Foi estudada a influência da imobilização, remobilização livre e remobilização com treinamento físico sobre as propriedades mecânicas do músculo esquelético. O limite máximo, limite proporcional, rigidez e resiliência foram obtidos a partir de ensaio de tração axial. Foram utilizados os músculos gastrocnêmio de 37 ratas fêmeas albinas, da variedade Wistar, divididas em 4 grupos. Um grupo foi utilizado como controle. Três grupos tiveram o membro posterior direito imobilizado com aparelho de gesso por três semanas. Destes, um grupo foi sacrificado logo após a retirada da imobilização, outro foi submetido a período de remobilização livre e o terceiro à remobilização com treinamento físico com natação por quatro semanas. Houve redução estatisticamente significante para os valores das propriedades mecânicas para os músculos submetidos à imobilização. A remobilização constituída por exercitação não produziu a recuperação da resiliência. Houve aumento estatisticamente significante da rigidez para os músculos submetidos ao treinamento físico. A remobilização livre devolveu ao músculo suas propriedades mecânicas. / The influence of immobilization, free remobilization and remobilization followed by physical training on the mechanical properties of the striated muscle was studied in rats. The right gastrocnemius was tested in traction and the following mechanical parameters were obtained: ultimate limit, elastic limit, stiffness and resilience. Thirty-seven adult female Wistar rats were used and divided in four groups. One group was kept as a control and consisted of non-immobilized animals. In the three other groups the right hind limb was immobilized in a spica cast during three weeks. In the last group the animals were exercised with swimming after the immobilization period, during four weeks. Our results showed that there was a remarkable decrease of mechanical parameters with the immobilization. In the group with immobilization followed by exercise there was not recovery for the resilience. All trained animals showed a statistically significant recovery of the stiffness. With the mobilization the muscle recovered the normal parameters.

El objetivo general de la presente tesis doctoral es el desarrollo de herramientas para la obtencion, produccion y aplicacion de dos enzimas glicosidicas: �¿-L-arabinofuranosidasa proveniente del hongo Aspergillus niger (Abf) y �À-D-glucosidasa (Bgl), proveniente de la levadura Candida molischiana. Estas hidrolasas se emplean en la liberacion de azucares en procesos de conversion de biomasa y en la industria alimentaria, pero tambien en la sintesis de aminoglicosidos, glicoconjugados y oligosacaridos, compuestos de alto valor anadido para la industria quimico-farmaceutica. Las enzimas se han expresado en la levadura metilotrofica Pichia pastoris, y se han purificado para caracterizar sus propiedades bioquimicas. Asimismo, se ha comprobado su capacidad para catalizar reacciones de transglicosilacion con alto rendimiento. En relacion a su produccion, se ha establecido y validado un modelo basado en restricciones del metabolismo de Pichia pastoris, evaluando su consistencia mediante analisis de flujos metabolicos posibilistico. El modelo permite estimar la tasa de crecimiento y la distribucion de flujos intracelulares a partir de unos pocos flujos extracelulares medidos experimentalmente. Adicionalmente, el modelo se ha extendido para estimar la productividad de proteina recombinante, y se ha empleado para analizar diferentes condiciones de cultivo de las cepas transgenicas que sobreproducen las enzimas Abf y Bgl. Finalmente, las enzimas se han inmobilizado en organosilicas bimodales de la familia UVM-7. Los biocatalizadores resultantes se han caracterizado bioquimica y fisico-quimicamente y se han evaluado en diferentes aplicaciones de interes biotecnologico. / Tortajada Serra, M. (2012). Process development for the obtention and use of recombinant glycosidases: expression, modelling and immobilisation [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/16800 / Palancia

