) is one of the advanced oxidatives process utilized due to several advantages of application: more efficiency on long term basis, production of lower tocity residues and high potencial at low cost of application. In this study a photocatalytic reactor was elaborated for collecting and treating environmental air samples from the Sanitization Laboratory located at UNICAMP, in Campinas, SP, from June 19th to July 28th of 2006. A total of 540 Petri dishes were analyzed in two different experiments, during morning, afternoon and night periods. At each experiment were utilized 135 dishes with culture media of Brain Heart Infusion Agar - BHI for bacteria and 135 other ones with Saboraud Dextrosis culture media for fungus. At each experiment the samples were submitted to four disinfection systems. In the Experiment 1 they were - S1: air collected from inside the reactor without TiO2/UV, S2: air collected from inside the reactor with one UV light and without TiO2 and S3: air collected from inside the reactor with two UV lights and without TiO2. In the Experiment 2 they were - S5: air collected from inside the reactor with TiO2 and without UV light, S6: air collected from inside the reactor with TiO2 and one UV light and S7: air collected from inside the reactor with TiO2 and two UV lights. Air samples were collected at ? 1, 2 and 3 minutes (t1, t2 and t3) time intervals for each experiment, with 15 replications (R) for each system. Bacteria colonies data were analyzed using qui-square tests for frequencies and non-parametric test of ?Friedman? at the levels of 1% and 5% of significance. Results from fungus colonies were analyzed by parametric test, with analysis of variance and tests of differences between average values, with previous transformation of data into ?N. Average data were compared by tests t and Tukey at the level of 5% of significance. Bacteria disinfection process of surrounding air is more efficient by using one or two UV-C lights inside the reactor with 1 and 3 minutes of exposition when are used heterogeneous photocatalysis (with TiO2) and photolysis (without TiO2) processes respectively. One minute time of exposition, with or without TiO2 inside the reactor is efficient for fungus removal, and germicide action was observed using one or two UV-C lights. The heterogeneous photocatalysis process (TiO2/UV) is an effective method for appropriate disinfection of microrganisms - fungus and bacteria, detected in the air of a restricted environmental from a laboratory, during one minute of exposition, with one or two UV-C lights
Mestrado
Saneamento e Ambiente
Mestre em Engenharia Civil
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Amadoro, Carmela. "Influenza della tecnologia di produzione sulle caratteristiche microbiologiche e chimiche della ventricina." Doctoral thesis, Università degli studi del Molise, 2010. http://hdl.handle.net/11695/66251.
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La ventricina, prodotta nell’area a confine tra Abruzzo e Molise, è un insaccato stagionato preparato con carne suina, abbondantemente speziata con peperone dolce e peperoncino piccante. La presente tesi di dottorato ha rivolto l’attenzione allo studio degli aspetti microbiologici di ventricine differentemente preparate, seguendo scrupolosamente la ricetta tradizionale o mediante triturazione meccanica della carne. In particolare, con tale ricerca è stata studiata l’influenza delle dimensioni dei pezzi di carne utilizzati ai fini della produzione sul processo di fermentazione delle ventricine oggetto di studio. I risultati hanno mostrato in entrambi i lotti una simile evoluzione della popolazione microbica. Tuttavia i ceppi isolati durante la maturazione hanno evidenziato differenti caratteristiche tecnologiche in relazione al lotto di appartenenza. Lactobacillus sakei e Streptococcus equorum sono risultate le specie maggiormente rappresentative in tutti i campioni durante tutto il periodo di maturazione. Mediante SDS-Page è stato possibile evidenziare come l’attività proteolitica sia una caratteristica propria del ceppo isolato. Tuttavia, è stata evidenziata una relazione tra l’effettiva attività sulle proteine sarcoplasmatiche e il tempo e/o il lotto da cui ogni ceppo è stato isolato. Pertanto è possibile asserire che la tecnologia di produzione potrebbe influenzare la selezione di ceppi che, pur se appartenenti alla stessa specie, sono dotati di caratteristiche tecnologiche diverse.Ventricina produced at the boundary between Abruzzo and Molise, two Italian regions, is a fermented sausage prepared with pork meat and fat chopped in cubes and abundantly spicy by sweet and hot pepper. This PhD thesis dealt with the study of microbiological aspects of two different type of Ventricina salami, produced with the traditional recipe or with meat grinding. In particular, the research was planned in order to investigate the influence of the meat size on the fermentation process. Results showed a similar microbial composition in the two type of Ventricina but strains isolated during the ripening evidenced different technological features, depending on the batch of isolation. Lactobacillus sakei and Streptococcus equorum were the main representatives species in all samples during all time of ripening. The SDS-Page evidenced that the proteolytic capability is a strain-specific character. Moreover, a relationship between the effectiveness of the action on sarcoplasmic proteins and the age and/or batch of isolation of each strain was found. Data obtained demonstrated that the technology of production could select strains belonging to the same species but characterised by different features.
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Bertaggia, Marco. "Ricerca di nuovi indici molecolari e microbiologici dello stato nutrizionale della vite." Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423016.
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The study of soil-plant relationships is a prerequisite for controlling production of the vineyard. In vineyard sites of Gambellara, we studied the relationship between productivity, the main physico-chemical properties of soils and some innovative indices for the diagnosis of the nutritional status of the grape as i) the biodegradation capacity of organic matter evaluated by means of degradation of filaments of vegetal and animal origin and ii) the expression of genes that could be involved in defence mechanisms of grape to abiotic stress. Large and significant differences (p<0.05) were observed for physical and chemical fertility parameters evaluated. The vineyards characterized by high productivity are those showing a neutral pH, good supply of organic matter and adequate C/N ratio. These soils also showed high biodegradation of organic matter determined through the soil filaments degradation. The ARISA analysis (Amplified Ribosomal Intergenic Spacer Analysis), carried out on DNA samples isolated through an automatic procedure, showed that sites Pio Paulsen and Pio Carenza, characterized by low biodegradation capacity of organic matter, have a reduced genetic similarity compared to sites Chiarafontana and Branco viceversa characterized by high biodegradation capacity. Furthermore, the number of ARISA peaks, index of the number of soil bacterial species, was was statistically lower (p <0.05) in sites Pio Paulsen and Pio Carenza with respect to sites Chiarafontana and Branco. Plants of Campilonghi’s vineyard which is characterized by acid pH, scarce organic matter content, low C/N ratio, limited degradation capacity of filaments and low leaves content of nitrogen and sulphur showed, with respect plants of Pio Paulsen involved as control, up-regulation of WRKY, SuSy, PAL and STS1 genes. In conclusion, the degradative capacity of filaments and the assays of expression of above genes seem to be valid indicators of soil fertility and grape nutritional status.Lo studio della relazione suolo-pianta è un presupposto fondamentale per il controllo vegeto-produttivo del vigneto. In siti vitati della zona D.O.C. di Gambellara, ci si è proposti di studiare la relazione fra la produttività, le principali caratteristiche fisico-chimiche del suolo e alcuni indici innovativi per la diagnosi dello stato nutrizionale della vite quali la capacità biodegradativa della sostanza organica valutata mediante la degradazione di fili di natura vegetale e animale inseriti nel suolo e la valutazione dell’espressione di geni che potrebbero essere coinvolti nei meccanismi di difesa della vite dagli stress abiotici. Ampie e significative differenze (p<0,05) sono state riscontrate fra i parametri di fertilità fisico-chimica esaminati. I vigneti caratterizzati da maggiore produttività sono quelli che evidenziano valori di pH neutro, buona dotazione di sostanza organica e un adeguato rapporto C/N. Questi suoli presentano, inoltre, elevata capacità biodegradativa della sostanza organica determinata in base alla degradazione dei fili immessi nel suolo. L’analisi ARISA (Amplified Ribosomal Intergenic Spacer Analysis), eseguita su campioni di DNA estratto da suolo in maniera automatizzata tramite la messa a punto di un nuovo protocollo, ha evidenziato che i siti Pio Paulsen e Pio Carenza, caratterizzati da bassa attività biodegradativa della sostanza organica, hanno una ridotta similarità genetica rispetto ai siti Chiarafontana e Branco caratterizzati viceversa da pronunciata attività biodegradativa. Inoltre, il numero di picchi ARISA, indice della numerosità delle specie batteriche presenti nel suolo, è risultato statisticamente inferiore (p<0,05) nei siti Pio Paulsen e Pio Carenza rispetto ai siti Chiarafontana e Branco. Nelle piante del sito Campilonghi che è caratterizzato da pH acido, scarsa dotazione di sostanza organica, basso rapporto C/N, limitata attività degradativa dei fili vegetali e da basso contenuto fogliare di azoto e zolfo è stata riscontrata la sovra-espressione, rispetto al sito di controllo Pio Paulsen, dei geni WRKY, SuSy, PAL e STS1. In conclusione, la capacità degradativa dei fili e la valutazione dell’espressione dei suddetti geni sembrano essere dei validi indicatori della fertilità del suolo e dello stato nutrizionale della vite.
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BERTOLINI, MARTINA. "GROUNDWATER BIOREMEDIATION: MICROBIAL POPULATIONS INVOLVED IN CHLOROETHENES AND BTEX CONTAMINATED AQUIFER PROCESSING." Doctoral thesis, Università degli Studi di Milano, 2021. http://hdl.handle.net/2434/809422.
