abstract: Many previous investigators highlight the importance of snowfall to the water supply of the western United States (US). Consequently, the variability of snowpack, snowmelt, and snowfall has been studied extensively. Snow level (the elevation that rainfall transitions to snowfall) directly influences the spatial extent of snowfall and has received little attention in the climate literature. In this study, the relationships between snow level and El Niño-Southern Oscillation (ENSO) as well as Pacific Decadal Oscillation (PDO) are established. The contributions of ENSO/PDO to observed multi-decadal trends are analyzed for the last ~80 years. Snowfall elevations are quantified using three methods: (1) empirically, based on precipitation type from weather stations at a range of elevations; (2) theoretically, from wet-bulb zero heights; (3) theoretically, from measures of thickness and temperature. Statistically significant (p < 0.05) results consistent between the three datasets suggest snow levels are highest during El Niño events. This signal is particularly apparent over the coastal regions and the increased snow levels may be a result of frequent maritime flow into the western US during El Niño events. The El Niño signal weakens with distance from the Pacific Ocean and the Southern Rockies display decreased snow level elevations, likely due to maritime air masses within the mid-latitude cyclones following enhanced meridional flow transitioning to continental air masses. The modulation of these results by PDO suggest that this El Niño signal is amplified (dampened) during the cold (warm) phase of the PDO particularly over Southern California. Additionally, over the coastal states, the La Niña signal during the cold PDO is similar to the general El Niño signal. This PDO signal is likely due to more zonal (meridional) flow throughout winter during the cold (warm) PDO from the weakening (strengthening) of the Aleutian low in the North Pacific. Significant trend results indicate widespread increases in snow level across the western US. These trends span changes in PDO phase and trends with ENSO/PDO variability removed are significantly positive. These results suggest that the wide spread increases in snow level are not well explained by these sea surface temperature oscillations. / Dissertation/Thesis / Ph.D. Geography 2011

L’océan Pacifique est le siège de variabilités climatiques interannuel et multi-décennale, El Niño Southern Oscillation (ENSO) et la Pacific Decadal Oscillation (PDO), dont les répercussions (via des téléconnections) peuvent être mondiales. Des impacts importants sur les populations, les activités socio-économiques et sur l’environnement ont été attribuées à ENSO. Il est alors primordial d’améliorer notre compréhension de la dynamique Pacifique et notamment du phénomène ENSO ainsique son évolution sous l’effet du changement climatique.Les mesures géochimiques (Sr/Ca et 818O) réalisées sur les coraux constituent des enregistrements paléoclimatiques de choix pour l’étude de l’évolution d’ENSO et sont essentielles pour mettre en perspective la dynamique actuelle du climat par rapport à sa dynamique passée. Après avoir évaluer la robustesse du paléothermomètre géochimique corallien (Sr/Ca), cette thèse a permis la reconstruction de température de surface océanique (SST) à partir de coraux de l’atoll de Clipperton (Pacifique tropical Est) et de l’archipel des Marquises (Pacifique tropical centre) couvrantplusieurs parties du dernier millénaire. Nos résultats suggèrent que la structure spatiale d’ENSO étaitplutôt stable au cours des deux derniers siècles, montrant majoritairement une structure de type ENSOcanonique (Est Pacifique) par opposition à l’ENSO Modoki (centre Pacifique). Bien qu’encore débattue, cette structure spatiale pourrait avoir évoluée très récemment, en liaison avec le changement climatique global (et cela pourrait continuer dans le futur). A l’échelle décennale, nos deux zones d’étude (centre et Est Pacifique) sont influencées par la PDO.Les résultats de cette thèse tendent également à suggérer que l’activité d’ENSO actuelle (sous l’effet du forçage anthropique) n’est pas atypique à l’échelle du dernier millénaire. En effet, son intensité et sa fréquence étaient plus fortes au début du petit âge glaciaire (LIA, 16ème siècle). La comparaison deces résultats avec un ensemble de simulations climatiques (PMIP3) montre que la variabilité ENSO estbien reproduite par ces modèles climatiques mais qu’ils échouent à reproduire correctement l’état moyen des températures du Pacifique. / The Pacific Ocean is the place of interannual and multi-decadal climate variabilities, namely the El Niño Southern Oscillation (ENSO) and the Pacific Decadal Oscillation (PDO). There can have globals impacts via teleconnections. Major impacts on populations, economic and environmental activitieshave been attributed to ENSO. It is therefore essential to improve our understanding of the Pacificdynamic, particularly