Phytocannabinoids are the active substances responsible for the medicinal and psychotropic effects of Cannabis sativa. Although the bioactivity of cannabis and its preparations have been known for millennia, several steps in the biosynthetic pathway leading to phytocannabinoids remain unclear. Phytocannabinoids are prenylated resorcylic acids which are formed in specialized plant organs called glandular trichomes. Following the analysis of a pre-generated cannabis trichome cDNA library, a type III polyketide synthase (tetraketide synthase; TKS) was identified and assayed, yielding three major compounds, hexanoyl triacetic acid lactone (HTAL), pentyl diacetic acid lactone (PDAL), and olivetol, yet no resorcylic acid was detected. This lack of resorcylic acid in enzyme assays has instigated the characterization of TKS and a search for putative cyclases in the cannabis trichome cDNA library, and involved protein pulldown, co-immunoprecipitation, and co-assay experiments. These experiments led to the discovery of a novel polyketide cyclase protein named olivetolic acid cyclase (OAC) responsible for the proper cyclization of a polyketide intermediate produced by TKS. This thesis shows that TKS assays conducted with OAC produce olivetolic acid (OA), an intermediate required during the biosynthesis of cannabinoids. The TKS/OAC spatial relationship was also investigated following the creation of fluorescent fusion proteins which show that the enzymes co-localized in vivo when viewed with confocal microscopy. Furthermore, yeast two-hybrid assays using TKS and OAC were performed to establish whether the enzymes physically interact. Finally, an attempt to determine the responsible amino acids involved in OAC’s mechanism was conducted by comparing the activity of single point OAC mutants with the wild-type OAC. Based on the available data, mechanisms for the production of HTAL, PDAL, olivetol, and OA are proposed.

La précision de tir d’une arme dépend de nombreux facteurs intervenant aux différentes étapes du parcours de la munition (balistique intérieure, intermédiaire et extérieure). Certains travaux ont démontré l’importance de l’influence de la phase de balistique intérieure, pendant laquelle la munition traverse le tube, sur les résultats à la cible. En effet, c’est cette phase qui détermine les conditions de sortie du tube de la munition et par conséquent son comportement au cours du vol. Les conditions d’entrée du projectile, la géométrie du tube et de l’arme, et les mouvements de ces derniers au cours du tir, sont autant de paramètres pouvant modifier l’interaction tube/projectile et ainsi entraîner un changement des vitesses angulaires et de translation de la munition au moment du largage. Cette étude a donc pour but de mettre en exergue les paramètres géométriques de l’arme et du tube qui influencent la justesse et la dispersion. Une analyse statistique a été réalisée à partir de la base de données des résultats de tir du 25 mm. Elle a permis de mettre en évidence l’influence de plusieurs paramètres dont la rectitude du tube. Par la suite, des essais ont été conduits en appareil de tir dans le but d’isoler la part de la géométrie du tube sur les écarts à la cible et ainsi de confirmer la contribution de la rectitude. Un modèle numérique tridimensionnel a été développé afin de faire varier ce paramètre et d’étudier son influence sur le comportement de la munition en phase de balistique intérieure. La représentativité du modèle a été vérifiée à l’aide d’accéléromètres embarqués dans la munition. Ces tirs ont nécessité le développement d’une solution innovante optoélectronique afin de transmettre les accélérations en temps réel. Les résultats expérimentaux obtenus ont permis de constater que les accélérations de la munition modélisée étaient représentatives. Le modèle permet maintenant de réaliser des études paramétriques et de déterminer les profils de tube les plus pénalisants pour la précision de tir. / Gun accuracy is influenced by several factors during the stages of the ammunition course (internal, intermediate and external ballistics). According to previous studies, internal ballistics are the major contributor to deviations from target. Indeed, this phase determines projectile exit conditions and, consequently, his behavior during the flight. The projectile entry conditions, the weapon and barrel geometry and their movements during firing, can modify the interaction tube / projectile and change ammunition angular rates and its transversal velocities. The purpose of this thesis is to determine the parameters related to barrel and gun geometry, which influence the bias and the dispersion. A statistical analysis was led thanks to the data base of the 25 mm firing results. It was found that some parameters, including barrel straightness, affect accuracy. Subsequently, tests were conducted with a firing appliance in order to isolate the barrel geometry influence on the deviations from the target and to confirm the straightness impact. A tridimensional numerical model was created in order to vary this parameter and to study its influence on the ammunition behavior during internal ballistics. The representativeness of the model was validated using accelerometers embedded in the ammunitions. The firing of these ammunitions has required the development of an optoelectronic system to transmit accelerations in real time. The comparison between experimental and numerical results has shown close amplitudes and similar shapes curves that proves the representativeness of the model. The model can be used now to lead parametric analysis and to determine the straightness shapes the most penalizing for gun accuracy.

