La clausine est une bactériocine sécrétée par la souche probiotique Bacillus clausii. Elle est active contre des bactéries multi-résistantes aux antibiotiques, comme le MRSA (Staphylococcus aureus résistant à la méticilline) et le VRE (Enterococcus résistant à la Vancomycine). Nous avons produit, purifié, caractérisé la structure de la clausine et effectué des expériences d'interaction par Résonance Magnétique Nucléaire (RMN) du liquide. La clausine s'est révélée être un nouveau peptide maturé de la famille des lantibiotiques. Il est composé de trois ponts thioéthers ou lanthionines, d'un pont aminovinyle et de trois résidus deshydratés. La clausine possède une structure tri-dimensionnelle bi-modulaire, similaire à la fois à la nisine et à la mersacidine. Comme ces dernières, elle s'est révélée se lier aux lipides bactoprénols, tels que le lipide II (LII) et l'UndécaprénylPyroPhosphate (UPP), empêchant ainsi la formation de la paroi bactérienne. Nous nous sommes ensuite focalisés sur le mode d'action de la clausine sur des modèles de membranes bactériennes. Dans ce but, nous nous sommes basés sur la composition phospholipidique de trois bactéries affectées par la clausine : Staphylococcus aureus, Micrococcus Luteus et Bacillus Megaterium. Nous avons utilisé la RMN du solide du 2H et du 31P pour caractériser l'effet de la clausine sur la dynamique membranaire, ainsi que l'éllipsométrie et l'épifuorescence avec le LII-dansylé pour visualiser son effet sur des monocouches lipidiques. La clausine seule a entraîné une désorganisation membranaire à fort ratio peptide/lipide et formé des domaines. Nous avons souligné l'importance de la tête polaire des bactoprénols sur la dynamique lipidique. Nous avons finalement observé un effet synergique de la clausine avec les bactoprénols, suggèrant la formation de domaines « raft-like » organisés en réseau avec l'UPP ou en fibre avec lelipide II. Nos résultats in vitro permettent d'expliquer les divisions cellulaires aberrantes observées in vivo par un mécanisme de séquestration des bactoprénols par les peptides lantibiotiques. / Clausin is a bacteriocin secreted by the probiotic strain Bacillus clausii. It is active against multi-resistant bacteria to antibiotics, such as MRSA (Methicillin-Resistant Staphylococcus Aureus) and VRE (Vancomycin-Resistant Enterococcus).We have produced, purified, characterized the structure of Clausin and performed binding studies with Liquid-state Nuclear Magnetic Resonance (NMR). Clausin has been shown to be a new mature peptide from the lantibiotic family. It consists of three thioether rings called lanthionines, an aminovinyl ring and three dehydrated residues. Clausin displays a bi-modular three-dimensional structure similar to nisin as well as to mersacidin. It binds as well to bactoprenol lipids, such asLipid II (LII) and UndecaprenylPyroPhosphate (UPP), blocking then bacterial cell wall formation.We have then focused our attention to the action mode of Clausin on bacterial membrane models. For this purpose, we have used the phospholipid composition of three bacteria affected by Clausin: Staphylococcus aureus, Micrococcus Luteus and Bacillus Megaterium. We have used 2H and 31P Solid-state NMR for characterizing the Clausin effect on lipid dynamics, as well as ellipsometry and epifluorescence with dansylated-LII for visualizing its effect on lipid monolayer. Clausin alone has led to membrane desorganization at a high peptide/lipid ratio and formed domains. We have further underlined the importance of the polar head-group of bactoprenols on lipid dynamics. We have finally observed a synergic effect of Clausin with bactoprenols on lipid dynamics suggesting the formation of « raft-like » domains organized in network with UPP or evenfiber with LII. Our in vitro results allow us to explain the aberrant bacterial cell division observed in vivo through a mechanism of bactoprenol sequestration by lantibiotic peptides.