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Groundwater plays an important role in water supply around the world. 2 billion of people use aquifers as drinking water. Consequently, contamination of groundwater has a great social and economic impacts.
The use of organisms (microorganisms and plants) to remediate contaminated matrices, called bioremediation, is becoming more and more frequent. These techniques are cheaper than chemical and physical remediation techniques.
Chloroethenes, aromatic and aliphatic hydrocarbons are widely contaminant compounds because of their intensive use in industrial activity. It is possible to lower their concentration in the environment by means of microbial biodegradation in anaerobic and aerobic conditions.
In this study, an aquifer (located near Porto Marghera, Venice, Italy) contaminated by a leaching from a former landfill was analyzed. The contamination comprised chlorinated solvents, benzene, toluene, ethylbenzene and xylenes (BTEX) and aliphatic hydrocarbons. In 1995, an intervention with a pump and treat reactor was installed. Due to low efficiency and high maintenance costs of the physic-chemical treatment, the installation of a biological treatment, based on two permeable reactive biobarriers, was planned. After preliminary characterization of the microbial community at the site in order to evidence the presence of natural microbial populations involved in decontamination processes, in February 2016 a first biobarrier was installed to stimulate bacterial anaerobic organohalide respiration to dechlorinate chloroethenes. The injection of a reducing substrate was set up to create strong reducing conditions to improve the activity of anaerobic bacteria. A second biobarrier was meant to stimulate bacterial aerobic biodegradation of BTEX and aliphatic hydrocarbons. Urea, ammonium phosphate and O2 were planned to be injected in the aquifer. Moreover, this treatment was also forecasted to be used for complete vinyl chloride aerobic biodegradation.
In order to define the presence of organo-halide respiring bacteria at the aquifer, laboratory-based anaerobic microcosm study was set up. The effect of the biostimulation intervention (i.e., the addition of a reducing substrate) was also monitored in comparison with natural attenuation processes. Chlorinated ethenes were analyzed through gas-chromatography coupled to mass spectrophotometry (GC-MS). At microcosms scale, the natural organohalide respiration activity was influenced by the presence of reducing substrate, showing an increase of dechlorination of highly chlorinated ethenes, with a concomitant accumulation of vinyl chloride. Landfill active microbial community composition was determined through Illumina 16S rRNA sequencing of cDNA from RNA extracted from groundwater samples. Active organo-halide bacteria were quantified by quantitative Real Time PCR (q-PCR). Phylogenetic bacterial biomarkers for Dehalococcoides, Geobacteriaceae, and functional biomarkers tceA and vcrA, coding for chlorinated ethenes reductases, were applied. The ability of aerobic biodegradation of vinyl chloride, BTEX, aliphatic hydrocarbons and chlorobenzene was studied by the Most Probable Number (MPN) technique and q-PCR of etnC and tbmD genes, coding for alkene and toluene-benzene monooxygenases, respectively.
Once established the presence of bacterial natural attenuation activities for all the compounds, chemical and microbiological analyses were performed at field scale in order to monitor the efficacy of the bioremediation treatments. Moreover, the microbial community composition of anaerobic biobarrier was analyzed before and after 22 months of treatment, by 16S rRNA Illumina sequencing. Reducing substrate addition affected the microbial community composition at the site, causing an increase of fermentative bacteria, mainly belonging to Archaea domain, whereas typically recognized bacteria involved in organohalide respiration were not displayed. These data, along with a decrease in chlorinated solvents measured at the site, suggest a possible presence of a still unexplored biodiversity of OHR bacteria and further culturomics efforts will help to elucidate this. At the plume fringe in the aerobic part of aquifer, BTEX, chlorobenzene and aliphatic hydrocarbon degrading bacteria were characterized. Moreover, microbial consortia able to use vinyl chloride as sole carbon and energy form were selected, demonstrating the feasibility to remediate the site from the carcinogenic intermediate of organohalide respiration.
The microbiological work carried out during this Doctorate, along with hydrogeochemical data, demonstrated that a bioremediation intervention could successfully decontaminate this historical and naturalistically important site. Since the beginning of 2020, a full-scale biobarrier plant has been established and it is expected to run for 30 years in order to completely remediate the aquifer.
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Aloisio, Irene <1980>. "Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4420/1/Tesi_Aloisio_Irene.pdf.
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Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics.
The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.
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Aloisio, Irene <1980>. "Therapeutic microbiology: characterization of Bifidobacterium strains for the treatment of enteric disorders in newborns." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4420/.
Full textAbstract:
Several studies support the use of probiotics for the treatment of minor gastrointestinal problems in infants. Positive effects on newborn colics have been evidenced after administration of Lactobacillus strains, whereas no studies have been reported regarding the use of bifidobacteria for this purpose. This work was therefore aimed at the characterization of Bifidobacterium strains capable of inhibiting the growth of pathogens typical of the infant gastro-intestinal tract and of coliforms isolated from colic newborns. Among the 46 Bifidobacterium strains considered, 16 showed high antimicrobial activity against potential pathogens; these strains were further characterized from a taxonomic point of view, for the presence and transferability of antibiotic resistances, for citotoxic effects and adhesion to non tumorigenic gut epithelium cell lines. Moreover, their ability to stimulate gut health by increasing the metabolic activity and the immune response of epithelial cells was also studied. The examination of all these features allowed to identify 3 B. breve strains and a B. longum subsp. longum strain as potential probiotics for the treatments of enteric disorders in newborns such as infantile colics.
The formulation of a synbiotic product with an appropriate prebiotic fiber capable of supporting the growth of the selected Bifidobacterium strains was also considered in this study. In this respect the ability of the 4 selected Bifidobacterium strains to use as the sole carbon source and energy source different polisaccharide fibers was investigated The last phase of the work has been dedicated to the evaluation of the gut microbial diversity in newborns whose mothers has been subjected to antibiotic therapy a few hours before the delivery because of a Streptococcus type B infection. These newborns can represent a possible target for the probiotic strains selected in this work.
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Albuquerque, Edilincon Martins de. "Avaliação do tratamento combinado de lixiviado de aterro sanitário e esgoto sanitário em sistema de lodos ativados." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/18/18138/tde-03092012-090610/.
Full textAbstract:
Um dos principais resíduos gerados nos aterros é o lixiviado, que possui elevada concentração de matéria orgânica biodegradável e refratária e de matéria inorgânica, como nitrogênio amoniacal e metais pesados. O tratamento combinado de lixiviado com esgoto sanitário tem sido utilizado em algumas estações de tratamento de esgoto (ETE) brasileiras. No entanto, o processo ainda sofre vários questionamentos e incertezas, especialmente sobre os efeitos da adição do lixiviado sobre o sistema de tratamento. Nesse contexto, o presente trabalho visou avaliar a eficiência do tratamento combinado de lixiviado/esgoto em sistema de lodos ativados, em diferentes condições. Na primeira etapa desta pesquisa, foram realizados experimentos de tratabilidade em escala de bancada (regime de batelada) utilizando as proporções volumétricas de 0% (controle), 0,2%, 2% e 5% de lixiviado em diferentes condições experimentais. Dentre eles, o Experimento 2 (lixiviado pré-tratado por alcalinização e air stripping) se mostrou mais viável tecnicamente, alcançando eficiências de remoção da DBO, da DQO e do COD acima de 97, 82, 60%, respectivamente, até a proporção de 2% de lixiviado pré-tratado. Na segunda etapa da pesquisa, foi avaliada a tratabilidade utilizando reatores em escala piloto (regime contínuo) tratando esgoto sanitário com 2% (P1) e 0% (P2-controle) de lixiviado pré-tratado, com relação microrganismo-alimento de 0,22 kgDBO/kgSSV.dia, tempo de detenção hidráulica de 24 horas e idade do lodo de 20 dias. Os resultados indicaram viabilidade do tratamento nas condições estudadas com 2% de lixiviado pré-tratado, cujas eficiências médias de remoção da DBO, da DQO, do COD foram de 93, 84 e 60%, respectivamente. Esta pesquisa foi desenvolvida com apoio financeiro (Processo N° 2010/51955-2) da Fundação de Amparo à Pesquisa de São Paulo (FAPESP).The leachate is one of the main wastes generated in landfills, it has a high concentration of biodegradable and refractory organic matter and inorganic matter, such as ammonia and heavy metals. The combined treatment of leachate with sewage has been used in various sewage treatment plants in Brazil. However, there are still many questions and uncertainties about the process, especially the effects of adding leachate on the treatment system. In this context, this study aimed at evaluating the efficiency of combined treatment of leachate/sewage in activated sludge under different conditions. In the first stage of this research, treatability experiments were carried out in a bench scale (SBR) using the volumetric proportions of 0% (control), 0.2%, 2% and 5% leachate under different experimental conditions. The Experiment 2 (leachate pretreated by alkalinization and air stripping) was more technically feasible, achieving efficiencies of removal of BOD, COD and DOC above 97, 82, 60%, respectively, until the proportion of 2% pre-treated leachate. In the second stage of the research was evaluated the treatability using pilot-scale reactors (continuous flow) for sewage treatment with 2% (P1) and 0% (P2-control) leachate pretreated. The operational parameters adopted were food-microorganism rate of 0.22 kgDBO/ kgSSV.d, hydraulic retention time of 24 hours and the sludge retention time of 20 days. The results indicated the viability of the combined treatment with 2% leached pretreated, whose average efficiency of removing BOD, COD, DOC were 93, 84 and 60% respectively. This research was developed with financial support (case No. 2010/2-51955) of Fundação de Amparo à Pesquisa de São Paulo (FAPESP).