ENSO activity and its evolution under recent climate change.Geochemical measurements (Sr/Ca and 818O) performed on corals are relevant paleoclimatic records for studying the evolution of ENSO and are essential to put into perspective the current climatedynamic in comparison to past climate.After an evaluation of the robustness of the coral geochemical paleothermometer (Sr/Ca), we present the reconstruction of sea surface temperature (SST) from Eastern tropical Pacific coral (Clippertonatoll) and central tropical Pacific coral (Marquesas archipelago) covering several parts of the last millennium. Our results suggest that ENSO spatial pattern was relatively stable over the past two centuries, mainly indicating an eastern Pacific ENSO pattern (canonical) in comparison to the centralPacific ENSO (Modoki). Although still debated, this spatial pattern could have recently changed dueto global climate change (and this could continue in the future). At the decadal timescale, both studiedareas (central and eastern Pacific) are influenced by the PDO.The results of this Phd thesis also suggest that the present day ENSO activity (under the influence ofanthropogenic forcing) is not atypical throughout the last millennium. The intensity and frequency of ENSO were stronger in the early Little Ice Age (LIA, 16th century). These results are compared withan ensemble of climate simulations (PMIP3) and indicate that ENSO variability is correctly reproduced by numerical climate models but that these models fail to correctly reproduce the mean temperature state of the Pacific.

A importância do Estado de São Paulo (ESP) é notável no desenvolvimento do Brasil, seja no setor econômico ou energético, o que justifica o estudo do comportamento do clima nessa região. O conhecimento da variabilidade da precipitação é imprescindível na gestão de recursos hídricos e possui grande impacto na agricultura e geração de energia por meio de fontes hidrelétricas. Estudos anteriores apontaram efeitos não-lineares do El Niño-Oscilação Sul (ENOS) sobre a precipitação no ESP; entretanto, nenhum estudo específico acerca da influência da Oscilação interdecadal do Pacífico (ODP) nesta área foi ainda realizado, embora haja alguns impactos conhecidos na América do Sul. Deste modo, este trabalho estudou a relação entre anomalias de precipitação no ESP e a ODP, no período de 1901 a 2007, de forma a auxiliar as pesquisas na linha da previsão climática nessa região do Brasil. Na primeira etapa, foram descritos os regimes de precipitação tanto para a América do Sul como localmente, para o ESP, onde se destacaram fatores como a topografia e a influência do Oceano Atlântico. Posteriormente, foram calculados quantis anuais e mensais que permitiram classificar cada evento quanto ao total de precipitação. Regiões pluviometricamente homogêneas foram determinadas no ESP com base na climatologia e nos quantis de precipitação. Notou-se a relação construtiva entre eventos ENOS e as fases da ODP, com máximo durante o verão austral. Os sinais da ODP são percebidos em todo o ESP principalmente na primavera e no verão austrais. Uma análise complementar mostrou que as fases da Oscilação Multidecadal do Atlântico (AMO) também contribuem para a precipitação no ESP durante o verão e a primavera austrais no litoral, durante o verão no interior, e ao longo da primavera na região da Serra da Mantiqueira. Aparentemente, não há relação entre os eventos ENOS e a AMO. / São Paulo State (SPS) is remarkably important to the development of Brazil, economically or energetically, and this justifies climate studies on that region. Knowing rainfall variability is essential to water resources management and it has a great impact on agriculture an power production by hydroelectric power plants. Previous studies have detected non-linear effects of El Niño-Southern Oscillation (ENSO) on SPS rainfall however no specific work deals with PDO influence in this area besides some impacts on South America are known. Therefore this work has studied the relationship between rainfall anomalies in SPS and PDO from 1901 to 2007 to contribute to the climate forecasting improvement. First it was described the rainfall regime in South America, and locally in SPS where topography and the Atlantic Ocean influences were of special importance. Then annual and monthly quantiles were calculated to allow the classification of events according to rainfall totals. Rainfall homogeneous regions were established in SPS using climatology and quantiles. It was observed the constructive relationship between ENSO events and PDO phases, mainly on austral summer. PDO signals were noticed all over the SPS mostly on austral spring and summer. An additional analysis showed that Atlantic Multidecadal Oscillation (AMO) phases also contribute to SPS rainfall during austral summer and spring at the coast, only on summer at the country and during spring at the Mantiqueira Slopes. Apparently, there is no relation between ENSO events and AMO phases.