Globally, male ungulate species are heavily managed for their sporting and trophy qualities. North American elk (Cervus canadensis) are typically managed using a male-biased harvest regime, placing increased chances of mortality on males in these hunted populations. To manage for trophy quality animals that typically represent older age classes, wildlife managers have implemented many age-biased harvest regulations, including spike-only tags and antler point restrictions. Many of these age-biased harvest regulations have fallen short of their desired goal of producing older bull elk. Consequently, the consensus has evolved to center on an overall reduction in harvest pressure. The state of Kentucky began an elk restoration project in 1997, with 1,553 elk released through 2002. As with other modern elk restoration projects, the male demographic received little research attention in the years immediately post restoration. The difficult logistics surrounding the transport of adult male elk and the reluctance of source states to part with potential trophy animals, led to few adult male elk receiving tracking collars to monitor this demographic. Hunter success rates indicated a growing male component to this population in light of the lack of a radio-marked cohort. With overall population numbers increasing in step with predictive models, so too did hunting tag numbers and hunting pressure. This rise in hunting pressure likely forced elk to become more cryptic, giving rise to the perception of a decline in the elk population, especially older age class male elk. This research represents the first in-depth look at the survival rates and habitat selection of adult male elk in Kentucky. Recent improvements in field methodology have allowed for the more efficient acquisition of a robust sample of adult male elk. I conducted a radio-telemetry study of adult male elk within southeast Kentucky to investigate the following: (1) survival and cause-specific mortality factors, (2) survival during the fall hunting period, (3) changes in survival following the implementation of a limited entry area (LEA) enclosing our study area, and (4) the associations of morphometric characteristics with the survival of adult male elk. Given the lack of information on the habitat use of male elk, a cohort of global positioning system (GPS) equipped elk were captured to investigate: (1) seasonal habitat use of male elk, (2) quantification of availability of male elk in readily viewable habitats, (3) changes to the percent of open land within the fall home range of adult male elk, and (4) the influence of open land on survival rates. To investigate the dispersal of male elk, I compared genetic relatedness to space use. Finally, in an attempt to better understand our existing capture methodologies, I analyzed drug induction and reversal metrics for the immobilization drug Carfentanil citrate. Survival analysis resulted in a 16.9% (CI = 12.2 – 23.7) three-year survival rate for adult male elk. An improvement in survival rate (p = 0.077) was noted after the implementation of an LEA system that limited the number of hunters in the study area. No morphometric characteristics were observed to have an association with survival, indicating that hunters indiscriminately harvest male elk. Predictive, habitat use models for male elk indicated a preference for grass habitats and use of habitats near grass patches. Seasonal variation in habitat use was observed with the greatest daily use of grass habitats occurring in the winter season. Adult male elk selected for open land at greater rates than is available across the study area. Over the course of three hunting seasons, elk were found to reduce their use of open land during daylight hours, and we anecdotally believe this to be a response to hunting pressure. A reduction in survival probability of male elk was directly related to use of open land in the final year of the project. Little home range overlap was observed between related male elk, indicating some level of dispersal and intra-specific competition. Predictive models for Carfentanil immobilization indicated an increase in efficacy of a shoulder injection as opposed to a hindquarter drug injection. Future management of elk in Kentucky should center on promoting the persistence of healthy grassland areas within the elk restoration zone and meeting hunter expectations. Hunter expectations should be gathered and management tailored to meet their desires and the objectives of the management agency. This research indicates that hunters harvest male elk regardless of trophy characteristics, yet we are not sure of the underlying reasons. The interaction of habitat and survival is complex and further complicated by the reclaimed coal mines that Kentucky elk live upon. Habitat management priorities should focus on a heterogeneous, yet healthy habitat that meets the needs of all species residing on these once-exploited lands.

