Uma das estratégias utilizadas no desenvolvimento de novas drogas envolve a descoberta de compostos que modulem a atividade de enzimas, importantes no processo infeccioso de patógenos. Uma abordagem interessante na triagem de novos ligantes é o uso de métodos baseados na imobilização de enzimas em suportes cromatográficos acoplados a sistemas de cromatografia líquida. O uso de IMERs (Immobilized Enzyme Reactors) como uma fase estacionária acoplado a sistemas de cromatografia líquida de alta eficiência consiste em uma estratégia para triagem de compostos rápida e eficiente e tem vantagens em relação ao uso de enzimas em solução. A enzima NTPDase-1 de Trypanosoma cruzi age como um facilitador da infecção do patógeno, inibindo assim a resposta imune do hospedeiro, permitindo uma infecção silenciosa, o que sugere seu uso como um bom alvo na busca por inibidores. Neste trabalho, a enzima NTPDase-1 foi imobilizada na parede interna de capilares de sílica fundida formando ICERs (Immobilized Capillary Enzyme Reactors). Estudos das condições de uso destes biorreatores juntamente com o desenvolvimento de um método cromatográfico multidimensional, foram realizados e validados. A otimização do método cromatográfico e sua validação, apresentaram ótimos resultados em relação aos valores obtidos para os parâmetros avaliados para métodos bioanalíticos. A imobilização da enzima foi realizada com sucesso, sendo possível a detecção da atividade catalítica no sistema cromatográfico (TcNTPDase1-ICER). Foi realizado também, o estudo cinético para ATP no TcNTPDase1-ICER, obtendo-se KM de 0,317 ± 0,044 mM, que comparado com estudos em solução, KM de 0,096 mM, ainda apresenta grande afinidade pelo substrato. / One of the strategies used in the development of new drugs involves the discovery of compounds that modulate the activity of enzymes, important in the infectious pathogens process. An interesting approach in the screening of new ligands is the use of methods based on immobilization of enzymes in chromatographic supports coupled to liquid chromatography systems. The use of IMERs (Immobilized Enzyme Reactors) as a stationary phase coupled to high performance chromatographic systems consist in a strategy to a fast and efficient compounds screening and it has advantages comparing to the use of enzymes in solution. The enzyme NTPDase-1 Trypanosoma cruzi acts as a pathogen infection facilitator, thus inhibits the host immune response allowing a silent infection, suggesting its use as a good target in the search for inhibitors. In this paper, the enzyme NTPDase-1 was immobilized for the manufacturing of ICERs (Immobilized Capillary Enzyme Reactors). Studies of conditions to the use of these bioreactors in the ligands screening along with the development of a multidimensional chromatographic method were performed and validated. The chromatographic method optimization and validation, presented excellent results, relating to the obtained values, from evaluated parameters in bioanalytical methods. The enzyme immobilization was successfully performed, being possible to detect the catalytic activity in the chromatographic system (TcNTPDase1-ICER). The kinetic study for the substrate ATP was also performed in the TcNTPDase1-ICER, obtaining KM of 0.317 ± 0.044 mM, which in comparison with studies in solution KM of 0.096 mM, still presents high affinity for the substrate.