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Introdução e objetivos: β-glicosidases da família GH1 das glicosil-hidrolases possuem um dobramento do tipo barril (β/α)8. Propõe-se que proteínas com este dobramento, que usualmente classificam-se como tendo um único domínio, na verdade são compostas por dois domínios, cada um deles correspondendo a um \"meio barril\" (β/α)4. Assim, as proteínas com dobramento barril (β/α)8 seriam provenientes de uma duplicação e fusão gênica de um ancestral \"meio barril\" (β/α)4. O objetivo geral deste projeto é investigar a existência de dois domínios (β/α)4, as metades N- e C-terminal, na estrutura (β/α)8 barril da β-glicosidase A de Thermotoga maritima (bglTm) e β-glicosidase B de Paenibacillus polymyxa (bglB) por meio de análise da desnaturação térmica e química dessas enzimas. Resultados: Para atingir esse objetivo foram introduzidas mutações que rompem contatos não covalentes entre os supostos domínios destas β-glicosidases. Os segmentos de DNA que codificam as enzimas selvagem e mutantes foram clonados em plasmídeo de expressão pLATE51 e as enzimas recombinantes expressas em Escherichia coli BL21(DE3). Foram purificadas com sucesso as enzimas selvagens e duas mutantes de bglTm, denominadas T1 e T2, que possuem 2 e 4 mutações respectivamente em resíduos na interface inter-metades. Para confirmar o enovelamento destas proteínas recombinantes foi empregada a análise de estruturas secundárias por dicroísmo circular, também o espectro de fluorescência intrínseca de triptofano e sua supressão por acrilamida e finalmente foram determinados parâmetros cinéticos. Observou-se que não houve mudanças significativas na exposição dos triptofanos das proteínas recombinantes, sugerindo que se encontram enoveladas, o que está de acordo com a observação de que as proteínas recombinantes mantêm sua atividade catalítica. Já pela análise de dicroísmo circular concluiu-se que a mutante T1 possui dobramento semelhante à selvagem bglTm e que T2 apresenta diferenças significativas, sendo as porcentagens de α-hélice de 21%, 22% e 10% para bglTm, T1 e T2, respectivamente. Em seguida demonstrou-se que T1 mantém a termo estabilidade semelhante à selvagem, enquanto que T2 tem termo estabilidade reduzida, apresentando kobs de 0,3 min-1 em 80°C e de 0,06 min-1 em 75 °C.. O cálculo de Tm através de Differential Scanning Fluorimetry foi feito para bglB e T2 (42 e 81.7 °C respectivamente), enquanto que bglTm e T1 mantiveram-se estáveis na faixa de temperatura analisada (até 95°C). Na análise do efeito da temperatura sobre a estrutura das mutantes T1 e T2 não se observou nenhuma evidência da presença dos dois supostos domínios (β/α)4. A análise da desnaturação por cloreto de guanidina mostrou que o c50 diminuiu para T2 (2,4 M), mas não mostrou alteração para T1 (4,5 M) em comparação à bglTm (4,3 M). Coerentemente, a estabilidade da enzima selvagem e de T1 na ausência de desnaturante é a mesma (ΔGH2O = 5,2 kcal/mol), mas se reduziu para a T2 (ΔGH2O = 3,5 kcal/mol). Os cálculos do parâmetro m mostraram uma cooperatividade semelhante na desnaturação de bglTm, T1 e T2, não evidenciando independência entre os dois supostos domínios (β/α)4. Em conclusão, a análise estabilidade da β-glicosidase bglTm frente à temperatura e ao cloreto de guanidina não revelaram a presença dos domínios (β/α)4 que correspondem às metades N- e C-terminal desta β-glicosidase. / Introduction and Aims: β-glucosidases from the family GH1 of the glycosil-hidrolases presents a (β/α)8 barrel folding. These proteins are usually classified as single domain, however it has been alternatively proposed that they actually are formed by two \"half barrel\" (β/α)4. Thus, (β/α)8 barrel proteins had evolved from an \"half barrel\" ancestor that underwent a duplication-fusion event. The general goal of this