Orientador: Rubens Monti / Banca: Eleonora Cano Carmona / Banca: Maristela de Freitas Sanches Peres / Resumo: Invertase ou β -D frutofuranosidase (EC 3.2.1.26) é uma enzima que catalisa a hidrólise do terminal não redutor do resíduo β- frutofuranosidase em frutofuranosídeos. A imobilização em suportes lignocelulósicos como resíduos bagaço de cana (BC) e resíduos de sabugo de milho (SM) são favoráveis, pois são suportes de baixo custo. A invertase comercial e extraída de Saccharomyces cerevisiae e os derivados foram caracterizadas enzimaticamente. O pH ótimo encontrado para os derivados foram bem próximas da encontrada para as invertases livres, em torno de pH 4,5 a 5,5. Os derivados apresentaram estabilidade térmica superior a das invertases livres e alguns derivados tiveram acréscimos na temperatura, como os derivados da invertase extraída obtido com pó BC e SM glutaraldeído e derivados da invertase comercial obtido com pó SM e BC amino e BC glioxil. A energia de ativação encontrada para a maioria dos derivados apresentou- se na faixa de 12 a 29 kJ/ mol, valores inferiores ao encontrados para as formas livres de invertase, sendo 32.02 kJ/ mol para invertase comercial e 34.94 kJ/ mol para invertase extraída.O derivado da invertase comercial obtido com pó sabugo de milho amino e o derivado invertase extraída obtido com pó de sabugo de milho glutaraldeído foram os derivados mais estáveis, apresentando capacidade de serem reutilizados em cinco ciclos subseqüentes. Estes derivados foram ensaiados em concentrações variadas de sacarose e os resultados mostraram que a invertase imobilizada apresenta inibição pelo produto glicose/frutose após 6 horas de ensaio. A utilização de resíduos da agroindústria como suportes para imobilização da invertase apresentou-se promissora para a aplicação em escala industrial. / Abstract: Invertase or β - D fructofuranosidase (EC 3.2.1.26) it is an enzyme that catalyzes hydrolysis of the non reducing terminal of the residue β- fructofuranosidase in frutcofuranosideos. Immobilization on lignocellulosic supports as residue sugarcane bagasse and residues of corn cobs are favorable, therefore they are matrices of low cost commercial invertase and extracted of Saccharomyces cerevisiae and the derivatives had been enzymatically characterized. The optimum pH found for the derivatives had been well next to the found one for free invertases, around pH 4.5 the 5.5. The derivatives had presented superior thermal stability of free invertases and some derivatives had additions in the temperature, as the derivatives of extracted invertase gotten with dust sugarcane bagasse and corn cobs glutaraldehyde and derivatives of commercial invertase gotten with dust corn cobs and sugarcane bagasse amine and sugarcane bagasse glyoxyl. The energy of activation found for the majority of the derivatives were between 12 the 29 kJ/mol, less values to the found ones for the free forms of invertase, being 32.02 kJ/mol for commercial invertase and 34.94 kJ/mol obtained with extracted invertase. The derivative of commercial invertase gotten with dust corn cobs amine and the derivative extracted invertase gotten with dust of corn cobs glutaraldehyde had been the derivatives most stable, presenting capacity to be reused in five subsequent cycles. These derivatives had been assayed in concentrations of varied sucrose. The results with derivates shown that invertase presented inhibition for the product glucose/ fructose after 6 hours of test. The use of residues of the agribusiness as supports for immobilization of invertase presented promising for the application in industrial scale. / Mestre

O objetivo deste estudo foi avaliar a influência da irradiação a laser de baixa intensidade, associada ou não a imobilização, nas propriedades mecânicas do músculo gastrocnêmio submetido a lesão experimental por mecanismo de impacto. Foram utilizadas 70 ratas da variedade Wistar, divididas em 7 grupos experimentais: grupo controle; grupo lesão; grupo lesão irradiação laser; grupo lesão-imobilização 24 horas-irradiação laser; grupo lesão-imobilização 72 horas-irradiação laser; grupo lesão-imobilização 24 horas; grupo lesão-imobilização 72 horas. A lesão foi provocada no músculo gastrocnêmio por mecanismo de impacto (trauma direto não invasivo). Os músculos foram submetidos a ensaios mecânicos na máquina universal de ensaio EMIC e, por meio da interface com microcomputador, geraram gráficos carga x alongamento a partir dos quais foram calculadas as respectivas propriedades mecânicas: carga e alongamento no limite de proporcionalidade e no limite máximo e rigidez. A lesão provocou diminuição da carga no limite de proporcionalidade e no limite máximo em relação ao grupo controle (p < 0,05). Os grupos submetidos a imobilização por 24 e 48 horas respectivamente e irradiados não apresentaram melhora das propriedades avaliadas quando comparados ao grupo lesão (p < 0,05). A análise mecânica mostrou que a lesão muscular foi efetiva e que a associação de imobilização gessada e irradiação laser não foi efetiva na melhora das propriedades mecânicas avaliadas / The aim of this study was to evaluate the influence of low power laser irradiation, associated or not with immobilization, on the mechanical properties of a gastrocnemius muscle submitted to experimental injury caused by impact mechanism. Seventy Wistar rats were used, and were divided into 7 experimental groups: 1-control group; 2-injury group; 3-injury–laser irradiation group; 4-injury–24 hour immobilization–laser irradiation group; 5-injury–72 hour immobilization–laser irradiation group; 6-injury–24 hour immobilization group; 7-injury–72 hour immobilization group. Injury was caused in the gastrocnemius muscle through impact mechanism (non-invasive direct trauma). Muscles were submitted to mechanical tests in the EMIC machine, and using computer interface they generated load x deformation graphs from which the mechanical properties of load and stretch were calculated: at the limit of proportionality, at the maximum limit and stiffness. The injury reduced load at the limit of proportionality and at the maximum limit in relation to control group (p < 0,05). Groups that were submitted to 24 and 48 hour immobilization respectively and were irradiated showed no improvement in the evaluated properties when compared to injury group (p < 0,05). A mechanical analysis showed that the muscular injury was effective, and that the association of plaster cast immobilization with irradiation was not efficient to improve the evaluated mechanical properties