A imobilização de membros e/ou articulações é prática utilizada na clínica no tratamento de diversas patologias, podendo variar em sua extensão e duração. Acometimentos ortopédicos comuns, como fraturas e lesões músculo-tendíneas, podem exigir como tratamento de dias a semanas de imobilização do segmento afetado. Além dos efeitos clínicos almejados, a imobilização desencadeia alterações negativas que afetam localmente o sistema musculoesquelético, mas que também trazem consequências para o sistema nervoso central (SNC) e sistema nervoso periférico. Tais consequências negativas implicam em perdas funcionais, que podem levar à dependência parcial ou completa para a realização de atividades da vida diária, como a higiene, a alimentação e a locomoção, com enormes custos individuais, familiares e sociais. É necessário, portanto, esclarecer a natureza de tais efeitos negativos e, em especial identificar o quão cedo se apresentam as alterações comportamentais e fisiológicas decorrentes do imobilismo, assim como quantificá-las e determinar a efetividade de estratégias de interferência neste processo. Neste trabalho, investigamos os efeitos de 24 horas de imobilização sobre diferentes tarefas motoras, como apertar botões, realizar a oposição do polegar em sequências variadas e fazer o alcance de alvos com o membro superior. Investigamos também, para a tarefa de oposição do polegar, o potencial terapêutico do treino motor imaginário durante o período de imobilização. Em nossos resultados, não encontramos efeitos estatísticamente significativos da imobilização sobre o comportamento motor nas tarefas pesquisadas. Adicionalmente, o treino motor imaginário na tarefa de oposição do polegar levou a uma piora no desempenho na tarefa de alcance. Esses resultados são relevantes no sentido de ressaltar que a imobilização, um recurso terapêutico importante, apresenta efeitos colaterais, mas esses não se instalam tão rapidamente quanto inicialmente hipotetizamos. É provável, ainda, que a imobilização atue de forma distinta sobre diferentes habilidades motoras, sendo umas prejudicadas com maior intensidade e precocidade do que outras. Entre as habilidades motoras afetadas pela imobilização, pode ser que encontremos, inclusive, a habilidade de imaginar cinestesicamente movimentos. O presente estudo investigou um tipo específico de treino motor imaginário, em voluntários saudáveis, submetidos a um período extremamente curto de imobilização. Possivelmente, os resultados de outros tipos de treino (variando, por exemplo, a intensidade do treino, a forma de admnistração e até mesmo a tarefa) podem ser diferentes. As sutilezas envolvidas na execução do treino motor imaginário provavelmente são a raiz da variabilidade nos resultados obtidos nos diversos experimentos da literatura. Os usos do treino motor imaginário devem, portanto, continuar a ser submetidos à experimentação, para que sua prescrição seja criteriosa e beneficie satisfatoriamente a função motora após a imobilização / The immobilization of body segments is frequently used for the treatment of orthopaedic injuries, such as fractures and soft tissue lesions. Immobilization may be as long as days or weeks, leading to several undesired side effects. The negative effects due to immobilization are felt not only in the immobilized limb, but also in the central nervous system. They lead to several functional impairments, compromising the independency of the patients in fulfilling their daily activities. Therefore, its necessary to determine the nature of such negative effects, and, specially, determine how early the functional and physiological impairments present themselves, as much as to quantify them and stabilish strategies for interference on them. In the present work, we investigated the effects of a 24-hour period of immobilization of the upper limb over several motor tasks, such as pressing buttons, oppose the thumb to the other fingers in several sequences and reaching targets. We also addressed the issue of the potential benefits of delivering a motor imagery training session, specific to the thumb opposition, during the immobilization period. Our results showed no significant effect of immobilization over the motor behavior in the evaluated tasks. Aditionally, the motor imagery training in opposing the thumb lead to an impairment on the reaching task. These results are relevant to reassure that immobilization is a safe therapeutic tool, for its side effects do not present themselves as early as hypothesized by us. Its possible, yet, that immobilization has distinct effects over different motor habilities and, in doing so, that some of the motor skills are more affected than others. It is possible that, between the motor skills affected by immobilization, we found the motor imagery hability. The present study investigated specific schedule of motor imagery training, in healthy volunteers, submited to a very short immobilization period. Its possible that other training schedules (varying the intensity, the way of administering it e even the task) might have other results then ours. The subtleties involved in motor imagery training may be the cause for the great amount of variance found in literature about it. The uses of motor imagery should be submitted to experimentation. In this way, it might be prescribed with criteria and benefit the restoration of motor function

