La tuberculose (TB) reste la maladie infectieuse la plus meurtrière au monde. L’absence de développement de médicaments accompagnée de l'émergence de la résistance aux molécules existantes se traduit principalement par l'échec du traitement et la mort. M. tb infecte l'homme par l'inhalation. Le patient peut rester en bonne santé sans aucun symptôme, on parle alors de tuberculose latente. Dans ce cas, la bactérie résider dans le macrophage. La principale caractéristique de M. tb est de rester cacher à l'intérieur de ces cellules, puis se réactiver lorsque le système immunitaire s’affaibli.Mon travail a été consacrée à l'étude de nouveaux analogues de Cyclophostin / Cyclipostins (CyCs) et Oxadiazolone. Nombreuses molécules présentaient des activités antibactériennes lorsque la bactérie était cultivée dans le milieu liquide ou à l'intérieur du macrophage sans aucun effet toxique sur les macrophages.En utilisant le meilleur inhibiteur de CyC17, nous avons identifié 23 protéines cibles potentielles, étant impliquées dans le métabolisme des lipides de M. tb et / ou dans la biosynthèse des lipides de la paroi cellulaire. Parmi ces cibles, Ag85C et TesA, ont été caractérisées biochimiquement et structurallemnent.De plus, nous avons établi que les CyC sont des inhibiteurs puissants et sélectifs de la croissance des mycobactéries. De manière intéressante, ces composés étaient également actifs contre de nombreuses souches cliniques de M. abscessus, un pathogène opportuniste également appelés « cauchemars pour les antibiotiques », ouvrant ainsi la voie à la recherche de solutions alternatives pour lutter contre les souches mycobactériennes résistantes. / Tuberculosis (TB) is one of the deadliest infectious diseases worldwide. The stagnation in drug development accompanied with the emergence of drug resistance mainly results in treatment failure and death. M. tb infect human through inhalation. Patient can remains healthy without any symptom in which case it is known as latent TB. In latent TB, the bacteria is known to be reside in the macrophage - a type of white blood cell of the immune system, that engulfs and digests infected microorganisms. The famous characteristic of M. tb is the ability to stay inside and avoid to be killed by these cells, then reactivate when the immune system is weak.My work was devoted to the study of two series of new compounds, namely analogs of Cyclophostin/Cyclipostins (CyCs) and Oxadiazolone (OXs). Many molecules exhibited antibacterial activities when the bacteria was grown in the liquid medium or inside the macrophage without any toxic effect to the macrophages.Using the best CyC17 inhibitor, we identified 23 potential candidate target proteins, being involved in M. tb lipid metabolism and/or in cell wall lipid biosynthesis. Among these targets, two important protein, i.e. Ag85C and TesA, were biochemically and structurally characterized.Moreover, we clearly established that the CyCs are powerful and selective growth inhibitors of mycobacteria, with no effect on Gram-negative or Gram-positive bacteria. Interestingly, these compounds were also active against numerous M. abscessus clinical strains, an opportunistic pathogens also called “nightmare for antibiotic”, thus opening the way to find alternative solutions to fight against resistant mycobacterial strains.

The objective of the study was to analyze the relationship of psychrotrophic lipolytic bacteria in bulk samples of cow?s raw milk and their metabolic activity based on the content of free fatty acids and identifying the factors that may influence their levels. The contents of free fatty acids (FFA) and counts of total bacteria, psychrotrophic lipolytic bacteria (PLiBC) and somatic cells were determined in 150 samples of cow?s bulk raw milk on 20 farms with three different milking technologies in South Bohemia during 2008?10. FFA were determined using an extraction-titration method. Within the compared technologies, the highest mean values of FFA (38,8 mmol?kg-1; P<0,001) and PLiBC (696 CFU?ml?1) were observed on farms with pipeline milking in stalls. The lowest mean FFA level (15,4 mmol?kg-1) was determined on farms with an automatic milking system. Medium values were determined on farms with parlour milking. From the aspect of the inhibition of an increase in psychrotrophic bacteria, and mainly in psychrotrophic lipolytic bacteria in conditions of cold storage of raw milk the temperature of 4°C seems optimum as it markedly inhibits the growth of mesophilic and psychrotrophic bacteria and at the same time the increase in the values of free fatty acids is slower at this temperature compared to the temperatures of 6,5 and 10°C.