This thesis describes the geomorphology and morphodynamics of two embayed, sandy, macrotidal beach-dune systems in the Cape St. James region of Gwaii Haanas National Park Reserve and Haida Heritage Site. Gilbert Bay beach is a small embayment with a southwest aspect that exhibits prograding dune ridges. Woodruff Bay beach, a larger system with a SE aspect, is characterized by large erosional scarps on the established foredune. Aspect to erosive conditions and embayment size control the distinct morphologic responses of these beach-dune systems. The morphodynamic regime at Cape St. James consists of high onshore sediment transport potential combined with an increasingly erosive water level regime that is forced by PDO and ENSO climatic variability events. Conceptual models of potential future responses of these beaches to sea level rise show a possible landward migration of up to 3.5 m at Gilbert Bay beach and up to 4 m at Woodruff Bay beach.

This thesis describes the geomorphology and morphodynamics of two embayed, sandy, macrotidal beach-dune systems in the Cape St. James region of Gwaii Haanas National Park Reserve and Haida Heritage Site. Gilbert Bay beach is a small embayment with a southwest aspect that exhibits prograding dune ridges. Woodruff Bay beach, a larger system with a SE aspect, is characterized by large erosional scarps on the established foredune. Aspect to erosive conditions and embayment size control the distinct morphologic responses of these beach-dune systems. The morphodynamic regime at Cape St. James consists of high onshore sediment transport potential combined with an increasingly erosive water level regime that is forced by PDO and ENSO climatic variability events. Conceptual models of potential future responses of these beaches to sea level rise show a possible landward migration of up to 3.5 m at Gilbert Bay beach and up to 4 m at Woodruff Bay beach.

A importância do Estado de São Paulo (ESP) é notável no desenvolvimento do Brasil, seja no setor econômico ou energético, o que justifica o estudo do comportamento do clima nessa região. O conhecimento da variabilidade da precipitação é imprescindível na gestão de recursos hídricos e possui grande impacto na agricultura e geração de energia por meio de fontes hidrelétricas. Estudos anteriores apontaram efeitos não-lineares do El Niño-Oscilação Sul (ENOS) sobre a precipitação no ESP; entretanto, nenhum estudo específico acerca da influência da Oscilação interdecadal do Pacífico (ODP) nesta área foi ainda realizado, embora haja alguns impactos conhecidos na América do Sul. Deste modo, este trabalho estudou a relação entre anomalias de precipitação no ESP e a ODP, no período de 1901 a 2007, de forma a auxiliar as pesquisas na linha da previsão climática nessa região do Brasil. Na primeira etapa, foram descritos os regimes de precipitação tanto para a América do Sul como localmente, para o ESP, onde se destacaram fatores como a topografia e a influência do Oceano Atlântico. Posteriormente, foram calculados quantis anuais e mensais que permitiram classificar cada evento quanto ao total de precipitação. Regiões pluviometricamente homogêneas foram determinadas no ESP com base na climatologia e nos quantis de precipitação. Notou-se a relação construtiva entre eventos ENOS e as fases da ODP, com máximo durante o verão austral. Os sinais da ODP são percebidos em todo o ESP principalmente na primavera e no verão austrais. Uma análise complementar mostrou que as fases da Oscilação Multidecadal do Atlântico (AMO) também contribuem para a precipitação no ESP durante o verão e a primavera austrais no litoral, durante o verão no interior, e ao longo da primavera na região da Serra da Mantiqueira. Aparentemente, não há relação entre os eventos ENOS e a AMO. / São Paulo State (SPS) is remarkably important to the development of Brazil, economically or energetically, and this justifies climate studies on that region. Knowing rainfall variability is essential to water resources management and it has a great impact on agriculture an power production by hydroelectric power plants. Previous studies have detected non-linear effects of El Niño-Southern Oscillation (ENSO) on SPS rainfall however no specific work deals with PDO influence in this area besides some impacts on South America are known. Therefore this work has studied the relationship between rainfall anomalies in SPS and PDO from 1901 to 2007 to contribute to the climate forecasting improvement. First it was described the rainfall regime in South America, and locally in SPS where topography and the Atlantic Ocean influences were of special importance. Then annual and monthly quantiles were calculated to allow the classification of events according to rainfall totals. Rainfall homogeneous regions were established in SPS using climatology and quantiles. It was observed the constructive relationship between ENSO events and PDO phases, mainly on austral summer. PDO signals were noticed all over the SPS mostly on austral spring and summer. An additional analysis showed that Atlantic Multidecadal Oscillation (AMO) phases also contribute to SPS rainfall during austral summer and spring at the coast, only on summer at the country and during spring at the Mantiqueira Slopes. Apparently, there is no relation between ENSO events and AMO phases.