No tecido muscular esquelético, a interface entre o sarcolema e a matriz extracelular é constituída por proteínas especializadas responsáveis pela transmissão de força transversal e longitudinal à miofibra. As adaptações do músculo esquelético às forças fisiológicas e patológicas, como a imobilização segmentar e exercícios de reabilitação, podem contribuir para a percepção celular dos sinais mecânicos e, consequentemente, induzir modificações na flexibilidade e na força muscular. Este estudo investigou as respostas teciduais do músculo sóleo de ratas submetido ao estresse longitudinal induzido pela associação do treinamento do tipo alongamento passivo com a livre movimentação pós-imobilização dos membros posteriores direitos. O membro posterior direito das ratas foi imobilizado por 10 dias em flexão plantar, a fim de manter o músculo sóleo em posição de encurtamento. Após a imobilização, os animais passaram por um período de alongamento passivo manual intermitente. Antes da imobilização e durante o período de alongamento e de livre movimentação, foi realizada análise funcional da marcha dos animais. Noventa e seis ratas Wistar adultas foram divididas em 8 grupos: Imobilizado (I); Imobilizado e alongado por 1 dia (IAL(1)); Imobilizado e alongado por 3 dias (IAL(3)); Imobilizado e alongado por 10 dias (IAL(10)); Imobilizado e livre por 1 dia (IL(1)); Imobilizado e livre por 3 dias (IL(3)); Imobilizado e livre por 10 dias (IL(10)); Controle do Imobilizado (C(Imob)). Após os procedimentos experimentais, o músculo sóleo foi removido e congelado, para o processamento de reação de coloração Hematoxilina-Eosina; imunoistoquímica para fibronectina, colágenos tipos I e III; imunofluorescência de laminina, distrofina e macrófagos; western blot de laminina e distrofina. Análises qualitativa e quantitativa em Microscópio de Luz e sistema de análise de imagens foram realizadas. As variáveis foram avaliadas inter- e intra-grupos pelo Modelo de Regressão Linear com Efeitos Mistos. Após a imobilização, os animais apresentaram perda de peso corporal e alterações no tamanho e formato das fibras. Ainda, a hipocinesia modificou as variáveis funcionais da marcha, reduziu a amplitude de movimento (ADM) de dorsiflexão, aumentou a relação fibras com fibronectina intracelular/número total de fibras (rFFI/NTF), a expressão de distrofina, de laminina e dos colágenos tipos I e III. Após três dias de remobilização, as alterações morfológicas estavam exacerbadas: intensa celularidade, núcleos centralizados, corpos de inclusão, necrose. Estes achados foram mais intensos no IAL(3). Os grupos IAL(3) e IL(3) também apresentaram comprometimento funcional, restrição de ADM, aumento da rFFI/NTF e da expressão do colágeno tipo III. Outros achados observados nestes grupos foram aumento da quantidade de macrófagos no tecido e de distrofina. As anormalidades relativas aos parâmetros da marcha e as alterações morfológicas geradas pela imobilização mostraram melhora no grupo IAL(10). A remobilização associada ao alongamento durante dez dias mostrou significativa efetividade na reversão das anormalidades musculoesqueléticas induzidas pelo desuso, especialmente nas variáveis funcionais. / The interface between sarcolemma and extracellular matrix in skeletal muscle tissue is constituted by specialized proteins that are responsible for the transversal and longitudinal forces transmission to the myofiber. Skeletal muscle adaptations to physiological and pathological forces, such as segmental immobilization and rehabilitation exercises, may contribute to cellular perception of mechanical signals and consequently induce alterations related to flexibility and muscular force. This study investigated the tissue responses of the soleus muscle in female rats caused by longitudinal stress induced by the association of passive stretching training with the free movement after immobilization of the right hind limb. The right hind limbs of female rats were immobilized during 10 days in a plantar flexion in order to keep the soleus muscle in a shortened position. After the immobilization, the animals were submitted to intermittent passive manual stretching. The animals gait was functionally analyzed before immobilization and during the period of stretching or free movement. Ninety-six adult female Wistar rats were divided into 8 groups: Immobilized (I); Immobilized and stretched for 1 day (IS(1)); Immobilized and stretched for 3 days (IS(3)); Immobilized and stretched for 10 days (IS(10)); Immobilized and free for 1 day (IF(1)); Immobilized and free for 3 days (IF(3)); Immobilized and free for 10 days (IF(10)); Immobilized control (C(Immob)). After the experimental period, the soleus muscle was removed, frozen and further processed by Hematoxylin-eosin stain; immunohistochemistry reactions for fibronectin, types I and III collagen; imunofluorescence of laminin, dystrophin and macrophages; western blot of dystrophin and laminin. Qualitative and quantitative results were obtained using a Light Microscope and a system of image analysis. The between- and within-group data were analyzed using Mixed-Effects Linear Models. After the immobilization, the animals presented loss of body weight and alterations in size and shape of the fibers. Furthermore, the hypokinesia changed the functional variables of gait, reduced the dorsiflexion range of motion (ROM), increased the intracellular fibronectin/total number of fibers ratio (FIF/TNFr), the expression of dystrophin, of laminin and of the types I and III collagen. After three days of remobilization, the morphological changes were exacerbated: intense cellularity, nuclear centralization, inclusion bodies and necrosis. These findings showed increased in the IS(3). The groups IS(3) and IF(3) also showed functional impairment, ROM restriction, increased FIF/TNFr and immunoreactivity of the type III collagen. Others findings observed in these groups were increase of the amount of macrophages in the tissue and of dystrophin. The abnormalities related to gait parameters and the morphological changes induced by immobilization were improved in the IS(10) group. The remobilization using stretching for ten days showed significant effectiveness and reverted the skeletal muscle abnormalities induced by disuse, especially concerning the functional variables.

