Au cours de ce travail, différents glucosylamines et aminodésoxyglucoses ont été synthétisés et caractérisés par différentes méthodes spectroscopiques dont l’IRTF, la RMN 1H, 13C et MALDI-Tof MS. L’étude des propriétés biologiques de ces molécules réalisée, d’une part, avec deux champignons du bois, Coriolus versicolor et Poria placenta, et d’autre part, avec trois microorganismes potentiellement rencontrés dans des aliments, Listeria innocua, Salmonella typhimurium et Fusarium proliferatum ont indiqué une contribution positive de la N-alkylation, du degré de N-substitution et de la quaternisation sur l’inhibition de leur croissance. Par ailleurs, l’impact sur la bioactivité, de la position du groupe amine sur le sucre, a été étudié. Il a été montré que la position du groupe amine sur le C-1 du glucose conduisait à une activité antifongique contre C. versicolor et P. placenta plus prononcée alors que la position C-3 du glucose était favorable à une activité antimicrobienne contre L. innocua et S. typhimurium. / In this study different glucosylamines and amino desoxyglucoses were synthesized and characterised using various spectroscopic methods including IRFT, both 1H and 13C NMR spectroscopy and MALDI-Tof MS. Biological assessment of these compounds realised with two wood decay fungi, Coriolus versicolor and Poria placenta on one hand, and with three food microorganisms Listeria innocua, Salmonella typhimurium and Fusarium proliferatum on other hand, indicated a positive impact of both N-alkylation and degree of N-substitution and quaternisation on their growth inhibition. Furthermore, a biological impact of the amine position on sugar was studied. It was found that amine function attached to the C-1 of glucose conducted to the best antifungal activity against both C. versicolor and P. placenta while that fixed on the C-3 of glucose was indicated for antibacterial activity against L. innocua and S. typhimurium.

No description available.

Ce travail a pour objectif de mettre au point un procédé d’oligomérisation de rutine et d’esculine par la laccase de Trametes versicolor. Un procédé de synthèse en parallèle et d’analyse en ligne par SEC-UV et par MALDI-TOF a été mis au point. L’analyse par MALDI-TOF a révélé la formation d’un simple pontage, allant jusqu’au degré d’oligomérisation 6 pour la rutine et 9 pour l’esculine. Un pontage par liaison éther a été observé par FTIR dans le cas des oligorutines. L’analyse par RMN a démontré la mise en place de liaisons tant C-C que C-O localisées sur la partie phénolique et la partie sucre des monomères. De faibles pH et températures favorisent l’allongement de la chaîne, alors que l’augmentation de la constante diélectrique du solvant ou de la température augmente la production des oligomères de rutine. La limitation de la masse de ces oligomères serait due à une inhibition de l’enzyme, provoquée par les capacités chélatantes des oligomères. Une diminution du pouvoir antioxydant et une augmentation du pouvoir inhibiteur de la xanthine oxydase ont pu être observées lors de l’accroissement de la masse des oligomères de rutine. Ces deux activités sont améliorées lors de l’accroissement de la masse des oligomères d’esculine. Pour ces deux types d’oligomères, la solubilité dans l’eau est fortement accrue. Dans le cas des oligorutines, cette forte augmentation a été corrélée à la mise en place d’un réseau dense de liaisons hydrogène observé par modélisation moléculaire. Globalement, l’approche par modélisation moléculaire dans le vide et dans le solvant a permis de dégager des relations structure-activité, reliant notamment le nombre de liaisons hydrogène à la solubilité / The aim of this work is the elaboration of rutin and esculin oligomerization process by the laccase from Trametes versicolor. A parallel synthesis process and on-line analysis of reaction media by SEC-UV and MALDI-TOF have been elaborated. The MALDI-TOF analysis has revealed the formation of simple bridges between rutin and esculin units, up to degree of oligomerization of 6 and 9 respectively. An ether bond has been observed by FTIR spectrometry for the rutin oligomers. Finally, the NMR analysis has revealed the formation of C-C and C-O bridges both on phenolic and the sugar parts of the flavonoids. At low pH and temperature, the elongation of the chain is favored, whereas increasing the dielectric constant of the solvent or the temperature favors the production of rutin oligomers. The limitation of oligomers mass is explained by the inhibition of the enzyme, probably due to the highest chelation properties of oligomers. In the case of oligorutin, a decrease of antiradical activity and an increase of xanthine oxidase inhibitory activity have been observed when the oligomers molecular mass increases. In the case of esculin oligomers, these two activities increase with the increase of the oligomers mass. For these two types of oligomers, the water solubility is considerably increased. For the oligorutins, this augmentation has been correlated to a dense network of H-bonds, which has been demonstrated by molecular modeling. Globally, the molecular modeling approach in vacuum and in solvent has allowed to establish structure-activity relationship