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Contador, Luciana. "Étude de la structure chimique et microbiologique de l'interface air-mer en Baie de Guanabara (Rio de Janeiro, Brésil)." Phd thesis, Université Pierre et Marie Curie - Paris VI, 2006. http://tel.archives-ouvertes.fr/tel-00122547.
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La microcouche de surface (SML) correspond au premier millimètre de la colonne d'eau. Située à l'interface air-eau, la SML est un environnement unique, riche en microorganismes, en matières organiques, en hydrocarbures et présentant des conditions extrêmes par l'impact des radiations UV et les fortes concentrations de polluants. Les échantillons ont été prélevés dans la SML et dans l'eau sous-jacente (UW) à 7 stations de la Baie de Guanabara, Brésil. Cette étude a permis de caractériser l'abondance et la diversité des bactéries et des hydrocarbures présents dans la SML de la baie. L'origine des hydrocarbures a été analysée par des marqueurs chimiques par chromatographie en phase gazeuse. Sur le plan microbiologique, 43 souches bactériennes ont été isolées et identifiées. Leur résistance aux radiations UV a été évaluée à l'aide d'un simulateur solaire. La SML de la baie est bien définie par rapport à l'UW et enrichie en hydrocarbures et en bactéries. Le bactérioneuston est plus diversifié que le bactérioplancton. La plupart des souches testées sont très résistantes aux UV et sont des espèces potentiellement intéressantes sur le plan biotechnologique.
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Contador, Luciana. "Etude de la structure chimique et microbiologique de l'interface air-mer en baie de Guanabara (Rio de Janeiro, Brésil)." Paris 6, 2006. http://www.theses.fr/2006PA066158.
Full textAbstract:
La microcouche de surface (SML) correspond au premier millimètre de la colonne d’eau. Située à l’interface air-eau, la SML est un environnement unique, riche en microorganismes, en matières organiques, en hydrocarbures et présentant des conditions extrêmes par l’impact des radiations UV et les fortes concentrations de polluants. Les échantillons ont été prélevés dans la SML et dans l’eau sous-jacente (UW) à 7 stations de la Baie de Guanabara, Brésil. Cette étude a permis de caractériser l’abondance et la diversité des bactéries et des hydrocarbures présents dans la SML de la baie. L’origine des hydrocarbures a été analysée par des marqueurs chimiques par chromatographie en phase gazeuse. Sur le plan microbiologique, 43 souches bactériennes ont été isolées et identifiées. Leur résistance aux radiations UV a été évaluée à l’aide d’un simulateur solaire. La SML de la baie est bien définie par rapport à l’UW et enrichie en hydrocarbures et en bactéries. Le bactérioneuston est plus diversifié que le bactérioplancton. La plupart des souches testées sont très résistantes aux UV et sont des espèces potentiellement intéressantes sur le plan biotechnologique.
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Guenoune, Yanis. "Exposition aux bactéries environnementales dans l’habitat : méthodes de mesure et impacts sur la santé des occupants." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1B061/document.
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La qualité de l’air des environnements intérieurs est essentielle pour la santé. Le manque de renouvellement d’air et l’humidité dans les habitats favorise la prolifération microbienne. Les effets sur la santé sont multiples et souvent associés à des maladies chroniques respiratoires, tel que l’asthme. Ces effets sont plus ou moins graves selon le niveau d’exposition et la vulnérabilité des occupants et le rôle des moisissures est pointé. Cependant, le manque d’outils valides permettant d’évaluer quantitativement l’exposition aux bactéries environnementales constitue une des principales difficultés pour mieux appréhender leur impact sur la santé humaine. Un protocole expérimental basé sur les techniques culturales a été développé et testé au laboratoire pour mesurer la survie des bactéries dans des poussières domestiques collectées au sol. L’analyse de ces poussières a permis de déterminer le temps de survie des bactéries testées. Cependant, les méthodes culturales actuelles sont limitées et n’apportent pas assez d’informations sur la composition de la flore bactérienne dans l’habitat. L’utilisation des méthodes moléculaires, tel que le séquençage haut débit, est nécessaire pour y remédier. Par ailleurs, les poussières domestiques pourraient constituer un substrat intégrateur de l’exposition chronique des occupants. Outre le développement, la standardisation, et la validation d’outils de mesure, une approche globale de sensibilisation et de prévention du risque d’exposition aux contaminants des environnements intérieurs est recommandée, en particulier chez les populations vulnérablesIndoor air quality is essential for health. Lack of ventilation and presence of humidity in habitats promotes microbial growth. The health effects are multiple and often associated with chronic respiratory diseases, such as asthma. These effects are more or less serious depending on the level of exposure and the vulnerability of occupants and the role of mold is pointed out. However, the lack of valid tools for quantitatively assessing exposure to environmental bacteria is one of the main difficulties in better understanding their impact on human health. An experimental protocol based on cultural techniques was developed and tested in the laboratory to measure the survival of bacteria in domestic dust collected on the ground. The analysis of these dusts made it possible to determine the survival time of the bacteria tested. However, current culture methods are limited and do not provide enough information on the composition of the bacterial flora in the habitat. The use of molecular methods, such as high throughput sequencing, is needed to address this. In addition, domestic dust could be an integrating substrate for chronic occupant exposure. In addition to the development, standardization, and validation of measurement tools, a comprehensive approach to raising awareness and preventing the risk of indoor exposure to contaminants is recommended, particularly for vulnerable populations
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Mine, Takashi. "Laser and plasma air decontamination." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/2276/.
Full textAbstract:
This research investigated novel decontamination methods of airborne microorganisms in enclosed public spaces. There are many ways the pathogenic micro-organisms can be transmitted from one body to another, which includes for example, physical contact between the contaminated surface to another, transfer of infected blood from a donor to another medium, or respiratory infections where the large droplets containing micro-organisms caused by talking, sneezing or coughing can infect another whether by direct or close contact, and airborne transmission where the tiny aerosol droplets containing the micro-organisms remain in the air for a long period of time thus spreading to wider areas, making this mode of transmission the most effective and thus dangerous. There are many technique and systems in the market today in the field of air cleaning, and many more under development, these include: ozone, plasma, UV, IR, microwave irradiation, passive solar exposure, pulsed light, electrostatic precipitation, photo-catalytic oxidation etc. However air decontamination using a laser is an unexplored approach. In general two different mechanisms are studied in detail in this research. The possibility of using radiation from the laser and also using plasma and its bi-products were investigated. Many variations and techniques were evaluated for both mechanisms to optimise each decontamination effect. Two types of lasers were used to investigate the concept of using lasers to decontaminate air: a CO2 laser producing a beam at 10.6 μm in the IR region and a KrF excimer laser producing a beam at 248 nm in the UV region. This research was to investigate and make use of the power that is available in the laser in a certain way to decontaminate the air. The effect of laser beam absorption in the presence of microorganisms was modelled in Matlab and this could be used to analyse any wavelength. Two variations of creating a plasma were investigated, one method used a Chang profiled, uniform field electrode and the other used an increased size flat electrode. The plasma produced from these systems emitted radiation around 200 nm to 900 nm. The Chang profiled electrode, which was manufactured in house, was originally designed to be used as a Nitrogen air laser. However, experiments with a purchased Nitrogen laser (detailed in Chapter 3) did not show any significant bacterial killing so the system was modified to be used as a plasma air decontamination device. The electrode was sized 60 mm x 10 mm, and the discharge volume was varied by altering the discharge gap. The effects of various parameters were investigated including: the discharge voltage, type of pre-ionisers to optimise the discharge and air flow shaping through the discharge region. Microbiological experiments conducted using air seeded with microorganisms was used to test the system’s decontamination efficiency. The second plasma system used larger 200 mm x 30 mm aluminium electrodes. Again various parameters were investigated to maximise the discharge stability which included, type of dielectric medium, type of power source, electrical circuit setup, use of laser marked electrodes, air flow shaping and using multiple electrode pairs running off the same power supply. Again, microbiological experiments conducted using air seeded with microorganisms was used to test the system’s decontamination efficiency. Two further systems were built using the results obtained from testing the 200 mm x 30 mm aluminium electrodes, an Industrial Based Air Decontamination Unit and a Ozone Shock Plasma System. Both systems were comprised with multiple pairs of laser marked electrodes with dielectric media and possible addition of flow shaping. The two systems were tested as before with good effect. The developed prototypes can be applied to most applications where air cleanliness is required.
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Thoendel, Matthew James. "Synthesis of the accessory gene regulator autoinducing peptide in Staphylococcus aureus." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2999.