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Made available in DSpace on 2016-06-02T19:55:29Z (GMT). No. of bitstreams: 1 3391.pdf: 9156638 bytes, checksum: 0de8724bcb52a60114f5d3639d17f212 (MD5) Previous issue date: 2010-08-11 / Financiadora de Estudos e Projetos / Molecular biology and genetic engineering have been deployed widely in recent years, several protocols have been established, presenting new methodologies capable of altering the genetics of bacterial strains industrial interest. This research had as main objectives: (1) the construction of the following plasmids: p&#61636;lat, pAMB4 and pAMB3RpoZneo, (2) the production of Streptomyces clavuligerus mutants by gene disruption, by insertion of plasmid integrative, and by replication of multi-copy plasmid in Streptomyces clavuligerus by conjugation and (3) study the effect of these mutations on clavulanic acid production and lipolytic activity. In Spain (University of Leon), six mutants Streptomyces clauligerus &#61636;lat::apr, Streptomyces clavuligerus cvm7P::neo, Streptomyces clauligerus &#61636;Lat::apr &#61636;cvm7P::neo, Streptomyces clavuligerus pRAneoZ, Streptomyces clavuligerus pAMB4 and Streptomyces clavuligerus pAMB3RpoZneo were produced. In Leon, the fermentations were performed with the SA culture medium and only with the mutants Streptomyces clavuligerus &#61636;lat::apr and Streptomyces clavuligerus pRAneoZ. In this case, there was no statistically significant difference at 5% probability by analysis of variance (ANOVA). In Brazil, the fermentations with all mutants in the culture medium-based oil and soybean meal, showed a different pattern in the production of clavulanic acid. The mutants Streptomyces clavuligerus pAMB3RpoZneo and Streptomyces clavuligerus pRAneoZ (&#61665;&#61472;= 5%) showed clavulanic acid titles higher when compared with the wild type. The double mutant Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, contrary to expectations, showed the lowest levels of clavulanic acid in relation to its parental strain. Streptomyces clavuligerus pRAneoZ mutant and the mutant Streptomyces clavuligerus pAMB4 control, showed the highest lipolytic activity at 5% probability. The double mutant in turn, had the lowest lipolytic activities. A direct relationship between levels of clavulanic acid and lipase production was observed. All mutants produced in this work could be fermented into bioreactor to assess production levels of clavulanic acid and lipase. The construction of a new double mutant named Streptomyces clavuligerus &#61636;lat::apr RpoZneo from existing ones mutant could be of great interest to investigate this new combination of mutations on clavulanic acid production and lipolytic activity. / A biologia molecular e a engenharia genética têm se desenvolvido muito nos últimos anos e vários protocolos foram estabelecidos, apresentando novas metodologias capazes de alterar a genética de linhagens bacterianas de interesse industrial. O presente estudo teve por objetivos principais: (1) a construção dos seguintes plasmídeos: p&#61636;lat, p&#61636;cmv7P, pAMB4 e pAMB3RpoZneo; (2) a produção de mutantes de Streptomyces clavuligerus por disrupção gênica, por inserção de plasmídeo integrativo, e por replicação de plasmídeo multi-cópia em Streptomyces clavuligerus por conjugação bacteriana; (3) o estudo do efeito destas mutações na produção de ácido clavulânico e na atividade lipolítica. Na Espanha (Universidade de León), seis mutantes foram produzidos: Streptomyces clavuligerus &#61636;lat::apr, Streptomyces clavuligerus &#61636;cvm7P::neo, Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, Streptomyces clavuligerus pRAneoZ, Streptomyces clavuligerus pAMB4 e Streptomyces clavuligerus pAMB3RpoZneo. Em León, as fermentações foram realizadas com o meio de cultura SA e somente com os mutantes Streptomyces clavuligerus &#61636;lat::apr e Streptomyces clavuligerus pRAneoZ. Nestas fermentações não houve diferença estatística significativa ao nível de 5% de significância, pela análise de variânica (ANOVA). No Brasil, as fermentações com todos os mutantes no meio de cultura a base de óleo e farinha de soja, mostraram um padrão diferenciado na produção de ácido clavulânico. Os mutantes Streptomyces clavuligerus pRAneoZ e Streptomyces clavuligerus pAMB3RpoZneo apresentaram títulos de ácido clavulânico superiores quando comparados com a linhagem selvagem (&#61665;&#61472;= 5%). O duplo mutante Streptomyces clavuligerus &#61636;lat::apr &#61636;cvm7P::neo, ao contrário do esperado, apresentou os níveis mais baixos de ácido clavulânico e em relação a sua linhagem parental. Os mutantes Streptomyces clavuligerus pRAneoZ e o mutante controle Streptomyces clavuligerus pAMB4, apresentaram a maior atividade lipolítica ao nível de 5% de significância. O duplo mutante por sua vez, apresentou as menores atividades lipolíticas. Uma relação direta entre os níveis de ácido clavulânico e a produção de lipase foi observada. Todos os mutantes produzidos neste trabalho poderiam ser fermentados em biorreator de bancada para se avaliar os níveis de produção de ácido clavulânico e de lipase. A construção de um novo duplo mutante denominado, Streptomyces clavuligerus p&#61636;lat::apr RpoZneo, a partir dos existentes, poderia ser de grande interesse para investigar esta nova combinação de mutação na produção de ácido clavulânico e na atividade lipolítica.