>Magister Scientiae - MSc / Current water treatment technologies do not remove many endocrine disruptor compounds (EDCs) such as 17α-ethynylestradiol (EE2) in its entirety, and the amount of these pollutants that continues to enter the aquatic environment through wastewater effluents is still capable of causing harmful health effects. Therefore the development of simpler and more sensitive biosensor system for detection of EE2 must be developed which have high responsiveness, low cost and easy handling. Therefore the aim of this study was to work towards the development of rapid test system of polyaniline-laccase on gold enzyme nanobiosensor (PANI-PSSA/Lac/Glu) for water estrogens. Preliminary studies were first done on the materials used in this study: estrogens, laccase, gold nanoparticles (AuNPs), and electropolymerized PANI-PSSA. Laccase was shown to be active towards EE2 and the enzyme could be stored for over three months. EE2 solution also could be used for over three months. Buffer used in this study was found to be suitable. Phosphoric acid (H3PO4) was a suitable electrolyte than hydrochloric acid (HCl) to be used for the electropolymerization of aniline and was used because it has same ions as the McIlvaine buffer (McIlB) which the post-deposition CVs indicated the formation of electrochemically very stable film. AuNPs were successfully synthesized and its size was identified to be less than 22 nm. McIlB used for testing electrochemical properties of AuNP. CVs of GC/PANI-PSSA and GC/PANIPSSA/ Au showed no difference before and after exposure to aq. EE2 solution, an indication of being re-usable and could also serve as stable immobilising platform in laccase biosensor. When interrogating with electrochemical impedance spectroscopy (EIS), the charge transfer resistance (Rct) of both GC/PANI-PSSA and GC/PANI-PSSA/Lac/Glu showed an average increase by about 2.4% and 21% before and after exposure of EE2, respectively. This shows that the GC/PANI-PSSA/Lac/Glu was a functional EE2 biosensor and showing a positive step towards achieving a re-usable biosensor for EE2 as a model water estrogen. Future work Page | vi will focus on exploring different ways of improving the biosensor’s surface regeneration and its sensitivity to EE2.

Memoria para optar al Título Profesional de Ingeniero de la Madera / Nothofagus obliqua (Mirb.) Oerst (roble o pellín), es una especie que se distribuye desde la Región de Valparaíso hasta la Región de Los Lagos; también habita en Argentina. Esta especie se caracteriza por la alta durabilidad natural que presenta su madera en individuos adultos. Sin embargo, para la madera proveniente de renovales, con diferentes características de color y densidad, no existen antecedentes sobre su comportamiento frente al ataque de hongos de pudrición u otros agentes de biodeterioro. Actualmente, la madera de renovales de roble es sometida a un tratamiento de “vaporizado”, el que tiene como objetivo igualar el color rosado a rojizo del duramen de la madera de renoval, con el rojo oscuro del duramen de roble pellín, mucho más apreciado en el mercado. Con el objetivo de proporcionar antecedentes sobre la durabilidad natural de la madera de renoval de roble y de la influencia del tratamiento de cambio de color (vaporizado), se evaluó la resistencia de estas maderas frente a la acción de hongos de pudrición mediante la pérdida de peso, solubilidad en soda y el % de lignina. Para realizar el estudio se utilizó madera de renoval de roble, empleándose 40 probetas de albura y 40 de duramen. Se aplicó el proceso de vaporizado a 20 probetas de cada grupo, de manera de compararlas con el comportamiento de la madera sin vaporizar frente al ataque del hongo de pudrición blanca Polystictus versicolor y de pudrición café Poria monticola, utilizando además, 20 probetas de roble adulto como testigo. La mitad de las probetas de cada conjunto fue evaluada a los 2 meses (T1) y el resto a los 4 meses (T2) desde el inicio del ataque fúngico. Los resultados indicaron que el vaporizado no tuvo influencia sobre la resistencia de la madera de renoval frente al ataque de P. versicolor, que generó el %PP más alto para todos los tipos de madera a los 2 meses de acción fúngica. Así mismo, el % de solubilidad en soda indicó que el hongo de pudricion blanca provocó el mayor biodeterioro en comparación con el hongo de pudricion café. Para el caso de P. monticola, se observó que el vaporizado tampoco tuvo efecto sobre la resistencia al ataque de pudrición, generándose el mayor biodeterioro a los 4 meses (T2) que a los 2 meses (T1). Esto señaló finalmente, que la madera de renoval de roble no es una madera