abstract: Phytoplankton comprise the base of the marine food web, and, along with heterotrophic protists, they are key players in the biological pump that transports carbon from the surface to the deep ocean. In the world's subtropical oligotrophic gyres, plankton communities exhibit strong seasonality. Winter storms vent deep water into the euphotic zone, triggering a surge in primary productivity in the form of a spring phytoplankton bloom. Although the hydrographic trends of this "boom and bust" cycle have been well studied for decades, community composition and its seasonal and annual variability remains an integral subject of research. It is hypothesized here that proportions of different phytoplankton and protistan taxa vary dramatically between seasons and years, and that picoplankton represent an important component of this community and contributor to carbon in the surface ocean. Monthly samples from the Bermuda Atlantic Time-series Study (BATS) site were analyzed by epifluorescence microscopy, which permits classification by morphology, size, and trophic type. Epifluorescence counts were supplemented with flow cytometric quantification of Synechococcus, Prochlorococcus, and autotrophic pico- and nanoeukaryotes. Results from this study indicate Synechococcus and Prochlorococcus, prymnesiophytes, and hetero- and mixotrophic nano- and dinoflagellates were the major players in the BATS region plankton community. Ciliates, cryptophytes, diatoms, unidentified phototrophs, and other taxa represented rarer groups. Both flow cytometry and epifluorescence microscopy revealed Synechococcus to be most prevalent during the spring bloom. Prymnesiophytes likewise displayed distinct seasonality, with the highest concentrations again being noted during the bloom. Heterotrophic nano- and dinoflagellates, however, were most common in fall and winter. Mixotrophic dinoflagellates, while less abundant than their heterotrophic counterparts, displayed similar seasonality. A key finding of this study was the interannual variability revealed between the two years. While most taxa were more abundant in the first year, prymnesiophytes experienced much greater abundance in the second year bloom. Analyses of integrated carbon revealed further stark contrasts between the two years, both in terms of total carbon and the contributions of different groups. Total integrated carbon varied widely in the first study year but displayed less fluctuation after June 2009, and values were noticeably reduced in the second year. / Dissertation/Thesis / M.S. Microbiology 2010

Arbuscular mycorrhizas (AM) are the plant root-fungus interactions that are most widespread mycorrhiza in nature. As classically defined, there are two major AM morphologies named after the plant genera in which they were first described: Arum- (intercellular hyphae with arbuscules mainly in inner root cortex), Paris- (extensive hyphal coils in outer root cortex), as well as intermediate morphotypes. In this study, dandelions and chives harvested in Saskatoon (SK, Canada) were examined for AM colonization and morphological types. A Multiple Quantitation Method (MQM) was used for assessing fungal colonization intensity using magnified epifluorescence images of lactofuchsin stained roots, plus details analyzed by high-resolution confocal fluorescence imaging. The results showed that host plants harbored diverse endorhizal fungi, including arbuscular mycorrhizal fungi (AMF), septate endophytes (SE) and fine endophytes (FE), with varying abundances. The soil properties were assessed with respect to P status, organic matter and pH, but there was no correlation with the fungal abundance in this study. Both dandelion and chive roots had Arum- and Paris-type AM. In order to assess the applicability of a current model, I studied quantitative relationship between the cell packing pattern and AM morphotype. Cross sections of host roots were analyzed with Image J software to calculate the proportion of air spaces. The abundance of arbuscules (Arum-type) and hyphal coils (Paris-type) were significantly different in chive and dandelion roots. However, there was no difference in the proportion of air spaces in the inner or outer cortex. Therefore, host root cell packing does not appear to influence AM morphotype at least in the samples in this study. AM fungal diversity was preliminarily investigated by nested PCR with group specific primers, showing multiple PCR bands within root samples, and indicating the potential complexity of AMF groups. Further work to sequence the PCR products is needed to elucidate the AMF groups present.

Arbuscular mycorrhizas (AM) are the plant root-fungus interactions that are most widespread mycorrhiza in nature. As classically defined, there are two major AM morphologies named after the plant genera in which they were first described: Arum- (intercellular hyphae with arbuscules mainly in inner root cortex), Paris- (extensive hyphal coils in outer root cortex), as well as intermediate morphotypes. In this study, dandelions and chives harvested in Saskatoon (SK, Canada) were examined for AM colonization and morphological types. A Multiple Quantitation Method (MQM) was used for assessing fungal colonization intensity using magnified epifluorescence images of lactofuchsin stained roots, plus details analyzed by high-resolution confocal fluorescence imaging. The results showed that host plants harbored diverse endorhizal fungi, including arbuscular mycorrhizal fungi (AMF), septate endophytes (SE) and fine endophytes (FE), with varying abundances. The soil properties were assessed with respect to P status, organic matter and pH, but there was no correlation with the fungal abundance in this study. Both dandelion and chive roots had Arum- and Paris-type AM. In order to assess the applicability of a current model, I studied quantitative relationship between the cell packing pattern and AM morphotype. Cross sections of host roots were analyzed with Image J software to calculate the proportion of air spaces. The abundance of arbuscules (Arum-type) and hyphal coils (Paris-type) were significantly different in chive and dandelion roots. However, there was no difference in the proportion of air spaces in the inner or outer cortex. Therefore, host root cell packing does not appear to influence AM morphotype at least in the samples in this study. AM fungal diversity was preliminarily investigated by nested PCR with group specific primers, showing multiple PCR bands within root samples, and indicating the potential complexity of AMF groups. Further work to sequence the PCR products is needed to elucidate the AMF groups present.