Tissue Engineering is a scientific discipline that aims to regenerate tissues and organs that are diseased, lost or congenitally absent. It encompasses the use of suitable synthetic equivalents of native extracellular matrix that may or may not be supplemented with cells or relevant growth factors. Such scaffolds are designed to reside at the site of implantation for a variable period of time during which they induce the regeneration of native tissue. During this time, they also provide a template for new cells to attach, infiltrate, differentiate into appropriate phenotype and eventually restore function of the concerned tissue. Among the factors that affect the outcome are the composition of scaffold, methods of fabrication, bulk properties of the scaffold and topography and architecture at the cellular level. Bone is unique in the body in that it is one of the few tissues capable of complete regeneration even in adults, as seen during fracture healing. However, certain conditions (non-union of fractures, congenital and acquired bone deficiencies) exist in which the regenerative capacities of bone are exceeded and appropriate intervention becomes necessary. Current treatment options include autologous bone grafts harvested from iliac crest or de-cellularized allografts or synthetic substitutes made from metals, ceramics and polymers. However these options have serious limitations: while autografts are limited in supply, necessitate second surgery and show inadequate vascularization, allografts can transmit viral infections. Metals, ceramics and polymers are in essence structural replacements without performing any biological function. Other problems associated with these synthetic materials include adverse immune reactions, corrosion, stress-shielding and secondary fractures due to inadequate osseo-integration. Bone tissue engineering is a specialized field of research that provides an alternative strategy to repair bone defects by exploiting the advances in engineering and better understanding of bone biology. Scaffold-based tissue engineering approach is a promising field that involves implantation of a biomaterial that is specifically matched in terms of biological and material properties to the tissue it replaces. This study explores the feasibility of using electrospinning as a potential fabrication strategy for bone tissue engineering applications, more specifically intended for cleft palate repair. This model represents a congenital deformity that affects both hard and soft tissues and presents unique challenges and opportunities. Among the challenges are: the need for the implant allow growth of the most complex areas of the facial skeleton, integrate and grow with the patient through adolescence, the ability of the implant to not interfere with vital functions including breathing and feeding. Further the implant should provide a flexible matrix that can effectively support erupting teeth. In spite of these extreme demands, maxilla is a non load-bearing membranous bone, a favorable consideration from materials engineering perspective. The present study is organized into three independent sections. The first section investigates developing strategies intended to improve the material properties of electrospun bone scaffold. Bone is composed of a high volume fraction (50%) of inorganic hydroxyapatite nanocrystals that is closely associated with collagen. The dispersal of brittle mineral is critical in not only strengthening the bone in compression but also contributes to the osteoconductivity of the matrix. Since loading of mineral in a bone scaffold is a serious limitation, we attempted to achieve improved loading of bone mineral by dual mineralization approach. We first incorporated nanocrystalline hydroxyapatite (nHA) directly into the scaffold by adding it to the electrospinning polymer solution. The second step involves inducing biomimetic mineralization of electrospun scaffolds by incubating them in simulated body fluid (SBF) for 2 weeks. The hypothesis was that the nanocrystalline hydroxyapatite seeded during electrospinning would act as sites for nucleation and further crystal growth when incubated in solution supersaturated with respect to calcium and phosphate ions. We tested this approach in two synthetic, biocompatible polymers-polydioxanone and poly (lactide: glycolide) and four formulations of SBF with differential loading of nHA (0-50% by wt. of polymer). A modified Alizarin Red S (ARS) staining that specifically binds to calcium was developed that allowed us to quantify the mineral content of 3D scaffold with great accuracy. Results indicated a unique combination