Orientador: Wilson de Figueiredo Jardim / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-24T02:49:05Z (GMT). No. of bitstreams: 1 Torres_CarolinaFerreira_M.pdf: 3163293 bytes, checksum: 169b47e5a4d52ba3e29d4ad8c88bec54 (MD5) Previous issue date: 2013 / Resumo: O uso de fotocatalisadores suportados tem sido a estratégia preferencial em fotocatálise heterogênea, uma vez que a separação do material fotoativo do meio reacional é difícil e dispendiosa. A quitosana é um biopolímero de disponibilidade ilimitada, fonte renovável, é biodegradável e possui alto poder de adsorção. Esferas de quitosana impregnadas com TiO2 P-25 foram obtidas pela técnica de coagulação, onde uma mistura de quitosana e TiO2 P-25 é dissolvida em meio ácido e gotejada sobre um banho alcalino formando as esferas. Depois de lavadas e secas estas foram caracterizadas quanto à sua morfologia, resistência à solução ácida e à fotodegradação, área superficial, DRX, refletância de radiação UV/Vis e absorção de radiação IV. A atividade fotocatalítica das esferas foi avaliada utilizando solução de ácido salicílico 50 mg L. Placas de Petri contendo 0,5 g de esferas com relação quitosana: TiO2 P-25 na proporção de 1:10 (m/m) e 25 mL de solução de AS foram iluminadas por radiação UV e em intervalos de 8 horas alcançou-se mineralização de 40%, determinado por medidas de COT e emissão de fluorescência. As esferas foram reutilizadas em mais dois ciclos de ensaios de fotodegradação sem que passassem por qualquer processo de limpeza e sem perderem sua atividade fotocatalítica. / Abstract: The use of supported photocatalysts has been the preferred strategy for heterogeneous photocatalysis, since the separation of photoactive material from the reactional mean is difficult and expensive. Chitosan is a biopolymer of unlimited availability; it is a renewable source, biodegradable and has a high adsorption power. Chitosan beads impregnated with TiO2 P-25 were obtained by the coagulation technique, where a mixture of chitosan and TiO2 P-25 is dissolved in an acidic mean and dripped onto an alkaline bath, forming the spheres. Once washed and dried, they were characterized as to their morphology and resistance to acid solutions and photodegradation, as well as to their surface area, XRD, UV/Vis radiation reflectance and IR radiation absorption. The photocatalytic activity of the spheres was evaluated using salicylic acid solution 50 mg L. Petri dishes containing 0.5 g of beads with chitosan proportion: TiO2 P-25 at a ratio of 1:10 (w/w) and 25 mL of SA solution were illuminated by UV radiation and within 8-hour intervals a mineralization of 40% was reached, as determined by TOC measures and fluorescence emission. The beads were reused in two other test cycles of photodegradation without undergoing any cleaning process and without losing its photocatalytic activity. / Mestrado / Quimica Analitica / Mestra em Química

Abstract The purposes of the present research were to compare the outcome after Achilles tendon rupture repair in two postoperative regimens, to compare Achilles tendon elongation in two postoperative treatment methods, to compare the effects of two postoperative methods on motor performance aspects such as simple reaction time, choice reaction time, speed of movement, foot tapping speed and coordination, to test the mechanical properties of the recently developed poly-L/D-lactide (PLDLA) sutures and Maxon® sutures when implanted in the Achilles tendons of rabbits, and to study the histological tissue reactions and biodegradation of these sutures under the same conditions. Isokinetic calf muscle strength scores at the last control check-up were excellent in 56% of the patients in the early motion group, good in 32%, fair in 8%, and poor in 4%, whereas the scores in the cast group were excellent in 29% of cases, good in 50% and fair in 21%. The ankle