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The accessory gene regulator (agr) quorum-sensing system is one of the major regulators of virulence factor production in the pathogen Staphylococcus aureus. Activation of the system depends on the production and sensing of a cyclic peptide signal called the autoinducing peptide (AIP). The biosynthesis of AIP depends on the coordinated action of the AgrB integral membrane endopeptidase and SpsB signal peptidase to process the peptide precursor AgrD into the final signal structure. The primary goal of this dissertation was to gain further insight on the role of AgrD and AgrB in the AIP biosynthesis mechanism. Studies in Chapter II were undertaken to better understand the role of AgrD domains in AgrB-mediated processing. A series of truncation and site-directed mutagenesis studies identified key residues in the AgrD C-terminus that were essential for AgrB processing and AIP production. In parallel, genetic manipulation of the N-terminal leader and AIP-encoding sequence revealed a role for these segments in AIP processing. For the first time, a complex of AgrD covalently linked to AgrB was identified, supporting proposals that this intermediate is an important precursor to AIP production. In Chapter III structure-function studies were performed on AgrB to gain further insight into the AIP biosynthetic mechanism. Initially, the agrBD genes were subjected to random mutagenesis and screened for deficiencies in AIP production. Single-site mutations at 20 different residues within AgrB and another 14 in AgrD were isolated. Interestingly, new mutations in the AgrD N-terminal leader were identified that affect AIP biosynthesis at different steps. In AgrB, most of the mutations blocked peptidase activity, but charge alterations to the K129-K131 region were defective in a later pathway step, separating the peptidase function from AIP ring formation and transport. To localize the AgrB mutations, we reevaluated the membrane topology using the substituted cysteine accessibility method. Our new model predicts four transmembrane helices and a reentrant loop, with both termini located outside of the cell. Finally, co-immunoprecipitation studies indicate that AgrB forms oligomeric structures within the membrane. Taken together, these findings provide a better understanding of the functional role of specific AgrD and AgrB regions in AIP biosynthesis.
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Delaby, Stéphane. "Étude expérimentale du transport des aérosols dans un espace clos ventilé et impact des principales stratégies d'épuration microbiologique de l'air sur l'exposition des occupants." Thesis, Paris Est, 2008. http://www.theses.fr/2008PEST0071.
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L’exposition aux aérosols microbiologiques présents dans les environnements clos est susceptible de provoquer, chez les occupants, diverses pathologies telles que des infections, des toxi-infections et des allergies. Pour s’en prémunir, diverses stratégies passant notamment par l’emploi de dispositifs épurateurs d’air, ont été développées et commercialisées par les industriels de la ventilation et du traitement de l’air. Cependant, à ce jour, aucune méthodologie d’évaluation y compris normative ne permet d’évaluer la pertinence de ces stratégies. Ce travail de recherche se propose, d’une part, d’appréhender le devenir des aérosols microbiologiques au sein des espaces clos : de la source à l’individu exposé, en explorant le rôle de la ventilation dans ce transport et, d’autre part, d’explorer le gain apporté par les nouvelles technologies de traitement microbiologique de l’air sur l’exposition des occupants. Pour ce dernier point de l’étude, une démarche globale d’évaluation en 3 volets a été adoptée avec l’étude de l’efficacité du ou des principes d’épuration mis en oeuvre, la détermination du rendement intrinsèque en condition dynamique de ces systèmes et l’évaluation du gain apporté par ces derniers sur l’exposition des occupants. Les travaux menés avec les dispositifs épurateurs (filtration et photocatalyse) ont montré que les efficacités intrinsèques des systèmes ne permettent pas de préjuger de leur gain vis-à-vis du niveau de l’exposition des individus lorsqu’ils sont mis en oeuvre en environnement intérieur. Les résultats obtenus ont également mis en évidence que la prise en compte des flux aérauliques et du transport des particules induit par la ventilation et le dispositif épurateur est indispensable à la définition d’une stratégie cohérente de traitement d’airExposure to bioaerosols in indoor environments is associated with a wide range of adverse effects on health including infectious diseases, acute toxic effects and allergies. In order to guard against this phenomenon, the ventilation and air treatment industry has developed and marketed many air control strategies. However, at present, there is no methodology adapted to the evaluation of the relevance of these strategies. The aim of this research work was to characterize, in a first time, the progress of microbiological aerosol from the original source, to their eventual inhalation by person exposed, considering their dissemination through the indoor environments. Secondly, the work consisted of determining the efficiency of air cleaner devices applied to control indoor air quality. For this point, a global approach of evaluation in 3 steps was adopted, consisting of studying the efficiency of the epuration principle implemented, determining the intrinsic performance of the systems in dynamic conditions and their impact on the exposure level of the exposed persons. The tests carried out with air cleaner devices (filtration and photocatalysis) have shown that the intrinsic performance wasn’t able to estimate the beneficial impact of these systems on the exposure level of people when there were applied in indoor environments. So the intrinsic performance of devices is not the single impact factor, the airflow promoted by the device is also a factor to consider. Moreover, the characterization of indoor airflows and airborne particles transport is essential to define a coherent strategy of air treatment
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Reinert, Cristina. "Genes de virulência agr-dependentes em cepas de Staphylococcus aureus resistentes a oxacilina isoladas no Brasil (OU) Genes de virulência agr-dependentes em cepas de Staphylococcus aureus resistentes a oxacilina SCCmec tipo IV isoladas no Brasil." Universidade de São Paulo, 2006. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-16082017-154145/.
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O Staphylococcus aureus é um patógeno extremamente versátil tanto em termos de resistência a antimicrobianos quanto em virulência. O S. aureus resistente a oxacilina (ORSA) adquire a resistência a toda a classe de beta-Iactâmicos através de um cassete cromossômico (SCCmec) que carrega o gene mecA, mas pode carregar outros genes de resistência. A soma desses genes de resistência e de virulência torna o S. aureus um grave problema para hospitais do mundo inteiro, que nos últimos vem se estendendo também à comunidade. Foram estudados 50 isolados de ORSA, dentre os quais 15 pertencentes ao clone endêmico brasileiro (CEB) e 3 cepas SCCmec tipo IV isoladas entre 1995 e 1999. Adicionalmente, 32 amostras ORSA SCCmec tipo IV isoladas no Hospital de Clínicas de São Paulo. As amostras foram analisadas quanto ao perfil de sensibilidade a antimicrobianos, classificação do tipo de SCCmec, perfil de virulência quanto a toxinas e adesinas, classificação do grupo agr (locus regulatório dos genes de virulência) e sua funcionalidade, avaliação da expressão dos genes de toxinas e genotipagens por PFGE e MLST. Observou-se que as cepas CEB são multiresistentes. Já as cepas SCCmec IV apresentam um perfil de sensibilidade maior, uma vez que possuem um tipo de SCCmec que não carrega outros genes de resistência além do gene mecA. As amostras CEB SCCmec IV não apresentaram grandes diferenças no conteúdo de toxinas e adesinas. Apenas as cepas SCCmec IV isoladas entre 1995 e 1999 apresentaram um maior conteúdo de genes de virulência que as isoladas no HC. As cepas SCCmec IV isoladas no Brasil não são altamente virulentas como descrito em outros países. Não possuem fatores de virulência como a Leucocidina Panton-Valentine, toxinas exfoliativas e enterotoxinas. Por outro lado, possuem a alfa-hemolisina e a leucocidina LukD-LukE, toxina ainda pouco estudada, que vem sendo apresentada em pesquisas como causa de lesões oculares graves e diarréias pós-antibioticoterapia. Não foi possível estabelecer uma relação entre o tipo de agr e o perfil de virulência das cepas, uma vez que os perfis foram muito semelhantes mesmo entre cepas de grupos agr diferentes.Staphylococcus aureus is an extremely successful pathogen for it is both highly resistant to antibiotics in addition to being virulent. Methicillin-resistant Staphylococcus aureus (MRSA) acquires resistance to the beta-Iactam antibiotics through the acquisition of a chromosomal cassette (SCCmec) which carries the mecA gene, and can carry other resistance genes. The presence of these genes in S. aureus makes it a serious problem in hospitaIs worldwide. In spite of usually being restricted to the nosocomial environment, over the last few years MRSA has been spreading throughout the community. Fifty nosocomial MRSA strains were studied, including 15 belonging to the Brazilian endemic clone (BEC), 3 type IV SCCmec strains isolated between 1995-1999, and 32 type N SCCmec isolates from the \"Hospital de Clínicas (HC) de São Paulo\". The isolates were analyzed as to their susceptibility profile, SCCmec type, virulence and expression profile (toxins and adhesins), agr group classification and functionality, PFGE and MLST profiles. BEC isolates proved to be multiresistant to antibiotics. Type IV SCCmec strains presented a susceptibility profile to a number of drugs of different antimicrobial classes. BEC and type N SCCmec strains did not present significant differences in their virulence profiles. Only the type IV SCCmec strains isolated in 1995-1999 presented a greater virulence profile than those isolated in the HC. Type IV SCCmec strains isolated in Brazil were not highly virulent as described in other countries. Brazilian isolates usually do not possess virulence factors such as the Panton-Valentine leukocidin, exfoliative toxins and enterotoxins. On the other hand, they usually possess alpha-hemolysin and the LukED leukocidin, which is still very poorly studied that have been presented in papers like cause of serious ocular lesions and post-antimicrobial therapy diarrhea. A relation between the agr type and the virulence profile was not established, for virulence profiles were very similar even between isolates belonging to different agr groups.
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Ciccazzo, S. "DYNAMIC OF BACTERIAL COMMUNITY COLONIZATION IN HIGH-ALTITUDE MOUNTAIN ENVIRONMENTS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229911.
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Glacier forelands after glacier retreat are composed of harsh environmental niches characterized by severe climatic regimes and barren substrate with low total carbon and nitrogen content. Moraines represent ideal sites to study primary succession because glacier retreat releases an incoherent mineral substrate, where the primary succession and plant community establishment are key events in soil formation and fertilization. The underlying mechanisms driving this ecological succession remain still to be deepened. The physical and biogeochemical weathering processes provide soluble plant nutrient elements and when the plant colonization on parent materials occurs, the development of glacier foreland into fertile soils starts through rhizodeposition, exudation of nutritive substances, and decaying biomass. In these conditions, pioneer plants can select rhizosphere microbial communities able to promote plant growth thanks to the interactions with the system nutrient cycling. Moreover the rhizosphere community importantly contributes to the ecosystem functioning and carbon sequestration.