of factors: PDO scaffolds containing 50% nHA incubated in 1x revised-SBF incubated under static conditions gave maximum mineralization over a period of two weeks. We then sought to exploit these findings to engineer a stiffer scaffold by stacking multiple layers together and cold welding them under high pressure. Electrospun scaffolds (1, 2 or 4 layered stacks) were either compressed before or after mineralizing treatment with SBF. After two weeks, scaffolds were analyzed for total mineral content and stiffness by uniaxial tensile testing. Results indicated while compression of multiple layers significantly increases the stiffness of scaffolds, it also had lower levels of mineralization partly due to increased density of fibers and loss of surface area due to fiber welding. However this can be offset to a reasonable degree by increasing the number of stacks and hence this strategy can be successfully adopted to improve the mechanical properties of electrospun scaffolds. The second section introduces a novel infrared imaging technique to quantify and characterize the biological activity of biomaterials, based on cell adhesion. Cells attach to the surface by the formation of focal contacts where multiple proteins including vinculin and talin assemble to signal critical processes like cell survival, migration, proliferation and differentiation. After allowing MG-63 osteoblasts to adhere to 2D biomaterial surface coated with extracellular matrix proteins (collagen, gelatin, fibronectin) cells were fixed and probed with antibodies for vinculin and talin. Secondary antibodies, tagged with infrared-sensitive fluorescent dyes, were used to quantify the molecules of interest. In addition, the kinetics of focal contact formation in these different substrates was followed. Successful quantification of focal contacts were made and further research revealed phosphorylation of vinculin at pY-822 as one potential mechanism for recruitment of vinculin to focal contacts. Hence it could represent a subset of vinculin and might serve as a specific molecular marker for focal contacts. As an extension, we evaluated the possibility of using such an assay to quantify 3D electrospun tissue engineering scaffolds. We fabricated scaffolds of graded biological activity by electrospinning blends of polydioxanone and collagen in different ratios. Vinculin and talin expressed by MG-63 cultured on these scaffolds for 24 hours were quantified in a similar manner. Results indicate that while talin does not show a significant difference in expression among different scaffolds, vinculin showed a positive correlation with increasing biological activity of scaffolds. In conclusion, we have identified vinculin as a reliable marker of focal contacts in 3D scaffolds while phosphovinculin (pY-822) was more specific to focal contacts in coated 2D substrates. In both instances, infrared imaging proved to be reliable in study of focal contacts. The third section aims to make the bone scaffolds osteoinductive- a property of a material to induce new bone formation even when implanted in subcutaneous and intramuscular heterotopic sites. Bone morphogenetic proteins (BMP) are potent cytokines that can induce migration, proliferation and differentiation of stem cells along osteoblastic lineage. The therapeutic efficacy of BMPs in the treatment of severe bone defects has been identified and is currently FDA approved for specific orthopedic applications. BMPs are clinically administered in a buffer form that not only makes the treatment expensive but less effective. Suitable delivery systems for BMP delivery have been an intense area of investigation. We rationalized electrospinning as a strategy to incorporate BMP within the scaffold and that would enable controlled release when implanted. One of the drawbacks of using electrospinning to deliver bioactive molecules is the potential denaturing effect and eventual loss of activity of BMPs. The final section of this dissertation tries to develop sensitive and relevant assays that could answer intriguing questions about solvent-protein interaction. We chose to use the BMP-2/7 heterodimer as the osteoinductive molecule of choice because of its superior potency compared to homodimer counterparts. We characterized the detection and quantification of BMP-2/7 using a slot blot technique. Further, we used a novel cell line (C2C12 BRA) to test the retention of activity of BMP-2/7 that has been exposed to organic solvents. Results indicate significant loss of activity when BMPs are exposed to organic solvents but complete recovery was possible by diluting the solvent with an aqueous buffer.