project is the search for the two putative (β/α)4 domains, which form the N- and C-terminal ends of the β-glucosidase A from Thermotoga maritima (bglTm) and β-glucosidase B from Paenibacillus polymyxa (bglB), detecting their presence through the thermal and chemical stability of these (β/α)8 barrel proteins. Results: Site-directed mutagenesis was employed to replace residues forming non-covalent interaction between the putative (β/α)4 domains. DNA segments coding for bglB, bglTm and mutant bglTm were cloned into the pLATE51 expression vector and produced as recombinant proteins in E. coli BL21(DE3). The bglB, bglTm and two mutant bglTm, hereafter called T1 and T2, with 2 and 4 mutations respectively on residues in the interface between the protein halves, were purified. They were stable folded as shown by detecting their catalytic activity upon two different substrates and also by circular dichroism (CD) and tryptophan fluorescence analysis. Nevertheless, T2 showed a decrease in the α-helix content (10 %) in the CD analysis, whereas bglTm and T1 are similar (22 %). The wild-type bglTm and T1 are thermostable, whereas T2 was inactivated after pre-incubation at high temperature (kobs = 0,3 min-1 at 80 °C and 0,06 min-1 at 75 °C). The Differential Scanning Fluorimetry experiments revealed Tm of 42 e 81.7 °C for bglB and T2, respectively, whereas wild-type bglTm and mutant T1 did not showed any thermal transition up to 95 °C. Indeed, the analysis of the thermal stability of T1 and T2 did not reveal any evidence of the putative (β/α)4 domains. Following that, the analysis of the protein denaturation by guanidine hydrochloride showed that the c50 for T2 was reduced (2.4 M), whereas no modification was observed for bglTm and T1 (4,3 and 4,5 M, respectively). In agreement the stability of bglTm and T1 (ΔGH2O = 5,2 kcal/mol) is similar, but it was reduced for T2 (ΔGH2O = 3,5 kcal/mol). The m parameter showed a similar cooperative denaturation for wild-type bglTm and mutants T1 and T2, but no evidence of the independent unfolding of the putative (β/α)4 domains was found. Conclusion: In conclusion, the analysis of the thermal and chemical stability of the bglTm did not reveal the presence of the putative (β/α)4 domains that form the N- and C-terminal end of these β-glucosidase.

Le Rhône, prolongé de la Saône, forme un axe de pénétration à l’intérieur de la Gaule emprunté à l’époque romaine pour mettre en relation la Méditerranée avec le nord de l’empire. De brèves connections terrestres (portages) permettaient de poursuivre les échanges vers les bassins de la Loire, de la Seine, du Rhin et les lacs alpins. Seul le quart sud-ouest semble avoir échappé à l’influence rhodanienne. Ce corridor formait très clairement l’artère majeure du réseau fluvial gaulois, et cela avant-même la conquête romaine. La voie fluviale était à l’époque le principal vecteur de transport de marchandises à l’intérieur des terres, et cela jusqu’à l’arrivée du chemin de fer. Le Rhône et la Saône auraient détenu à eux seuls près de la moitié du trafic fluvial de toute la Gaule. Ces cours ont par ailleurs clairement contribué au développement des villes riveraines correspondant souvent à des haltes fluviales et des nœuds de redistribution. L’intérêt du sujet est donc de toute première importance. Les intentions de ce travail sont triples. Tout d’abord, présenter une synthèse de la navigation du Rhône et de la Saône actualisée des grandes séries de découvertes des années 2000 (épaves lyonnaises et arlésiennes) ; ensuite réhabiliter l’image du « marin d’eau douce » antique qui souffre d’un double a priori, l’archaïsme technique antique, et la facilité de la navigation en milieu fluvial ; enfin, l’intention principale est de montrer les spécificités de la navigation sur le Rhône et la Saône à l’ époque romaine. Pour y parvenir