Main purposes of this study were: i) to characterize bacterial communities involved in soil neo-genesis processes under the environmental harsh condition and nutrient scarcity typical of high mountain moraines; ii) to assess the diversity, structure and role of bacterial communities associated to pioneer plant grown in a deglaciated alpine environment.
We studied first the Lobuche glacier moraines (Mount Everest area, Khumbu Valley, Nepal, about 5100 m a.s.l.), where we identified environmental niches characterized by different stages of biotic colonization, from bare mineral substrate to complex biological soil crusts (BSC). Seven sites of mineral proto-soil covered by BSC evidenced the ongoing soil development. The sites differed in several environmental parameters which could indicate different soil quality and developmental stages. Automated ribosomal intergenic spacer analysis (ARISA) showed distinctive bacterial community per each site and differences between the BSC and the below mineral substrate within the same site.
The second studied site was the Weisskugel glacier foreland in the upper Matsch valley within the upper Vinschgau Valley (South Tyrol). Upper Matsch valley showed a wide range of ecosystems: grasslands, bogs and poor fens, vertical rocky walls colonized by lichens, rocky glaciers, isolated pioneer plants, loosely organized floristic proto-communities, transition and mature grassland stages. The study area is below a glacier foreland at 2400 m a.s.l. The vegetation colonization was evolved since 1840 when the Matschter glacier began to retreat. The bacterial communities associated to different environmental matrices i.e. rock surfaces, proto-soils, riparian sediments, lichen thalli, and water springs biofilms were investigated by three molecular techniques with different taxonomic resolutions: denaturing gradient gel electrophoresis (DGGE), length heterogeneity-PCR (LH-PCR), and ARISA. Bacterial communities were mainly composed of Acidobacteria, Proteobacteria, and Cyanobacteria but variations occurred among the sites. Proteobacteria were more represented in sediments, biofilms, and lichens whereas Acidobacteria were mostly found in proto-soils and Cyanobacteria on rocks. Firmicutes and Bacteroidetes were mainly found in biofilms. UniFrac P values confirmed a significant difference among different matrices. Significant differences (P < 0.001) in beta diversity were observed at the genus–species level, except for lichens and rocks which showed a more similar community structure, while two distinct bacterial communities between lichens and rocks were found at deep taxonomic resolution.
In the same alpine ecosystem we investigated the effect of plant species on the rhizobacterial communities of 33 plant individuals belonging to 13 different pioneer species. We compared these bacterial communities to those from similar non vegetated patches as control. To obtain a culture-independent picture of the samples, metagenomic DNA was extracted from both rhizosphere and bulk soil samples and analyzed by ARISA and DGGE. ARISA fingerprinting showed different genetic structure per each plant species, extremely different from bulk soil bacterial communities, and the DGGE analysis showed rhizosphere bacterial communities mainly composed by Acidobacteria and Proteobacteria. Unifrac P values of DGGE results confirmed the rhizosphere effect exerted by the different plant species (P < 0.05). We concluded that in early primary succession pioneer plant species can select distinct rhizobacterial communities. Moreover, the rhizosphere effect on the bacterial communities associated to 21 alpine plants belonging to 14 pioneer species within three floristic communities (RW, FI, M sites) was studied. When little sites surrounded by big stones (safe sites) are filled up of stone debris or mud, opportunistic pioneer plant species settle down and form new floristic consortia strongly influenced by the chemistry of the lytic materials. While RW site was a safe site of early developmental stage, FI site represented an intermediate stage and M site could be considered a later stage where floristic consortia are matured. ARISA fingerprints showed different bacterial genetic structure per each patchy floristic communities which differed also from the bulk soil (BS site) bacterial communities. ANOSIM and PERMANOVA analyses indicated as significant the differences among the ARISA profiles of the sites (P < 0.0001). When plants of the same species occurred within the same site, almost all their rhizosphere bacterial communities clustered together. The Unifrac significance value (P < 0.05) revealed significant differences between BS site and the vegetated sites with a weak similarity to the RW site. The intermediate plant colonization stage, FI site, did not differ significantly from the RW and the M vegetated sites. These results highlighted the peculiar effect of the different floristic communities rhizospheres on their soil bacterial communities. The percentage of N and C in the four soils confirmed a different developmental stage per each of the sites with a kind of gradient from BS through RW, FI up to the M site.
The biodiversity, structure and role of both the overall and the active rhizobacterial communities of the most common floristic associations in the valley (Cetrario Loiseleurenion, Nardion strictae, Festucetum halleri) were studied during the early and late growing season at two different time points of glacier retreat characterized by particular environmental parameters. ARISA and 16S rRNA gene pyrosequencing were applied to metagenomic DNA and RNA. The presence of nitrogen cycle key-players was detected with specific 16S rRNA gene PCR-DGGE and nifH gene pyrosequencing. The overall rhizobacterial community structure of the two transects in the early and late growing seasons were significantly different from the correspondent active rhizobacterial communities (PERMANOVA P < 0.0001). Within the overall bacterial communities, each plot differed significantly according first to the sampling season and then to the soil age, whereas within the active bacterial communities the plots clustered mainly according to the season. A marked shift in active Proteobacteria, Acidobacteria, Planctomycetes highlighted the different between the different vegetation plots, growing seasons and soil ages. Moreover, most of the rhizobacterial communities involved to N-cycle exhibited specific diversity according to the growing season. In high-altitude mountain environment, niches with different soil developmental stages coexist in the same area and different environmental constraints (growing season, site position, plant species) lead to the selection of specific pioneer bacterial communities characterized by peculiar taxonomic patterns and functional diversity.
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Sloan, Tim J. "Agr polymorphisms and exotoxin production in Staphylococcus aureus." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/14075/.
Full textAbstract:
Staphylococcus aureus is a highly successful human pathogen capable of colonising and spreading easily between humans while causing a wide range of infections, including life threatening bacteraemias, endocarditis and pneumonia. Virulence gene expression is regulated primarily by the staphylococcal accessory gene regulator (agr) in a cell density-dependent manner termed quorum sensing. Strains of S. aureus, particularly community-associated methicillin resistant S. aureus (CA-MRSA), carrying the genes for a pore forming exotoxin, the Panton Valentine Leukocidin (PVL), have been spreading worldwide over the last 10 to 15 years. As high level production of exotoxins has been implicated in the success of the CA-MRSA clone USA300, a collection of PVL producing clinical isolates from Nottingham were studied with the aim of understanding why PVL production might vary between strains. Sequence typing of the Nottingham strain collection revealed that high PVL producing ST22 agr group 1 and low PVL producing ST30 agr group 3 strains were the most common types. PVL production was stimulated by addition of the type specific exogenous autoinducing peptide (AIP), which activates the agr sensor AgrC, and inhibited by the AgrC antagonist (ala5)AIP-1 if added before a critical cell population density was reached. Analysis of the pvl promoter identified predicted binding sites for RNA polymerase and the SarA protein family of regulators. Promoter pull down experiments confirmed the binding of several staphylococcal regulators to both the agr and pvl promoters including SarA, SarS, Rot and MgrA indicating that these are likely to play a role in the regulation of PVL production. Analysis of the agr locus of high and low PVL producing strains found that a low PVL producing ST22 agr group 1 strain TS13 had a single nucleotide polymorphism (SNP) conferring an N267I substitution in the cytoplasmic domain region of agrC not present in a high PVL producing ST22 strain TS14. Whole genome sequencing of these strains revealed them to be closely related to each other, differing by less than 200 SNPs across their core genomes. While not possessing an SCCmec element, they carried phages encoding pvl and the immune evasion complex (IEC) comprising staphylokinase, the chemotaxis inhibitory protein (CHIPS) and the staphylococcal complement inhibitor (SCIN). AgrC N267I reduced sensitivity to AIP in a bioluminescent reporter for agr which responds to but does not produce AIP. Introducing AgrC N267I into an agr reporter which both produces and senses AIP resulted in delayed autoinduction. This appeared to explain the delay in AIP autoinduction observed in TS13 as compared with TS14 with resulting low PVL production. Other naturally occurring agrC cytoplasmic mutations including T247I and I311T/A343T reduced AIP sensitivity and were associated with delayed autoinduction and reduced exotoxin production. PVL production is closely regulated by agr with expression mediated by several staphylococcal regulators in a cell population density dependent manner. Mutations in agrC occur naturally, delay autoinduction, can have a marked impact on exotoxin production and may be a form of adaptation to niches where high level agr expression is not required.
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Barlow, Peter George. "The effects of air pollution particles on clearance mechanisms within the lung." Thesis, Edinburgh Napier University, 2004. http://researchrepository.napier.ac.uk/Output/1052591.