L’objet de cette thèse est de comprendre le rôle du paysage dans l’analyse de l’ancrage territorial des produits d’AOC en s’appuyant sur l’exemple des AOC fromagères de moyennes montagnes d’Auvergne et de Franche-Comté. La recherche a été menée à partir d’une approche multiscalaire, du territoire d’AOC jusqu’à l’exploitation agricole, lieu de production des objets paysagers mis en image et valorisés dans les supports de communication des filières.Une première hypothèse a été émise, selon laquelle les liens entre produits et paysages sont une fausse évidence. Pour la vérifier, nous avons analysé, à l’échelle des territoires retenus pour l’étude, les modèles paysagers véhiculés dans les documents promotionnels et les règles des cahiers des charges ayant un impact direct ou indirect sur le paysage. Cette première entrée nous a finalement conduit à relever la présence de liens de nature différente entre les régions d’Auvergne et de Franche-Comté.Les analyses menées à l’échelle de l’exploitation agricole se fondent sur une notion de paysage à l’interface des dimensions matérielles (objets) et idéelles (images), et des pratiques agricoles individuelles et collectives. Elles s’appuient en outre sur l’hypothèse de l’existence d’une sensibilité au paysage des agriculteurs qui conditionne les pratiques mises en œuvre dans les exploitations, et, par conséquent, l’absence ou la présence d’objets paysagers emblématiques des produits. Pour que cette sensibilité s’exprime, nous avons élaboré une méthode d’entretien particulière, fondée sur le principe de déconstruction - reconstruction des paysages attachés à des lieux. Au final, la compréhension de ce point de vue paysager et des déterminants des pratiques des agriculteurs nous a conduit à identifier des postures d’agriculteurs relatives à leur métier et à la place de l’animal dans le système. Ces dernières expliquent la force ou la faiblesse des liens entre les images promues et la réalité des systèmes de production. / The objective of this PhD thesis is to analyse the role of the landscape to understand the links between cheeses with a protected designation of origin (PDO) and their area of production. This research was conducted employing a comparative approach between seven typical cheeses from mountainous areas of medium altitude in central and eastern France. We developed a multi-scale analysis,reaching from territory down to farm scale. The farm is the place where the landscape elements which are promoted to the consumers are shaped.The first hypothesis was that seemingly evident relations between landscapes and products are in fact delusive. To verify this, we analysed landscape models presented in promotional documents as well as rules in the product specifications which could have an impact on landscapes, e.g. as a consequence of applied agricultural practices. Results representative on a territorial scale show existing links but the respective underlying mechanism differs between the regions of study.Analysis at farm scale are based on a diversified conception of landscape which takes into account both concrete and symbolic dimensions. At this scale we hypothesised that farmers have a rich perception of the landscapes that influences their practices and, consequently, the presence or absence of emblematic landscape elements on their farm. To analyse these perceptions, we created a specific method of interviews which helped farmers to talk about the landscapes of their farm and about the landscape of the PDO. This method allowed us to show that several farming styles exist, which explain the different strength of links between the cheeses and their area of production.