L'arme blanche apparaît comme un des instruments le plus fréquemment rencontré en matière judiciaire. Or, selon le principe de transfert de traces de Locart, une arme entrant en contact avec un corps laisse une signature lésionnelle. L'analyse des lésions osseuses engendrées devient alors cruciale afin de rechercher tout indice permettant de remonter à l'agent vulnérant utilisé pour commettre le crime voire à l'identité de l'auteur. Ces travaux de thèse se sont attachés à répondre à cette problématique à travers un nouvel outil d'analyse non délabrant pour l'os, le macroscope à épifluorescence. A travers des études expérimentales et de validation basées majoritairement sur du matériel osseux humain, nous avons pu déterminer des modèles lésionnels sommant les différentes caractéristiques de chaque instrument et apporter des éléments d'orientation permettant de reconstituer le mécanisme de production de la lésion. L'orientation des écailles, des fissures et la compression osseuse du sillon apparaissent comme des éléments fondamentaux permettant de reconstituer la manière dont le coup a été porté dans l'espace ainsi que les positions de l'assaillant et de la victime. Il a été également possible de mettre en évidence des signatures diagnostiques individuelles qui, sous couvert de comparatifs lésionnels, relie de manière exclusive une lésion à un agent vulnérant. Néanmoins, face à la demande croissante de fiabilité et de niveau de preuve, il est devenu nécessaire pour les experts d'apporter une estimation de la validité et du taux d'erreur de leurs observations et conclusions. C'est pourquoi nous avons conduit une étude de validation, réalisée sur les instruments tranchants. Elle a permis d'estimer la valeur du diagnostic lésionnel anthropologique dans le cadre de telles expertises. Nous avons montré que les valeurs prédictives positives et négatives étaient élevées, mais étroitement corrélées au degré d'expérience de l'observateur. Un œil expérimenté s'avère indispensable pour aboutir à une expertise fiable. Enfin, ces différents travaux ont été mis en pratique dans des cas concrets à la fois médico-légaux actuels et archéologiques. Ces applications nous ont permis de confirmer la validité de ce type nouveau d'analyse microscopique avec comme perspective principale l'intégration progressive de cet outil dans des expertises tant contemporaines qu'archéologiques. / Sharp force trauma generated by knives is one of the most common forms of armed violence encountered in judicial procedures. According to Locart's principle of "transfer of traces", a weapon can leave a lesion imprint on the bone. That's why cut mark analyses are important as they allow identification of the weapon class or even the perpetrator of the crime. This thesis addresses this problem by using a new tool that does not alter the bone, i.e. the epifluorescence macroscope. Through experimental and validation processes using mainly human bones, we have been able to identify lesion models exemplifying the characteristics per weapon class, allowing us to model kerfs for each weapon. By the means of fluorescence and comparative analysis, our study also demonstrates that it is possible to highlight special characteristics identifying the unique blade responsible for the imprints observed on a bone. Moreover, we demonstrate for the first time microscopic criteria of directionality, using fluorescence excitation. Orientation of cracks, flakes, and lateral pushing back especially help determine the trajectory of the blow as well as the positions of the aggressor and victim. Nevertheless, scientific evidence is admissible if it is based on a scientific technique accepted as reliable within the scientific community and with judges. The admissibility of expert testimony under this new standard requires an estimation of the validity of the data and proof reliability (the rate of error in observations and conclusions). Our validation study demonstrated a high level of negative and positive predictive value of such diagnoses, but showed that this is closely linked to the observer's level of experience. A highly-experienced regard is essential for a reliable expertise. Thanks to these studies, we have had the opportunity to examine forensic and archaeological cases. These applications have confirmed that this technique could provide a useful preliminary tool for nondestructive examination in dismemberment cases, whether on ancient or contemporary bones.