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The effects of inhaled air pollution particles on lung clearance mechanisms is an important factor in understanding how the mammalian lung deals with such pollutants and, as such, how exposure to these pollutants can be regulated. The nanoparticle(diameter S lOOnm) and transition metal components of PMIO (particulate matter with a diameter less than lO~m) have been implicated as playing major roles in the impairment of alveolar macrophage function and the subsequent retention of particles in the respiratory system. The aim of this study was to investigate the effects of components of PMIO on macrophage functions both directly, by examining macrophage phagocytosis and migration, and indirectly, by studying peripheral factors affecting macrophagefunction such as recruitment by type II cells and complement based mechanisms. We hypothesised that the alveolar epithelial type II cell line would release leukocyte chemoattractants in response to particle exposure and that this could be measured by use of a macrophage migration assay. A sub-toxic dose (125 ~g/ml)of surrogate air pollutionparticles (fine and nanoparticle carbon black and titanium dioxide) was established by measuring LOH release from a murine alveolar macrophage cell line (1774.2) and an alveolar epithelial type II cell line (L-2) in response to particle exposure. Optimisation ofa chemotaxis assay and measurement of macrophage migration towards conditioned medium obtained from the particle-exposed type II cells was conducted and it was determined that carbon black nanoparticles induced type II cells to secrete a chemoattractant that resulted in significant increases in macrophage migration compared to the negative control. This was in contrast to other particle types tested in this study which did not induce any increases in macrophage migration. It was also hypothesised that complement proteins could be involved in macrophage recruitment to sites of particle deposition and, as such, the migration of macrophages towards particle exposed blood serum was examined in vitro. Foetal bovine serum (FBS) was exposed to fine and nanoparticle caroon black and titanium dioxide (l-Smg/ml) for 2 hours. It was found, in accord with the previous study involving type II cells, that carbon black nanoparticles could activate the generation of chemotactic factors in serum that could subsequently induce significant increases (p < 0.001) in macrophage migration when serum was diluted to 10% using serum-free RPMI 1640 culture medium. This effect could be ameliorated by co-incubating the particle-treated serum in the presence of the antioxidant Trolox suggesting that oxidative stress played a role in the generation of the chemoattractant molecules. However, incubation of the serum with a pure oxidant at a range of doses did not result in the generation of chemotactic molecules suggesting that another factor could be involved in the chemoattractant generation. Further investigation to determine the exact molecular mechanism behind the chemoattractant generation is warranted. In contrast to the previous studies, we have also found evidence that components of PM₁₀ can cause decreased efficacy of macrophage clearance mechanisms in vivo and in vitro. It was hypothesised that PM₁₀ instillation would result in a decrease in macrophage phagocytic potential and an increase in chemotactic potential ex vivo. Rats were instilled with 125 and 250μg of PM₁₀ collected from North Kensington, London or sterile saline (negative control). Post-instillation (18 hours), significantly elevated concentrations of TNFa were detected in the BAL fluid together with a significant increase in the number of BAL neutrophils. Phagocytosis and chemotaxis assays conducted with BAL macrophages ex vivo showed that macrophage migration towards a positive chemoattractant, Zymosan Activated Serum (ZAS), was significantly lower than the macrophages obtained from the negative control rats. Macrophage phagocytosis of latex beads ex vivo was also found to be significantly decreased when PM₁₀ was visible inside the cell. An in vitro study where a macrophage cell line (J774.Al) was exposed to a low dose of nanoparticle carbon black (31.25μg) together with varying concentrations (100μM - 100nM) of zinc chloride (ZnCl₂) was also conducted. Exposure of macrophages to nanoparticle carbon black and zinc chloride alone induced a decrease in macrophage phagocytosis. It was found that when macrophages were co-exposed to nanoparticle carbon black and ZnCl₂, there was an additive decrease in macrophage phagocytic potential. The results contained within this manuscript demonstrate that the components of PM₁₀ can induce adverse effects on specific aspects of macrophage clearance mechanisms, but that nanoparticles can also stimulate the production of chemoattractants to aid in the recruitment of phagocytes and subsequent particle clearance. Although a contrary relationship appears to exist between these findings, the recruitment of leukocytes in response to particulate exposure is a mechanism that supports particle clearance. However, the retardation of phagocytic and chemotactic mechanisms in particle exposed macrophages may help to explain the increased toxicity, inflammation and retention time observed with nanoparticle inhalation.
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Samaké, Abdoulaye. "Processus de transfert vers l'atmosphère et de l'impact sanitaire des émissions biogéniques particulaires." Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAU025/document.
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Les particules en suspension dans l’air (notées « PM » pour « Particulate matter ») sont aujourd’hui au cœur des préoccupations sociétales en raison de leur impact majeur sur la santé publique et leur forte participation au changement climatique. La matière organique (MO) représente généralement la première composante en masse des PM mais reste encore très mal appréhendée, en particulier la fraction organique d’origine biogénique primaire (PBOA). Des sucres primaires sont proposés comme des traceurs moléculaires pour étudier les processus de transport atmosphérique ainsi que pour estimer la contribution des PBOAs à la masse totale des PM. Cependant, les connaissances sont encore très limitées sur leurs distributions spatiales et temporelles (i.e., cycles journaliers, saisonniers et annuels), leurs principales sources d’émissions, ou encore les facteurs environnementaux qui déterminent leurs concentrations atmosphériques. Par ailleurs, si la comprehension du potentiel oxydant (PO) —proxy de l’effet sanitaire des PM— inhérent à la composante chimique des aérosols a relativement bien avancé ces dernières années, la contribution de cette fraction PBOA est encore est très mal connue. Ces différents aspects constituent les objectifs de ce travail de thèse. D’un point de vue méthodologique, nos questions ont été abordées par une approche interdisciplinaire, qui a impliquée l’exploitation statistique d’une large base de données et le couplage de campagnes de terrain spécifiques avec la mise en œuvre d’une stratégie expérimentale novatrice développée pour l’étude simultanée des caractéristiques chimiques et microbiologiques des échantillons prélevés.Dans un premier travail basé sur l’exploitation d’une large base de données, nous avons montré que les PBOAs constituent une fraction très importante des PM en France, independamment de la typologie de l’environnement, contribuant en moyenne annuelle à 13 ± 4 % de la MO dans les PM10. On met en évidence une similitude entre les évolutions temporelles de concentrations et de ratios entre sucres primaires pour des sites localisés dans une même région géographique (jusqu’à une distance inter-sites d’environ 200 km). Ces observations indiquent que la source PBOA est très homogène spatialement sur des distances cohérentes avec celle de grands types d'écosystèmes. Cette observation a ensuite été validée par une expérimentation basée sur deux échantillonnages annuels de terrain qui nous a permis de démontrer (i) que les évolutions journalières des concentrations atmosphériques en sucres primaires sont déterminées par seulement quelques taxons microbiens atmosphériques, variables d’un point de vue regionale ; et (ii) que ces taxons proviennent respectivement de la flore locale et régionale pour les sites d’étude qui sont directement influencés et non par les activités agricoles. Enfin, dans le cadre d’étude de PO, nos résultats ont permis de démontrer (i) que tous les bioaérosols modèles testés possèdent un PO intrinsèque significatif, comparable pour certaines espèces à celui de composants chimiques atmosphériques modèles connus pour leur forte reactivité redox ; et (ii) qu’ils sont capables d’influencer significativement le PO des PM chimiques modèles ou collectées en condition réelle.Ces travaux apportent un nouveau regard sur l’importance massique des PBOAs et des nouvelles connaissances sur les sources et processus dominants conduisant à leur introduction dans l’atmosphère, ainsi que l’influence des facteurs environnementaux sur ces processus. L’ensemble des résultats de ce travail plaide pour une prise en compte systematique des PBOAs dans les modèles de chimie atmosphérique pour une meilleure prédiction de la qualité de l’airAirborne particles (called « PM » for Particulate matter") are nowadays at the core of societal concerns because of their major impact on public health and their strong participation in climate change. Organic matter (OM) generally represents the first mass component of PM but it is still poorly understood, in particular the organic fraction from primary biogenic origin (PBOA). Some specific primary sugars are proposed as molecular tracers to study the atmospheric transport processes as well as to estimate the contribution of PBOAs to the total mass of PM. However, knowledge is still very limited about their spatial and temporal distributions (i.e., daily, seasonal and annual cycles), their main emission sources, or the environmental factors that drive their atmospheric concentrations. Moreover, although the understanding of the oxidative potential (OP) —a proxy of the health effect of PM— inherent in the chemical component of aerosols has progressed quite well in recent years, the contribution of this PBOA fraction is still very poorly understood. These aspects constitute the main objectives of this thesis work. From a methodological point of view, our questions were addressed by an interdisciplinary approach, which involved the statistical exploitation of a large database and the coupling of specific field campaigns with the implementation of an innovative experimental strategy developed for the simultaneous study of the chemical and microbiological characteristics of the samples collected.In a first work based on the exploitation of a large database, we showed that PBOAs constitute a very important fraction of PM in France, regardless of the typology of the environment, contributing on average to 13 ± 4% of the annual MO in PM10. We observed a synchronous temporal trends in both concentrations and ratios between primary sugars species for sites located in the same geographical region (up to an inter-site distance of about 200 km). These observations indicate that the PBOA source is very spatially homogeneous over distances consistent with those of large ecosystem types. This observation was then validated by an experimental approach based on two annual field sampling studies that allowed us to demonstrate (i) that daily changes in atmospheric concentrations of primary sugars are drived by only a few regionally variable atmospheric microbial taxa; and (ii) that these taxa come from local and regional flora for study sites that are directly influenced and not by agricultural activities, respectively. Finally, in the framework of the OP study, our results demonstrated (i) that all the tested model bioaerosols have a significant intrinsic OP, which is comparable for some species to the model atmospheric chemical components known for their high redox reactivity; and (ii) that they can significantly influence the OP of chemical PM models or sampled under real ambient conditions.This work provides a different look into the mass importance of PBOAs and new insights into the dominant sources and processes leading to their introduction into the atmosphere, as well as the influence of environmental factors on these processes. Alltogether these results argue for a systematic consideration of PBOAs in atmospheric chemistry models for better prediction of air quality
38
RIPARI, VALERY. "Pasta madre: tradizione e modernità. Caratterizzazioni microbiologiche, aromatiche e birrificatrici di paste madri naturali." Doctoral thesis, Università Politecnica delle Marche, 2014. http://hdl.handle.net/11566/242876.