SiC is a hard man-made material and has emerged as an excellent material for a wide range of applications which are exposed to extreme conditions such as high temperatures and harsh chemical environments. These applications range from SiC being used as an abrasive, to a refractory material, to a semiconductor material for high power and high frequency electronic devices. The properties of the material for each application is different, with the semiconductor grade material for electronic devices being the most refined. SiC, with its excellent thermal properties and high resistance to harsh chemical environments, lends itself to being an ideal support for catalyst systems. Various characterisation & analysis techniques such as Scanning Electron Microscopy (SEM), Energy Dispersive Spectroscopy (EDS), X-ray Diffraction (XRD), Fourier Transform Infrared Spectroscopy (FTIR) and Gas Chromatography (GC) are used in this thesis to investigate the suitability of single crystal SiC for high temperature catalytic systems. Low temperature oxidation of methane was used to investigate the catalytic activity of: Porous and standard 4H-SiC with and without Pd Porous and Standard 6H-SiC with and without Pd. Nanocrystalline Beta-SiC powder with and without Pd. Part of the samples were impregnated with Pd using Palladium Nitrate (Pd (NO3)2) which is a common precursor for Pd. Activation treatments which were investigated were oxidation and reduction. Oxidation was generally better in activating the catalyst, as was expected, since the PdO phase is known to be more active in oxidising methane. A mixed set of Pd and PdO were observed by SEM and EDS which were the main characterisation techniques used to analyze the structure of the catalysts before and after the reaction. The Beta-SiC showed by far the best activity which could be attributed to the micro-crystalline powder format in which it was used, where as all other catalysts studied here were derived from crushed wafer pieces. Type II porous 4H-SiC was another of the samples which registered impressive results, vis-à-vis catalytic activity.

Cette thèse traite de la biodégradation par les bactéries d'une catégorie de polluants appelés hydrocarbures aromatiques polycycliques ou HAP. La bactérie Mycobacterium sp. 6PY1 a été isolée d'un sol pollué pour sa capacité à utiliser un HAP à 4 cycles, le pyrène, comme seule source de carbone et d'énergie. Pour comprendre comment le pyrène est métabolisé, l'identification des enzymes impliquées a été entreprise par une approche protéomique. Cette approche a notamment mis en évidence dans la souche 6PY1 deux arène dioxygénases, enzymes susceptibles de catalyser l'attaque initiale du pyrène. L'objectif de cette étude consistait à cloner les gènes de structure des arène dioxygénases identifiées chez Mycobacterium 6PY1, à surexprimer ces gènes en système hétérologue pour faciliter la purification des enzymes correspondantes, et à déterminer les propriétés biochimiques et catalytiques de ces enzymes. Les gènes pdoA1B1 codant la composante terminale d'une dioxygénase ont été clonés et surexprimés chez E. coli. Les propriétés catalytiques de cette enzyme, appelée Pdo1, ont été déterminées in vivo en dosant les produits d'oxydation de HAP composés de 2 à 4 cycles par chromatographie en phase gazeuse couplée à la spectrométrie de masse (CPG/SM). L'analyse de la sélectivité de l'enzyme, déterminée de cette façon montre que Pdo1 oxyde préférentiellement des HAP à 3 ou 4 cycles, comme le phénanthrène et le pyrène, mais n'attaque pas les di-aromatiques comme le naphtalène et le biphényle. La protéine Pdo1 s'est révélée instable et n'a été purifiée que sous forme inactive. Les gènes de structure d'une seconde composante dioxygénase ont été repérés dans un locus comportant deux autres gènes cataboliques. Les gènes pdoA2B2 codent pour une enzyme, appelée Pdo2, montrant une spécificité étroite vis-à-vis des HAP à 2 ou 3 cycles aromatiques et une nette préférence pour le phénanthrène. La protéine Pdo2 purifiée est un hexamère de type α3β3, possédant des centres [2Fe-2S] de type Rieske qui lui confèrent un spectre d'absorbance caractéristique. Des gènes susceptibles de coder pour une troisième dioxygénase ont été découverts dans le génome de Mycobacterium sp. 6PY1. Ces gènes sont très proches en séquence de ceux codant pour Pdo1, suggérant que la bactérie synthétise deux iso-enzymes capables d'oxyder le pyrène. Le fonctionnement des arène dioxygénases requiert deux protéines transporteurs d'électrons de type ferrédoxine et réductase. Celles qui sont associées aux enzymes Pdo1 et Pdo2 n'ont pas été identifiées. Cependant, l'activité des deux dioxygénases de la souche 6PY1 a été stimulée in vivo en co-exprimant dans E. coli des gènes accessoires recrutés d'autres bactéries. Enfin, des expériences d'immunodétection à l'aide d'anticorps spécifiques ont montré que les enzymes Pdo1 et Pdo2 sont co-induites en présence de HAP mais ne sont pas régulées de la même manière en fonction des conditions de croissance.