nous procédons en trois temps. Une première partie consacrée aux paramètres de la navigation (marchandises, emballages, embarcations, corporations, milieu fluvial) permettant de cerner en détail les différentes variables de la question. Une seconde partie est dédiée aux conditions de la navigation (conditions climatiques, conditions morphologiques – tirant d’eau, tirant d’air – conditions dynamiques – modes de propulsion autorisés –, conditions juridiques, religieuses, risque d’attaque, etc.). La dernière partie est consacrée aux possibilités de la navigation. Il s’agit en fait d’un outil permettant de simuler des situations de transport. Le lecteur a la possibilité de « composer » un scénario de transport en sélectionnant les variables de la première partie, et en le confrontant aux conditions de navigation de l’axe fluvial défini en seconde partie. Ainsi, il est possible de prendre position sur un certain nombre de sujet comme la remonte du fleuve à la rame ou à la voile, les tirants d’eau autorisés, la présence des navires maritimes ou des navires à dolia en amont d’Arles, les moments propices à la navigation pour les chalands, les vitesses et les temps de transport, etc. / The Rhone River, in conjunction with its tributary the Saone River, form an axis of penetration inside the Gaul used during the Roman period to link the Mediterranean sea to the north of the empire. Short land connections enabled the trades to flourish over the basins of the Loire, the Seine, the Rhine and the alpine lakes. Short land connections have allowed the pursuit of the trades over the basins of the Loire, the Seine, the Rhine and the alpine lakes. Only the southwest quarter seems to have escaped the influence of the Rhone. This corridor formed the major artery of the Gallic river network, even before the Roman conquest. The waterway at the time was the primary means for transporting goods inland, up until the arrival of the railway. The Rhône and Saone rivers alone would have conveyed nearly half of river traffic of all Gaul. These waterways have also clearly contributed to the development of riverside towns often corresponding to river stops and points of cargo redistribution. The interest of the subject is of paramount importance. The intention of this work is threefold. First, provide an overview of the navigation of the Rhone and the Saone as evidenced by series of discoveries of the 2000s (Lyon and Arles wrecks) ; then disprove two commonly held misconceptions of the Roman "landlubber": the use of archaic navigation technique and the ease of the river environment. Finally, the main intention is to show the specifics of navigation on the Rhone and Saone in Roman times. This will be covered in three parts. A first part related to the navigation parameters (goods, packaging, crafts, corporations, river environment) to identify in detail the characteristics and issues related to each of them. A second part is dedicated to given navigation conditions : weather, boat morphology (draft, air-draft), dynamic conditions (possible propulsions), legal and religious conditions, risk of attack, etc. Based on the navigation parameters and navigation conditions presented, the last part describes the possibilities of navigation that were offered at that period. It is a tool to simulate transport situations. The reader has the option to "compose" a transport scenario by selecting the parameters of the first part, and by confronting them to the navigation conditions of the fluvial axis defined in the second part. In the end, it is possible to to give answers to several questions such as the use of oar or sail against the current, the possible drafts that could be used, the presence of marine ships or dolia ships upstream of Arles, the most favorable conditions to barge navigation, the speed or travel time.