This research presents the results on an experimental investigation to identify the effect of polyphosphoric acid (PPA) on aging characteristics of an asphalt binder. Addition of PPA to asphalt binders is said to improve performance of flexible pavements. Asphalt binder PG 64-22 in modified and unmodified conditions was subjected to aging in the laboratory using a regular oven and also simulated short term aging using rolling thin film oven (RTFO) test. Aging experiments were conducted to analyze the extent of oxidation in terms of changes in molecular structure of the asphalt binder. These changes were appraised using Fourier transform infrared (FTIR) spectroscopy, dynamic shear rheometer (DSR), and epifluorescence microscopy tests. FTIR was used to determine the changes in major bands with addition of PPA. Stiffness and viscoelastic behaviors of asphalts were determined from the DSR test. The stiffness is measured by calculating the shear modulus, G* and the viscoelastic behavior is measured by calculating the phase angle, sin δ. Epifluorescence microscopy is a tool used to study properties of organic or inorganic substances. The morphological characteristics of PPA modified asphalt samples were observed through epifluorescence microscopy. Epifluorescence microscopy reveals the polymer phase distribution in the asphalt binders. Results of this investigation show PPA addition to asphalt binders improve G*/sin δ characteristics of asphalt binders. In addition, presence of PPA in polymer containing asphalt did not adversely affect aging of the binders.

No description available.

The abiotic oxidation of pyrite requires the supply of oxygen and water only. In abiotic systems, pyrite oxidation may proceed via several paths, with multiple steps in each of the paths. Defect sites (S-deficient, Fe3+ bearing sites) on the pyrite surface have been shown reported to be the initial reaction sites on pristine pyrite surfaces. In neutral to slightly acidic solutions (3.5<pH<7), ferric iron hydroxide patches will form on the surface. These patches have been shown to be the predominant sites for electron exchange. Efforts were undertaken to suppress the electron transfer at these sites to inhibit pyrite oxidation. It has been shown that pyrite oxidation can be controlled by exposing the pyrite to phosphate under relatively high pH values (pH above 5.0). However, phosphate ceases to function as an inhibitor under lower pHs. The use of two-tail phospholipids instead of phosphate to inhibit the pyrite oxidation proved to be very effective under abiotic conditions. The purpose of the present study is to determine if the use of two-tail phospholipids can be extended to systems that have bacteria present. Batch experiments were conducted in which pyrite slurries were treated with two-tailed lipid either before or after exposure to bacteria. Iron release into the solution was used as a reaction progress variable and Atomic Force Microscopy was used to study the distribution of lipids and bacteria on the pyrite surface. AFM images showed that the formation of 7nm lipid bilayers contributes to the majority of lipid structures on pyrite surface. The bilayers render the pyrite surface hydrophobic and inhibit the reaction of water with the surface, which is known to be a critical reactant. AFM images also showed that phospholipids are capable of displacing a large fraction of bacteria attached to pyrite, reducing the oxidation rate of the mineral. However, addition of heterotrophic bacterial (Acidiphilum acidophilum) to the system resulted in the increase in pyrite oxidation rate again. Cross-linking of the 23:2 dyne phospholipids by exposing the lipid to UV light greatly enhanced the stability of the lipid in the presence of the heterotrophic bacteria. UV pretreated lipid layers reduced pyrite oxidation in the presence of heterotrophic bacteria for up to 30 days. / Chemistry

La maîtrise de l'hygiène des surfaces demeure une préoccupation constante dans de nombreux secteurs d'activité. En effet, la contamination microbiologique des surfaces peut être à l'origine de problèmes de santé publique plus ou moins sévères dans les industries agro-alimentaires (toxi-infections alimentaires) ou le milieu médical (infections nosocomiales). L'efficacité des agents désinfectants restant variable d'une application à l'autre (notamment sur des micro-organismes adhérant), il s'avère nécessaire d'améliorer les formulations désinfectantes et/ou les procédures de désinfection qui y sont associées. Cette amiélioration passe par la mise en place de méthodes tests permettant d'évaluer rapidement l'activité antimicrobienne de produits commercialisés ou non, dans des conditions proches de la réalité. Ainsi, au cours de ce travail, nous avons défini un protocole pour déterminer l'activité létale des produits désinfectants sur cellules adhérentes, protocole composé de quatre étapes principales : i) choix des micro-organismes et standardisation de leurs conditions de conservation et de croissance, ii) mise en place d'un protocole d'adhésion reproductible sur supports conditionnés ou non, iii) test de désinfection sur cellules adhérentes et iv) optimisation de ce protocole dans le caractère in situ et le délai d'obtention des résultats par l'utilisation de marqueurs fluorescents, indicateurs de la viabilité cellulaire.

No description available.