Full textAbstract:
L’uso della pasta madre mostra un continuo interesse in ambito scientifico, commerciale e privato a causa dei miglioramenti che il suo uso apporta al prodotto finito. L’uso artigianale della pasta madre naturale è un processo che richiede attenzioni, cure e passione. Abbiamo avuto l’occasione di seguire processi produttivi di pane artigianale con l’uso della pasta madre e ne abbiamo potuto apprezzare tutti gli aspetti più importanti. Inoltre, abbiamo campionato 41 paste madri tradizionali e le abbiamo caratterizzate microbiologicamente attraverso l’uso di diversi metodi molecolari (DGGE; sequenziamento del 16S e del 28S). Abbiamo analizzato la popolazione batterica per verificare se vi fossero ceppi capaci di produrre EPS, oltre che portatori del gene pdc capace di codificare per l’enzima che decarbossila gli acidi fenolici. La degradazione degli acidi fenolici nella farina genera fenoli volatili che rendono il pane più appetibile. I ceppi pdc+ sono stati impiegati in terreni sintetici con aggiunta di acido ferulico e in impasti modello, per valutare la reale capacità di degradare gli acidi fenolici.
Abbiamo, poi creato da zero, secondo metodi tradizionali, alcune nuove paste madri, denominate da noi “ex novo”, utilizzando diversi materiali vegetali come fonte di inoculo iniziale. Abbiamo così potuto valutare come la popolazione microbica si è selezionata nel tempo e se questa possa essere stata influenzata dal materiale di partenza utilizzato. A tale scopo, inoltre, abbiamo impiegato una nuova tecnica molecolare (HRMqPCR) e abbiamo messo a punto il primo metodo HRMqPCR per le analisi di una popolazione fungina a partire dal DNA totale estratto dalle paste madri ex novo.
Utilizzando 4 paste madri, due delle quali collezionate e le altre due ottenute ex novo, abbiamo inoculato del mosto d’orzo per ottenere birre acide di tipo Lambic. Di esse abbiamo monitorato alcuni parametri tecnologici e altri microbiologici.
Le analisi gascromatografiche delle paste madri e delle birre, infine, hanno messo in luce risultati molto interessanti.Interest in sourdoughs has been steadily growing in recent years, whether biologically, technologically, commercially or family oriented. Handcrafted natural sourdoughs require care, concern and dedication. Here we describe handcrafted baking processes, making use of natural sourdoughs, investigated from a microbiological point of view. After sampling 41 traditional sourdoughs, we characterized them microbiologically, by different molecular methods (DGGE; sequencing). We then analyzed our bacterial populations, in order to evaluate if EPS+ strains, as well as their related pdc genes were present. Phenolic acid degradations in flour generates volatile phenolic acids, yielding more desirable bread aromas. pdc+ strains have been used in synthetic-media + ferulic acid, and in model-doughs, in order to evaluate the actual phenolic-acid degrading capacity of each pdc+ strain. According to traditional methods, various dough samples were prepared from scratch, and denoted “ex novo” sourdoughs (PMEN), always making use of various plant materials and organs (natural microbial sources, SNM). We have been able to evaluate microbial population dynamics, and to reflect on possible relations between SNM and final populations. A recent quantitative PCR technique (HRMqPCR) has been optimized for our PMEN microbial populations, possibly for the first time so far. Utilizing 4 sourdoughs (2 collected and 2 ex novo), we also inoculated some malt barley must, in order to obtain sour beers. These fermentations, monitored microbiologically and technologically, showed very interesting results.
39
D'Aimmo, Maria Rosaria <1975>. "Uso di simbiotici e di nitrati come alternativa agli antibiotici in allevamenti zootecnici." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/142/1/Tesi_pdf.pdf.
Full text
40
D'Aimmo, Maria Rosaria <1975>. "Uso di simbiotici e di nitrati come alternativa agli antibiotici in allevamenti zootecnici." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/142/.
Full text
41
Stefanini, Ilaria <1972>. "Uso di probiotici e prebiotici quale barriera a patogeni enterici in suinetti in svezzamento." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/144/1/Tesi_Ilaria_Stefanini.pdf.
Full text
42
Stefanini, Ilaria <1972>. "Uso di probiotici e prebiotici quale barriera a patogeni enterici in suinetti in svezzamento." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/144/.
Full text
43
Sado, Kamdem Sylvain Leroy <1973>. "Effect of diet supplementation in unsaturated fatty acids on meat keeping qualities: study of selected fatty acids antimicrobial properties and inhibition mechanism on Staphylococcus aureus." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/418/1/Manuscript_Tesi_di_Dottorato_Sado_Sylvain.pdf.
Full text
44
Sado, Kamdem Sylvain Leroy <1973>. "Effect of diet supplementation in unsaturated fatty acids on meat keeping qualities: study of selected fatty acids antimicrobial properties and inhibition mechanism on Staphylococcus aureus." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/418/.
Full text
45
Saracino, Pasquale <1977>. "New signalling molecules in some foodborne bacteria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/421/1/Saracino_Pasquale_tesi_dottorato.pdf.
Full text
46
Saracino, Pasquale <1977>. "New signalling molecules in some foodborne bacteria." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2007. http://amsdottorato.unibo.it/421/.
Full text
47
Carri, Simone <1980>. "Attività antagonistica di batteri lattici isolati da salami verso muffe e lieviti." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/697/1/Tesi_Carri_Simone.pdf.
Full text
48
Carri, Simone <1980>. "Attività antagonistica di batteri lattici isolati da salami verso muffe e lieviti." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/697/.
Full text
49
Nissen, Lorenzo <1977>. "Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/790/1/Tesi_Nissen_Lorenzo.pdf.
Full textAbstract:
The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to
select the best Bifidobacterium longum strains suitable to set the basis of our study.
We were looking for strains with the abilities to colonize the intestinal mucosa and
with good adhesion capacities, so that we can test these strains to investigate their
ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are
the two process that we want to study to better understand the role of an adhesion
protein that we have previously identified and that have top scores homologies with
the recent serpin encoding gene identified in B. longum by Nestlè researchers.
Bifidobacterium longum is a probiotic, known for its beneficial effects to the human
gut and even for its immunomodulatory and antitumor activities. Recently, many
studies have stressed out the intimate relation between probiotic bacteria and the GIT
mucosa and their influence on human cellular homeostasis. We focused on the
apoptotic deletion of cancer cells induced by B. longum. This has been valued in
vitro, performing the incubation of three B.longum strains with enterocyte-like Caco-
2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains
tested were defined for their adhesion properties using adhesion and autoaggregation
assays. These features are considered necessary to select a probiotic strain. The three
strains named B12, B18 and B2990 resulted respectively: “strong adherent”,
“adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to
investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted
positive in DNA fragmentation test, only when adherent strains were used (B12 and
B18). These results indicate that the interaction with adherent B. longum can induce
apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the
gastrointestinal tract and in restoring the ecology of damaged colon tissues. These
results were used to keep on researching and the strains tested were used as recipient
of recombinant techniques aimed to originate new B.longum strains with enhanced
capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new
goal it was decided to clone the serpin encoding gene of B. longum, so that we can
understand its role in adhesion and apoptosis induction. Bifidobacterium longum has
immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell
line. It secretes a hypothetical eukaryotic type serpin protein, which could be
involved in this kind of deletion of damaged cells. We had previously characterised a
protein that has homologies with the hypothetical serpin of B. longum (DD087853).
In order to create Bifidobacterium serpin transformants, a B. longum cosmid library
was screened with a PCR protocol using specific primers for serpin gene. After
fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia
coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B.
longum transformation were performed and the best efficiency was obtained using
MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot
assay to detect antigens presence against anti-antitrypsin polyclonal antibody.
The best signal was produced by one starin that has been renamed B. longum BLKS
7. Our research study was aimed to generate transformants able to over express serpin
encoding gene, so that we can have the tools for a further study on bacterial apoptotic
induction of Caco-2 cell line.
After that we have originated new trasformants the next step to do was to test
transformants abilities when exposed to an intestinal cell model. In fact, this part of
the project was achieved in the Department of Biochemistry of the Medical Faculty
of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship
Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic
ability of some bacterial strains using intestinal cells from a 6 years old pig. The use
of intestinal mammalian cells is essential to study this symbiosis and a functional cell
model mimics a polarised epithelium in which enterocytes are separated by tight
junctions.
In this list of strains we have included the Bifidobacterium longum BKS7
transformant strain that we have previously originated; in order to compare its
abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight
Lactobacillus strains of different sources were co-cultured with porcine small
intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell
filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb.
plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian
coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess
reaction, H202 by tetramethylbenzidine reaction and O2
- by cytochrome C reduction.