performance scores were excellent or good in 88% of the patients in the early motion group, fair in 4% and poor in 8%, whereas the scores in the cast group were excellent or good in 92% of cases and fair in 8%. No significant differences were seen between the two groups at 3 months and at the last control checkups with regard to pain, stiffness, subjective calf muscle weakness, footwear restrictions, range of ankle motion, isokinetic calf muscle strength or overall outcome. The complications included 1 re-rupture in the early motion group and 1 deep infection and 2 re-ruptures in the cast group. AT elongation occurred in both groups, but was somewhat less marked in the early motion group. The AT elongation curves rose at first and then fell slowly in both groups. The patients who had less AT elongation achieved a better clinical outcome. AT elongation did not correlate significantly with age, body mass index or isokinetic peak torques. The recovery of motor performance functions such as simple reaction time, choice reaction time, speed of movement, foot tapping speed and coordination did not depend on the two postoperative regimens. The motor functions of the operated leg had obviously recovered to the level of the non-operated leg 12 weeks after the operation. Sutures made of PLDLA were used successfully for Achilles tendon repair in rabbits. There was no significant difference between the in vitro and in vivo tensile strength retention of the sutures. By comparison with Maxon®, PLDLA was found to have a lower initial tensile strength but more prolonged strength retention. The breaking strength values of the Achilles tendons repaired with sutures of these types were not significantly different at 6 weeks. Intratendinous PLDLA sutures formed a thinner fibrous capsule during the 12-week follow-up period than did Maxon® sutures of the same diameter. The suture materials had not been totally absorbed by 12 weeks.

Abstract Biomass, such as wood, binds CO2 as it grows, and is thus considered an environmentally friendly alternative fuel to replace coal. In Finland, biomass is typically co-combusted with peat, and also municipal waste is becoming more common as a fuel for power plants. Wood, peat and waste-based fuels are typically burned in fluidized bed combustion (FBC) boilers. Ash is the inorganic, incombustible residue resulting from combustion. The annual production of biomass and peat ash in Finland is 600 000 tonnes, and this amount is likely to increase in the future, since the use of coal for energy production will be discontinued during the 2020s. Unfortunately, FBC ash is still largely unutilized at the moment and is mainly dumped in landfills. The general aim of this thesis was to generate information which could potentially improve the utilization of FBC ash by alkali activation. The specific objective was to produce geopolymer aggregates by means of a simultaneous alkali activation-granulation process. It was shown that geopolymer aggregates with physical properties comparable to commercial lightweight expanded clay aggregates (LECAs) can be produced from FBC fly ash containing heavy metals. Although the ashes were largely unreactive and no new crystalline phases were formed by alkali activation, a new amorphous phase was observed in the XRD patterns, possibly representing micron-sized calcium aluminate silicate hydrate-type gels. The heavy metal immobilization efficiency of alkali activation varied with the type of fly ash. Good stabilization was generally obtained for cationic metals such as Ba, Pb and Zn, but in common with the results obtained with alkali activation of coal fly ash, anionic metals became leachable after alkali activation. The efficiency of immobilization depended on the physical and chemical properties of the fly ash and was not related to the total content of the element. All