durable, considerando su resistencia frente al ataque de hongos de pudrición. / Nothofagus obliqua (Mirb.) Oerst (roble o pellín), is a specie allocated from Valparaiso Region to Los Lagos Region, Chile, also it can be found in Argentina. This kind is characterised by its high natural durability of its wood in adults members. Nevertheless, there are not history, for wood from secondary forests, with different characteristics of color and density, about its behaviour against the fungal attack or other biodeterioration agents. Nowadays, the roble wood from secondary forests is subjected to a “steaming” treatment, which aims to equalize pink to reddish heartwood color of the wood from secondary forests, with the dark red of the roble pellin heartwood, much more appreciated in the market. In order to provide history about the natural durability of the roble secondary forest wood, and of the influence of the color change treatment (steaming), the resistance of these woods against the action of fungal through weightloss, soda solubility and the lignin percentage was evaluated. Roble wood (renoval) was used to carry out the research, using 40 test tubes for sapwood and 40 test tubes for heartwood. Steaming process was tested in 20 test tubes of each group to do the comparison with those samples that were not under the process but under the white rot fungi attack Polystictus versicolor and the brown fungi Poria monticola. Also 20 test tubes of old roble were used as reference sample. Half of the samples of each group was checked at two months (T1) and the remaining samples at four months (T2) since the attacks of fungi started. The results shown that the steaming did not affect the wood resistance against the attack of P. versicolor produced the highest %PP for all the samples at 2 months of the test. Moreover, the %S indicated that the white fungi produced a bigger biodeterioration than the brown one. In the case of P. Monticola, it was observed that the steaming had no effect neither about the resistance of fungal attack, generating the greatest deterioration at 4 months (T2) than 2 months (T1). This finally pointed that the roble secondary forest wood is not a durable one, considering its resistance against the fungal attack

In this work is discussed about material Lignamon, which has been industrially produced in the former Czechoslovakia since the 1970s. The production of this material, mainly from beechwood, combines plasticization with gaseous ammonia, densification of material by hydraulic press and heat stabilization at 180 ° C. The aim of this work is to determine the effect of these processes on the resulting resistance to wood-decaying fungi. Lignamon has a very high resistance to wood-decaying fungi and is classified as very durable according to EN 350 - 1. For each phase of the Lignamon process, samples were produced to best illustrate the changes that take place during the given phase in the wood. In particular, sample sets treated with ammonia gas, densified samples, heat-treated samples, and samples on which a combination of ammonia gas impregnation and thermal modification were performed. The resistance to wood-decaying fungi have been tested according to EN 113, including a set of Lignamon samples. The results of this test were statistically evaluated and it was found that ammonia treatment has the greatest influence on the durability of Lignamon, as well as the heat treatment and not so significantly densification. Individually, however, none of these modifications cause wood resistance to wood-decaying fungi as their combination in the production process of Lignamon.

In this work endocrine disrupting potential of Delor 103, a commercial mixture of PCB congeners, was studied along with its effect on production of laccase by the ligninolytic fungus Trametes versicolor. Using a gene-reporter yeast assay for evaluation of hormonal activity Delor 103 showed an androgenic activity with an EC50 value of 2.29. 10-2 mg/l. Chlorbenzoic acids, Delor 103 potential metabolites resulting from microbial degradation, displayed on the other hand an estrogenic activity, indicating possible changes in hormonal activity of Delor 103 during its microbial degradation. The addition of Delor 103 to mineral medium T. versicolor cultures resulted in an up to 257times higher laccase activities detected in fungal cultures. Delor 103 induced enzymes showed different pI values from those of control cultures. In a complex malt-extract glucose medium (MEG) the stimulation effect of Delor 103 was kept down. Further, the production of laccase and synthesis of different pI forms depended strongly on the growth phase of fungal cultures. Exponencially growing cultures of T. versicolor were able to produce up to 7 different pI forms of laccase in responce to Delor 103 whereas stationary cultures produced only 4 enzyme forms with higher pI values. Stimulation of laccase activities in T. versicolor,...