Abstract Enzymes are potentially superior as catalysts for many industrial chemical processes because of their high specificity, selectivity, minimum energy requirement and environmental friendliness. However, many challenges remain in order to exploit fully the potential of industrial enzymes. The qualities which are needed are catalytic proficiency, availability in high quantities, low price, low product inhibition, and high activity and stability under process conditions. Directed evolution and rational design are the most common strategies to produce enzymes with the desired properties. The TIM barrel is the most frequent and most versatile fold among naturally occurring enzymes. In all known TIM barrel enzymes, the catalytically active residues are located at one end of the barrel structure, while residues maintaining the stability of the fold are found on the opposite end of the barrel. This special architecture of the TIM barrel proteins makes it possible to change catalytic activity of the protein without compromising its stability, which is a perfect start for protein engineering studies. In this research project, a monomeric triosephosphate isomerase (TIM) variant with an engineered binding groove (A-TIM) was created by using a rational design approach. The major aims of this work were (i) to find novel binders and (ii) characterize the new, bigger binding groove using X-ray crystallographic methods. These studies have discovered that monomeric A-TIM can bind compounds completely different from the natural substrate. Studies on three different classes of binder molecules are reported: (i) true substrate analogues of wild type TIM, (ii) substrate analogues that have an extended hydrophobic tail, and (iii) more extended, phosphate containing substrate analogues. In addition to this, the A-TIM active site was shown to be competent. In general these studies illustrate the importance of protein crystallography for characterizing the binding properties of enzyme variants being studied in enzyme discovery projects. / Tiivistelmä Entsyymit voivat toimia ylivoimaisina katalyytteinä monissa kemianteollisuuden prosesseissa johtuen niiden hyvästä spesifisyydestä, valikoimiskyvystä, alhaisesta energiantarpeesta ja ympäristöystävällisyydestä. Näistä ominaisuuksista huolimatta entsyymien kaikkien mahdollisuuksien hyödyntämisen esteenä on monia haasteita. Tarvittavia ominaisuuksia ovat katalyyttinen tehokkuus, saatavuus suurina määrinä, alhainen hinta, alhainen tuoteinhibitio sekä korkea aktiivisuus ja stabiilisuus prosessiolosuhteissa. TIM-tynnyrirakenne on yleisin ja monipuolisin proteiinien laskostumisrakenne luonnossa esiintyvissä entsyymeissä. Tässä rakenteessa katalyyttisesti aktiiviset aminohappotähteet ovat sijoittuneet tynnyrirakenteen toiselle puolelle, kun taas stabiilisuuden kannalta tärkeät aminohappotähteet ovat sijoittuneet kokonaan toiselle puolelle. Tämä erityinen rakenne antaa mahdollisuuden muokata proteiinin katalyyttistä aktiivisuutta vaikuttamatta haitallisesti sen stabiilisuuteen. Tämä on täydellinen lähtökohta proteiininmuokkaukselle. Tässä tutkimusprojektissa käytettiin ns. järkiperäistä suunnittelua monomeerisen trioosifosfaatti-isomeraasivariantin (A-TIM) luomisessa. Tämän tutkimustyön pääasialliset tavoitteet olivat (i) uusien sitoutujien löytäminen ja (ii) uuden, suuremman sitoutumistaskun ominaisuuksien määrittäminen röntgenkristallografisilla menetelmillä. Tässä tutkimuksessa havaittiin, että A-TIM kykenee sitomaan yhdisteitä, jotka ovat täysin erilaisia luonnolliseen substraattiin verrattuna. Tässä tutkimuksessa kuvaillaan kolmenlaisia sitoutujia: (i) todelliset villityypin entsyymin substraattianalogit, (ii) substraattianalogit, joihin on liitetty hydrofobinen hiilivetyketju ja (iii) villityypin substraattia suuremmat sokerifosfaatit. Tämän lisäksi A-TIM:n aktiivisen keskuksen todistettiin olevan toimintakykyinen. Yleisellä tasolla tämä tutkimus osoittaa röntgenkristallografisten menetelmien tärkeyden entsyymienmuokkausprojekteissa, joissa entsyymivarianttien ominaisuuksien määritys on tärkeää.