Les enzymes lipolytiques sont solubles en phase aqueuse mais hydrolysent des substrats insolubles. Leurs activités lipolytiques dépendent donc fortement de l’organisation des substrats lipidiques présents sous forme de structures interfaciales telles que des émulsions, des micelles, des liposomes, ou des bicouches lipidiques. Les propriétés cinétiques et la spécificité de substrat de ces enzymes résultent de l’étape initiale d’adsorption à l’interface lipide-eau et des interactions entre le substrat et le site actif. Dans le cadre de ce travail de thèse, la technique des films monomoléculaires a été utilisée pour étudier en détail les étapes séquentielles d’adsorption, de catalyse et d’inhibition de l’enzyme à l’interface lipide-eau. Dans une première partie, nous avons réalisé la caractérisation physico-chimique de la lipase gastrique de chien (DGL), avec l’étude :  de son adsorption sur un film non substrat de dilauroylphosphatidylcholine ; ‚ de l’hydrolyse interfaciale de la 1,2-dicaprine dans des films mixtes en présence d’Orlistat. Concernant l’étape de catalyse, nous avons étudié l’effet du propeptide sur la spécificité de substrat et l’activité interfaciale de la phospholipase A2 sécrétée de groupe X de souris. Enfin, dans une troisième partie, nous avons comparé les propriétés interfaciales de la lipase YLLIP2 de la levure Yarrowia lipolytica qui serait un bon candidat pour l’enzymothérapie de substitution chez les patients atteints d’insuffisance pancréatique exocrine (IPE), la lipase pancréatique humaine et la DGL. Nos résultats ont confirmé le rôle d’YLLIP2 en tant qu’excellent « substitut » non seulement de la HPL en cas d’IPE, mais aussi de la DGL. / Lipolytic enzymes are water-soluble whereas their substrates are insoluble in water. Their lipolytic activities depend strongly on the organization of the lipid substrates present in interfacial structures such as oil-in-water emulsions, micelles, liposomes, and membrane bilayers. The kinetic properties and substrate specificity of these enzymes result from both their adsorption at the lipid-water interface, and the interactions occurring between the substrate and the active site. In this thesis work, the monomolecular film technique was used to study in details the sequential steps of adsorption, catalysis and inhibition of model enzymes at the lipid-water interface. In a first part, we performed the physico-chemical characterization of the dog gastric lipase (DGL), by studying:  its adsorption onto a dilauroylphosphatidylcholine non-substrate film; ‚ its interfacial hydrolysis of 1,2-dicaprin in mixed films with various amounts of Orlistat. Regarding the catalysis step, we studied the effect of the propeptide on the substrate specificity and interfacial activity of the murine group X secreted phospholipase A2. A model of this enzyme with its propeptide was built from the available 3D structure of the corresponding mature human enzyme. Finally, in the third part, we compared the interfacial kinetic properties of YLLIP2 lipase of the yeast Yarrowia lipolytica which has been identified as a good candidate for enzyme replacement therapy for patients with exocrine pancreatic insufficiency (EPI), human pancreatic lipase and DGL. Our results confirmed the role of YLLIP2 as an excellent "substitute" not only for HPL in case of PEI, but also for the DGL at acidic pH values.

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.