Pharmaceuticals have been detected in natural waters for more than forty years, but with improvements in sample preparation procedures and analytical instrumentation, the number of scientific publications on the issue has increased significantly. Even though the concentration of pharmaceutical residues in surface and drinking water is not critical for human health according to the present level of knowledge, the consequences for the environment are not clear. Wastewater treatment plants (WWTPs) have been identified as the primary route of pharmaceuticals to the environment, with households as the major source point for most of the over-the-counter and prescription drugs. In this thesis, the first aim was to study the occurrence, fate and removal of 43 pharmaceuticals during conventional wastewater treatment. The target compounds were selected on the basis of their high consumption in Spain or/and frequently reported detection in wastewaters and the possibility to be analyzed under the same experimental conditions. They belong to different therapeutic classes, i.e. nonsteroidal anti-inflammatory agents and analgesics, lipid modifying agents, psycholeptic and antiepileptic drugs, beta-blocking agents, beta-2-adrenoreceptor agonists, H2-receptor antagonists, antibiotics, angiotensin converting enzyme agents, diuretics and antidiabetic drugs. A wide variation in removal efficiencies was observed even for individual compounds, and across therapeutic classes and treatment processes, without clear conclusion on the removal of any particular compound. According to mass balance calculations and estimated partition coefficients, the loss of the selected pharmaceuticals during biological wastewater treatment can be fully attributed to biodegradation/biotransformation. None of the studied compounds was entirely biodegraded and/or transformed during biological wastewater treatment, but the measured concentrations were below the levels of concern according to available toxicity data. Conventional WWTPs cannot be expected to be the only mechanism for controlling the entry of pharmaceuticals into the environment because they were neither designed nor can provide their complete removal. Therefore, the challenge is to look for solutions that would be the most economical and effective means of preventing further pollution of natural waters by pharmaceuticals. More appropriate management of sewage waters before they enter treatment plants as well as a stricter control of effluent discharges, along with an in-depth investigation on the development of new designs and strategies for the improvement of existing wastewater treatments should be considered. In light of this, as the second objective of this thesis, alternative approaches for the removal of the antiepileptic carbamazepine (CBZ) were studied in aqueous media in two laboratory scale experiments: a) biodegradation using white rot fungus T. versicolor in an air-pulsed fluidized bioreactor operated in batch and continuous modes, and b) advanced oxidation using TiO2-heterogeneous photocatalysis under simulated solar and UV-A irradiation, and under the combined use of ultrasound and UV-A irradiation (sonophotocatalysis). We selected CBZ as a representative example of compounds that are found to be refractory to biological treatment and ubiquitous in various environmental matrices. Both, the fungal and UVA-driven TiO2-photocatalytic treatments, very different in their nature, have been shown to be very effective in degrading carbamazepine in aqueous media. The fungal treatment resulted in average removals of 54 and 96% in batch and continuous reactor, respectively. Acute toxicity test using the bioluminescent marine bacterium Vibrio fischeri showed a decrease in toxicity during the treatment in both types of bioreactor. In the other study, initial CBZ concentrations were reduced for 95% during 120 min of the UV-driven photocatalytic experiment and sonophotocatalysis, while only 10 % of CBZ was photodegraded during under solar irradiation. A slight increase in toxicity in Daphnia magna acute toxicity testing was observed over the time-course of the photocatalytic experiments, which can be associated with the formation of transformation products of CBZ. Ultraperformance liquid chromatography coupled to a quadrupole-time-of-flight mass spectrometry was used for a tentative identification of the transformation products of CBZ formed during the performed experiments. Most of the tentatively identified intermediates exhibited only slight modifications of the CBZ molecular structure. The fungal as well as the photocatalytic processes yielded oxygenated transformation products. In the biological treatment using T. versicolor, transformation products were formed by enzymatic epoxidation and hydroxylation of seven-membered heterocyclic ring of the carbamazepine molecule. During the photocatalytic experiments, CBZ-related transformation products emerged from hydroxylation and further oxidation of different parts of the molecule of carbamazepine. The generated transformation products appeared to be more persistent than their parent compound, as they were present, although at low concentration, until the end of the experiments. The results of the thesis contribute to a