La fermentation malolactique permet une désacidification et une amélioration de la qualité du vin. Elle est réalisée par des bactéries lactiques principalement de l’espèce Oenococcus oeni : cette espèce est privilégiée pour son efficacité et ses intérêts organoleptiques. La capacité de O. oeni à résister au stress du vin et à garder son activité fermentaire est un sujet d’intérêt majeur. Les prélèvements des fûts de chêne ont montré que O. oeni adhère au bois et est capable de persister plusieurs mois dans le vin. Or, dans la majorité des habitats naturels, les microorganismes se développent attachés à un support, au sein d’un écosystème structuré appelé biofilm. Sous cette forme de vie, les cellules bénéficient d’une résistance accrue au stress. Ces deux propriétés que sont l’adhésion à une surface ainsi que la résistance au stress ont donc été étudiées pour O. oeni. Des observations par microscopie électronique à balayage ont ainsi permis de mettre en évidence la formation de biofilms par O. oeni. Puis, nous avons évalué la capacité de résistance au stress du vin de O. oeni sous forme de biofilm. L’intérêt pour le biofilm est croissant dans les industries agroalimentaires et biotechnologiques, en raison de la conservation de son activité métabolique en milieu stressant. C’est pourquoi la fermentation malolactique avec un biofilm de O. oeni a été suivie dans le vin. Enfin, l’influence du biofilm de O. oeni sur les transferts de composés aromatiques entre le bois et le vin a été étudiée. Cette étude est la première caractérisation du biofilm de O. oeni, de sa résistance au stress du vin et de son potentiel fermentaire / Malolactic fermentation improves wine quality, mainly by decreasing acidity. It is carried out by lactic acid bacteria, mainly Oenococcus oeni. This species is favored for its efficiency and its organoleptic outcome. O. oeni ability to withstand wine stress and to keep its fermentation activity is a subject of major interest.Samples of oak showed that O. oeni adheres to wood and is able to persist for several months in wine. However, in the majority of natural habitats, microorganisms grow attached to a surface, within a structured ecosystem called biofilm. In this form of life, cells benefit from an increased stress resistance.These two properties, adherence to a surface and stress resistance, were studied for O. oeni. Observations by scanning electron microscopy have highlighted biofilm formation by O. oeni. Then, we evaluated the wine stress resilience of O. oeni biofilm. Biofilm is a growing interest in food and biotechnology industries, due to the conservation of its metabolic activity in stressful environment. Therefore, malolactic fermentation with an O. oeni biofilm was monitored in wine. Finally, the influence of O. oeni biofilm on transfers of aromatic compounds between wood and wine was studied.This study is the first characterization of O. oeni biofilm, its wine stress resistance and its fermentation potential.

Tato práce popisuje výzkum zaměřený na studium vertikálních teplotních profilů a fluktuací v turbulentní Rayleighově-Bénardově konvekci. Experiment byl proveden v "Barrel of Ilmenau" obsahující válcovou experimentální celu s průměrem 7,15 m a výškou 4,7 m ("the aspect ratio" = 1,5) naplněnou suchým vzduchem. Teplotní profily a fluktuace byly studovány podél vertikální osy cely mezi horní a spodní deskou spodní deskou v rozmezí Rayleighova čísla (Ra) 1E11 4E12. Teplotní profily byly změřeny novou metodou využívající systém s optickým vláknem Luna ODiSI-B, který byl pořízen týmem z Ilmenau. Systém umožňuje měřit teplotní profil ve všech bodech podél celého vlákna současně s prostorovým rozlišením 5 mm.

Diploma thesis deals with the design of steam turbine of PBS ENERGO, a.s. company design concept. Condensing turbine is on low steam parameters and of barrel concept. Turbine has two unregulated steam extractions, regulation stage with action blading and seven stages with reaction blading type, which was computed with ca/u method on mean blading diameter. The thesis contains also design of thermic diagram, calculation of axial force on rotor, calculation of steam seals and basic design of longitudinal turbine cross section.

The state plays a prominent role in the process of regional development. State interventions might bring radical influences into the entire socioeconomic system, to which the other actors of regional development have to adapt. Importantly, the state administrates a large amount of funds, creates large numbers of jobs, and it is led by "elites" which might influence the other people's opinions. The need for studying of the role of the state and the expenditure side of its budget for an understanding of the mechanisms of regional development is recognized by many authors. However, deep discussions of the function of the state could be hardly found in regional development theories. The aim of this dissertation thesis is to contribute to understanding of the role of the state as one of major actors in regional development via investigation of the regional impacts of public support programs. To provide concrete scrutiny, parliamentary grants (so-called "pork barrel") were depicted through which deputies annually sent large sums of money from the state budget during its approval process in the Chamber of Deputies on projects with local impact. The topic of parliamentary grants is a traditional topic for international studies within the concept of the so-called pork barrel politics, which includes all activities...