Cytotoxic effect by crystal violet staining and induction on metabolic activity by
MTT cell proliferation assay were tested too. Transepithelial electrical resistance
(TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to
observe epithelium permeability, decrease during pathogenesis and tissue becomes
permeable to ion passive flow lowering epithelial barrier function. Probiotics can
prevent or restore increased permeability. Lastly, dot-blot was achieved against
Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI
C1 increased slightly after co-culture not affecting mitochondrial functions. No strain
was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as
measured by the release of NO2, H202 and O2
- by PoM2 and PSI C1. During coculture
TER of polarised PSI C1 was two-fold higher comparing with constant TER
(~3000 ) of untreated cells. TER raise generated by bacteria maintains a low
permeability of the epithelium. During treatment Interleukin-6 was detected in cell
supernatants at several time points, confirming immunostimulant activity. All results
were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as
controls. In conclusion we can state that both the list of putative probiotic bacteria
and our new transformant strain of B. longum are not harmful when exposed to
intestinal cells and could be selected as probiotics, because can strengthen epithelial
barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell
model. Indeed, we have found out that none of the strains tested that have good
adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains
tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we
have assayed even the capacity of producing certain citokynes that are correlated with
immune response. The detection of Interleukin-6 was assayed in all our samples,
including B.longum transformant BKS 7 strain, this result indicates that these bacteria
can induce a non specific immune response in the intestinal cells. In fact, when we
assayed the presence of Interferon-gamma in cells supernatant after bacterial
exposure, we have no positive signals, that means that there is no activation of a
specific immune response, thus confirming that these bacteria are not recognize as
pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The
most important result is the measure of Trans Epithelial Electric Resistance that have
shown how the intestinal barrier function get strengthen when cells are exposed to
bacteria, due to a reduction of the epithelium permeability. We have now a new strain
of B. longum that will be used for further studies above the mechanism of apoptotic
induction to “damaged cells” and above the process of “restoring ecology”. This
strain will be the basis to originate new transformant strains for Serpin encoding gene
that must have better performance and shall be used one day even in clinical cases as
in “gene therapy” for cancer treatment and prevention.
50
Nissen, Lorenzo <1977>. "Study of apoptotic deletion mediated by Bifidobacterium longum with construction of recombinant strains for Serpin encoding gene and phenotypes comparison in a pig cell model." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2008. http://amsdottorato.unibo.it/790/.
Full textAbstract:
The first part of the research project of the Co-Advisorship Ph.D Thesis was aimed to
select the best Bifidobacterium longum strains suitable to set the basis of our study.
We were looking for strains with the abilities to colonize the intestinal mucosa and
with good adhesion capacities, so that we can test these strains to investigate their
ability to induce apoptosis in “damaged” intestinal cells. Adhesion and apoptosis are
the two process that we want to study to better understand the role of an adhesion
protein that we have previously identified and that have top scores homologies with
the recent serpin encoding gene identified in B. longum by Nestlè researchers.
Bifidobacterium longum is a probiotic, known for its beneficial effects to the human
gut and even for its immunomodulatory and antitumor activities. Recently, many
studies have stressed out the intimate relation between probiotic bacteria and the GIT
mucosa and their influence on human cellular homeostasis. We focused on the
apoptotic deletion of cancer cells induced by B. longum. This has been valued in
vitro, performing the incubation of three B.longum strains with enterocyte-like Caco-
2 cells, to evidence DNA fragmentation, a cornerstone of apoptosis. The three strains
tested were defined for their adhesion properties using adhesion and autoaggregation
assays. These features are considered necessary to select a probiotic strain. The three
strains named B12, B18 and B2990 resulted respectively: “strong adherent”,
“adherent” and “non adherent”. Then, bacteria were incubated with Caco-2 cells to
investigate apoptotic deletion. Cocultures of Caco-2 cells with B. longum resulted
positive in DNA fragmentation test, only when adherent strains were used (B12 and
B18). These results indicate that the interaction with adherent B. longum can induce
apoptotic deletion of Caco-2 cells, suggesting a role in cellular homeostasis of the
gastrointestinal tract and in restoring the ecology of damaged colon tissues. These
results were used to keep on researching and the strains tested were used as recipient
of recombinant techniques aimed to originate new B.longum strains with enhanced
capacity of apoptotic induction in “damaged” intestinal cells. To achieve this new
goal it was decided to clone the serpin encoding gene of B. longum, so that we can
understand its role in adhesion and apoptosis induction. Bifidobacterium longum has
immunostimulant activity that in vitro can lead to apoptotic response of Caco-2 cell
line. It secretes a hypothetical eukaryotic type serpin protein, which could be
involved in this kind of deletion of damaged cells. We had previously characterised a
protein that has homologies with the hypothetical serpin of B. longum (DD087853).
In order to create Bifidobacterium serpin transformants, a B. longum cosmid library
was screened with a PCR protocol using specific primers for serpin gene. After
fragment extraction, the insert named S1 was sub-cloned into pRM2, an Escherichia
coli - Bifidobacterium shuttle vector, to construct pRM3. Several protocols for B.
longum transformation were performed and the best efficiency was obtained using
MRS medium and raffinose. Finally bacterial cell supernatants were tested in a dotblot
assay to detect antigens presence against anti-antitrypsin polyclonal antibody.
The best signal was produced by one starin that has been renamed B. longum BLKS
7. Our research study was aimed to generate transformants able to over express serpin
encoding gene, so that we can have the tools for a further study on bacterial apoptotic
induction of Caco-2 cell line.
After that we have originated new trasformants the next step to do was to test
transformants abilities when exposed to an intestinal cell model. In fact, this part of
the project was achieved in the Department of Biochemistry of the Medical Faculty
of the University of Maribor, guest of the abroad supervisor of the Co-Advisorship
Doctoral Thesis: Prof. Avrelija Cencic. In this study we examined the probiotic
ability of some bacterial strains using intestinal cells from a 6 years old pig. The use
of intestinal mammalian cells is essential to study this symbiosis and a functional cell
model mimics a polarised epithelium in which enterocytes are separated by tight
junctions.
In this list of strains we have included the Bifidobacterium longum BKS7
transformant strain that we have previously originated; in order to compare its
abilities. B. longum B12 wild type and B. longum BKS7 transformant and eight
Lactobacillus strains of different sources were co-cultured with porcine small
intestine epithelial cells (PSI C1) and porcine blood monocytes (PoM2) in Transwell
filter inserts. The strains, including Lb. gasseri, Lb. fermentum, Lb. reuterii, Lb.
plantarum and unidentified Lactobacillus from kenyan maasai milk and tanzanian
coffee, were assayed for activation of cell lines, measuring nitric oxide by Griess
reaction, H202 by tetramethylbenzidine reaction and O2
- by cytochrome C reduction.
Cytotoxic effect by crystal violet staining and induction on metabolic activity by
MTT cell proliferation assay were tested too. Transepithelial electrical resistance
(TER) of polarised PSI C1 was measured during 48 hours co-culture. TER, used to
observe epithelium permeability, decrease during pathogenesis and tissue becomes
permeable to ion passive flow lowering epithelial barrier function. Probiotics can
prevent or restore increased permeability. Lastly, dot-blot was achieved against
Interleukin-6 of treated cells supernatants. The metabolic activity of PoM2 and PSI
C1 increased slightly after co-culture not affecting mitochondrial functions. No strain
was cytotoxic over PSI C1 and PoM2 and no cell activation was observed, as
measured by the release of NO2, H202 and O2
- by PoM2 and PSI C1. During coculture
TER of polarised PSI C1 was two-fold higher comparing with constant TER
(~3000 ) of untreated cells. TER raise generated by bacteria maintains a low
permeability of the epithelium. During treatment Interleukin-6 was detected in cell
supernatants at several time points, confirming immunostimulant activity. All results
were obtained using Lactobacillus paracasei Shirota e Carnobacterium divergens as
controls. In conclusion we can state that both the list of putative probiotic bacteria
and our new transformant strain of B. longum are not harmful when exposed to
intestinal cells and could be selected as probiotics, because can strengthen epithelial
barrier function and stimulate nonspecific immunity of intestinal cells on a pig cell
model. Indeed, we have found out that none of the strains tested that have good
adhesion abilities presents citotoxicity to the intestinal cells and that non of the strains
tested can induce cell lines to produce high level of ROS, neither NO2. Moreover we
have assayed even the capacity of producing certain citokynes that are correlated with
immune response. The detection of Interleukin-6 was assayed in all our samples,
including B.longum transformant BKS 7 strain, this result indicates that these bacteria
can induce a non specific immune response in the intestinal cells. In fact, when we
assayed the presence of Interferon-gamma in cells supernatant after bacterial
exposure, we have no positive signals, that means that there is no activation of a
specific immune response, thus confirming that these bacteria are not recognize as
pathogen by the intestinal cells and are certainly not harmful for intestinal cells. The
most important result is the measure of Trans Epithelial Electric Resistance that have
shown how the intestinal barrier function get strengthen when cells are exposed to
bacteria, due to a reduction of the epithelium permeability. We have now a new strain
of B. longum that will be used for further studies above the mechanism of apoptotic
induction to “damaged cells” and above the process of “restoring ecology”. This
strain will be the basis to originate new transformant strains for Serpin encoding gene
that must have better performance and shall be used one day even in clinical cases as
in “gene therapy” for cancer treatment and prevention.