better understanding of a) the magnitude of the selected pharmaceuticals that reach the environment through the wastewater and sludge discharge, b) the efficiency of typical conventional wastewater treatment plants regarding the removal of these compounds from raw wastewater, and c) possible developments of alternative technologies for their enhanced elimination. / Desde hace más de cuarenta años se ha detectado la presencia de fármacos en el ciclo de aguas, sobre todo debido a los avances en la química analítica que han permitido el desarrollo de nuevas metodologías analíticas para la determinación de estos compuestos de modo fiable y a bajas concentraciones. Las estaciones depuradoras de aguas residuales (EDARs) han sido identificadas como la ruta principal de entrada de fármacos de origen humano en el medioambiente. Por tanto, el principal objetivo de esta tesis ha sido el estudio de la presencia, destino y eliminación de 43 fármacos seleccionados, durante el tratamiento convencional realizado en las EDARs. Los compuestos estudiados fueron seleccionados en base a los índices de consumo en España, a la frecuencia de detección en aguas residuales y además en base a la posibilidad de ser analizados bajo las mismas condiciones experimentales. Estos compuestos pertenecen a diferentes clases terapéuticas, i.e. antiinflamatorios no esteroideos , los agentes que reducen los lípidos séricos, ansiolíticos y antiepilépticos, los agentes bloqueadores beta-adrenérgicos, agonistas β2 adrenérgico, antagonistas H2 , antibióticos, inhibidores de la enzima convertidora de angiotensina, diuréticos y antidiabéticos. Para el análisis cuantitativo se utilizó la cromatografía de líquidos acoplada a espectrometría de masas en tándem (LC-MS/MS), empleando un sistema híbrido triple quadrupolo/trampa de iones lineal. Como segundo objetivo hemos considerado evaluar tratamientos avanzados alternativos al tratamiento convencional para la eliminación de un fármaco antiepiléptico, la carbamazepina, uno de los compuestos más recalcitrantes al tratamiento biológico convencional. Se procedió a estudiar su degradación en medio acuoso mediante dos procedimientos a escala laboratorio: a) biodegradación utilizando el hongo ligninolitico Trametes Versicolor en un reactor fluidizado por pulsos de aire operando en modo batch y continuo, y b) oxidación avanzada mediante un tratamiento fotocatalitico en presencia de TiO2 bajo irradiación UV-A y solar, y aplicando la radiación UV en combinación con ultrasonidos (sonofotocatálisis). Para la evaluación de los tratamientos alternativos, se identificaron los productos de transformación de la carbamazepina, y se evaluó la toxicidad de las muestras tratadas. Se utilizó la LC-MS/MS con analizador de tipo cuadrupolo-tiempo de vuelo. También se evaluó la toxicidad de las muestras tratadas.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.

A number of fungi produce spalted wood, which is characterized by accumulation of black pigment in fine demarcation lines, often accompanied by discoloration or staining on the wood fibers. Specific spalting fungi were identified by molecular analysis. From a total of 19 isolates and 140 clones studied, 11 fungal species were identified. The two Chlorociboria species from North America were investigated and their anamorphs were unambiguously identified for the first time. Fungal pigment formation under the influence of moisture content and pH variation was investigated in sugar maple, American beech and agar inoculated with spalting fungi. Maximum pigment production occurred at treatment with pH 4.5 for sugar maple and beech inoculated with Trametes versicolor. Xylaria polymorpha produced external pigmentation in beech treated with buffer at pH 5 and sugar maple at pH 4.5. Fungal pigmentation by Trametes versicolor and Xylaria polymorpha was stimulated at low moisture content in both wood species tested. Melanin production by Inonotus hispidus and Polyporus squamosus was stimulated above 22-28% and 34-38% moisture content in beech and in sugar maple respectively. Fomes fomentarius and Polyporus brumalis produced maximum pigmentation in beech at 26 - 41% and in sugar maple at 59 - 96% moisture content. The variation of the moisture content and pH values of wood substrates can stimulate the intensity of pigmentation of specific fungi in wood. To investigate melanin synthesis from a variety of melanin precursors, experimental research on three spalting fungi tested their reaction to catechol and L-Dopa melanin precursors in wood and agar substrate. The results indicate multiple biosynthesis pathways for melanin assembly in Trametes versicolor, Xylaria polymorha and Inonotus hispidus, and catechol produced most pigmentation in all spalting fungi investigated. Microscopic analysis by light, fluorescence, electron and confocal microscopy also indicates a bi- or multi-modal activity of melanin production and assembly by several spalting fungi. Possible variations of melanin assembly were identified based on fungal and wood species. Immunofluorescence and immunogold labelling with Mab 6D2 melanin antibody confirmed the melanin nature of the pigments produced by Oxyporus populinus, Trametes versicolor, Xylaria polymorpha, Fomes fomentarius, and Inonotus hispidus.