Les enzymes sont des biocatalyseurs de plus en plus utilisés pour la transformation de molécules organiques (chimie fine, bioconversions, dépollution, chimie du pétrole) car elles possèdent de très bonnes sélectivité et réactivité, générant rapidement de larges quantités de produit. Cependant, la fragilité des enzymes, notamment en solution, limite souvent leur utilisation. Il est donc crucial de les immobiliser et de les stabiliser dans des supports adaptés. Une grande variété de matrices d’immobilisation (organiques ou inorganiques) a déjà étudiée, mais aucune ne satisfait pleinement aux critères nécessaires pour le développement de bio-réacteurs (accessibilité au site actif de l’enzyme, relargage de l’enzyme, diffusion des réactifs, recyclabilité, stabilité..). En outre, la majorité de ces matrices présente une porosité désordonnée, inadaptée pour une immobilisation homogène. L’utilisation de matériaux hybrides, cristallins et poreux de type Metal-Organic Frameworks (MOFs) a été récemment proposée comme alternative avec des applications en biocatalyse et en biodétection.Le travail de cette thèse a consisté à associer des matériaux de type Metal-Organic Frameworks à une mini-enzyme, la microperoxidase 8 (MP8), afin d’obtenir des matériaux multifonctionnels. Dans une première partie, le MOF mésoporeux, MIL-101(Cr), a été utilisé pour encapsuler la MP8, ce qui a conduit à une amélioration de son activité catalytique dans des conditions qui ne sont pas adéquates pour l’activité enzymatique (conditions acides, forte concentration en H2O2), démontrant ainsi le rôle protecteur du MOF vis-à-vis de l’enzyme. De plus, il a été possible de recycler le biocatalyseur. Cette approche a également permis d’améliorer considérablement la sélectivité de la MP8 pour la dégradation d’un colorant organique toxique négativement chargé, le méthyl orange, grâce à son adsorption sélective par interaction électrostatique avec les particules de MIL-101(Cr). La seconde partie a été consacrée à l’utilisation de matériaux MIL-101(Cr) fonctionnalisés. Tout d’abord, l’influence de la fonctionnalisation du ligand (avec un groupement –NH2 ou –SO3H) sur l’encapsulation de la MP8 ainsi que sur son activité catalytique pour des réactions de sulfoxydation a été étudiée. Il a été montré que l’activité catalytique et la réactivité de la MP8 sont affectées par le microenvironnement spécifique des pores du MOF, notamment pour des réactions de sulfoxydation mettant en jeu des dérivés thioanisole. Ensuite, un MOF à métal mixte (MIL-101(Cr/Fe)) choisi pour ses propriétés catalytiques stables, a été synthétisé et caractérisé. Enfin, la dernière partie de cette thèse a été consacrée à la synthèse in-situ d’un MOF (le microporeux MIL-53(Al)-FA) en présence de biomolécules (BSA) dans des conditions compatibles avec la préservation de la structure protéique (en solution aqueuse à température ambiante). Les matériaux hybrides obtenus ont été caractérisés en couplant de nombreuses techniques. Cette méthode d’encapsulation a conduit à des taux d’immobilisation extrêmement élevés. Une étude préliminaire a été initiée avec l’enzyme, Horseradish Peroxidase , qui conserve son activité catalytique après immobilisation. / The use of enzymes in biocatalytic processes has been a challenging goal over the years. While enzymes present exceptional catalytic properties, their fragility hinders their industrial application. Their stabilization and protection are therefore of paramount importance. This can be effectively addressed through their immobilization within host solid matrices. Traditional materials (silica, clays, polymers, biopolymers, porous carbons…) have been widely studied as supports. Their pure organic or inorganic nature often requires a compromise between affinity with enzymes and robustness of the matrix. Besides, most of them have non-ordered porosity, with non-homogenous pore size distributions, unsuitable for homogeneous immobilization. Metal-Organic Frameworks (MOFs) have been recently introduced as alternative supports, thanks to their hybrid nature and their crystalline and highly porous structures.The aim of this PhD was to combine Metal-Organic Frameworks (highly porous and chemically stable polycarboxylate MOFs) and a mini-enzyme, microperoxidase 8 (MP8) to obtain multifunctional biocatalysts. In a first part, the mesoporous MIL-101(Cr) was used as a host matrix to encapsulate MP8. The encapsulation led to an increased catalytic activity under conditions (acidic conditions, high concentration of H2O2) detrimental to the catalytic activity of MP8, thereby demonstrating the protecting effect of MIL-101(Cr) matrix. The biocatalyst was also efficiently recycled. The selectivity of MP8 for the degradation of the harmful negatively charged organic dye methyl orange was also enhanced, thanks to the charged-based selective adsorption of the dye in MIL-101(Cr) porosity. A second part of the work was devoted to the use of functionalized MIL-101(Cr) analogs. First, functionalized ligands (bearing –NH2 and –SO3H groups) were used, and their influence on MP8 encapsulation was evaluated. The catalytic activity toward sulfoxidation reactions was also studied. The successful encapsulation of MP8 was strongly dependent on charge matching between the enzyme and the MOFs particles, while its catalytic activity was affected by the specific microenvironment of the pores. The MOF frameworks also modified the reactivity of MP8 toward different thioanisole derivatives. Then, a mixed metal MOF (MIL-101(Cr/Fe)), selected for its stable catalytic properties, was synthesized and characterized. Finally, the last part was devoted to the in-situ synthesis of MOFs (microporous MIL-53(Al)-FA) in presence of biomolecules (BSA) under compatible conditions with the preservation of the protein’s quaternary structure (aqueous media and room temperature). The resulting hybrid materials were thoroughly characterized and presented high loadings of BSA. A preliminarily study was performed with the enzyme, Horseradish Peroxidase, which retained its catalytic activity after immobilization.

Hierarchical systems that integrate nano- and macroscale structural elements can offer enhanced stability over traditional immobilization methods. Microparticles were synthesized using interfacial assembly of lipase with (CLMP-N) and without (CLMP) nanoparticles into a crosslinked polymeric core, to determine the impact of the highly ordered system on lipase stability in extreme environments. Kinetic analysis revealed the macrostructure significantly increases the turnover rate (kcat) following immobilization. The macrostructure also stabilized lipase at neutral and basic pH values, while the nanoparticles influenced stability under acidic pH conditions. A greener solvent, choline chloride and urea, was applied to produce sugar ester surfactants. Microparticles exhibited decreases in the turnover rate (kcat) and catalytic efficiency (kcat/Km) following exposure, but retained over 60% and 20% activity after exposure at 50 ºC and 60 ºC, respectively. CLMP and CLMP-N outperformed the commercially available lipase per unit protein in the production of sugar esters. The utilization of greener solvent systems with hierarchical immobilized enzyme systems has the potential to improve processing efficiency and sustainability for the production of value-added agricultural products.

In this work the influence of alkaline activator on immobilization of lead and copper in alkali-activated blast furnace slag was investigated. A total of five activators were used; sodium water glass, potassium water glass, sodium hydroxide, potassium hydroxide and sodium carbonate. The leaching test according to ČSN EN 12457-4 was used to evaluate the level of immobilization of heavy metals, the leached solutions were analyzed by ICP-OES. For a better understanding of immobilization, the selected samples were characterized by analytical methods (FTIR, XRD, SEM, XPS). It was found that the degree of immobilization Pb2+ and Cu2+ in AAS was very high, regardless of the type of alkaline activator used.

This diploma thesis deals with the preparation of solid hydrophilic sponges made of acetalized PVAl. The sponges were produced by foaming by gaseous reaction by-product. The experimental work also includes the study of active compound immobilization into the sponge (finely ground lignite as a heavy metal sorbent).

This thesis deals with preparation of geopolymeric binders for inhibition of toxic elements, especially arsenic. It studies influence of content progress of different phases in prepared binders on leachability of As during the setting. The aim of this study is to determine the type of inhibition or the cause of leachability of As in prepared binders respectively. Fly ash from high-temperature coal combustion, slag, cement kiln dust and sodium hydroxide solution were used for experimental works. FT-IR, TG-DTA, XRD analyses after different hydration times were used. The leachates were analyzed by ICP-MS. Calorimetric measurements of tested mixtures fresh pastes were implemented to learn about setting process. Hardened binder specimens were tested for bending and compressive strength.