the geopolymer aggregates met the criteria for a lightweight aggregate (LWA) as defined by EN standard 13055-1. Their strength depended on the reactivity and particle size distribution of the fly ash. Mortars and concretes prepared with such geopolymer aggregates had higher mechanical strength, higher dynamic modulus of elasticity and higher density than concrete produced with commercial LECA, while exhibiting similar rheology and workability. / Tiivistelmä Biopolttoaineet, esimerkiksi puu, ovat ympäristöystävällinen vaihtoehto kivihiilelle, koska ne sitovat hiilidioksidia kasvaessaan. Suomessa biopolttoaineita poltetaan tyypillisesti turpeen kanssa, ja nykyään myös jätteen hyödyntäminen polttoaineena on yleistynyt. Puu, turve ja jätepolttoaineet poltetaan tyypillisesti leijupetipoltto-tekniikalla. Tuhka on polton epäorgaaninen, palamaton jäännös. Puun ja turpeen tuhkaa tuotetaan Suomessa 600 000 tonnia vuodessa ja määrän odotetaan kasvavan, sillä kivihiilen poltto lopetetaan 2020-luvulla. Leijupetipolton tuhkaa ei tällä hetkellä juurikaan hyödynnetä ja tuhka päätyykin pääasiassa kaatopaikoille. Tämän tutkielman päämääränä oli tuottaa tietoa, joka parantaisi leijupetipolton tuhkien hyödyntämistä alkali-aktivaatiolla. Erityisesti tavoitteena oli valmistaa geopolymeeriaggregaatteja yhtäaikaisella alkali-aktivaatiolla ja rakeistuksella. Tutkielmassa osoitettiin, että raskasmetalleja sisältävistä tuhkista valmistettujen geopolymeeriaggregaattien fysikaaliset ominaisuudet ovat vertailukelpoiset kaupallisten kevytsora-aggregaattien (LECA) kanssa. Vaikka tuhkien reaktiivisuus oli matala, ja uusia kidefaaseja ei muodostunut alkaliaktivaatiolla, uusi amorfinen faasi havaittiin XRD-mittauksissa. Uusi amorfinen faasi oli mahdollisesti mikrometrikokoluokan kalsium-aluminaatti-silikaatti-hydraatti-tyyppinen rakenne. Raskasmetallien stabiloinnin tehokkuus vaihteli tuhkien välillä. Kationiset metallit, kuten barium, lyijy ja sinkki, stabiloituivat pääasiassa hyvin, mutta anionisten metallin liukoisuus kasvoi alkali-aktivoinnin myötä. Stabiloinnin tehokkuus riippui tuhkien fysikaalisista ja kemiallisista ominaisuuksista, mutta raskasmetallin kokonaispitoisuudella ei ollu vaikutusta. Kaikki geopolymeeriaggregaatit olivat kevytsora-aggregaatteja standardin EN 13055-1 mukaisesti. Aggregaattien lujuus riippui tuhkan reaktiivisuudesta ja partikkelikokojakaumasta. Geopolymeeriaggregaateilla valmistettujen laastien ja betonien mekaaninen lujuus, Youngin moduuli ja tiheys olivat korkeampia kuin kaupallisella kevytsora-aggregaateilla valmistetut, vaikka niiden reologia ja työstettävyys olivat samanlaisia.

Charles University, Faculty of Pharmacy in Hradec Králové Department of: Pharmaceutical Technology Consultant: PharmDr. Ondřej Holas, Ph.D. Student: Michaela Šitnerová Title of Thesis: Protein macromolecules immobilization onto polymer carriers Enzymes are unique biocatalysts because of their properties. They are highly specific, selective and functional even under mild reaction conditions. The method of immobilization is used to increase their operational stability, activity and possible reuse. This process allows the wide use of enzymes in industry, for example in the food industry, analytical chemistry, chemical synthesis and in the pharmaceutical industry. The aim of my thesis was immobilized enzyme acetylcholinesterase (AChE) on the surface pellets of microcrystalline cellulose (MCC). Used method was simple sorption, immobilization using glutaraldehyde, and TEMPO oxidation using MCC. Well known Ellman's method served to measure the activity of AChE. The absorbance of the solution with the immobilized AChE was measured spectrophotometrically at 412 nm.

Cette étude vise à produire deux enzymes amylolytiques (α-amylase et pullulanase) thermostables par des levures contaminant le blé récolté dans des zones semi arides et arides (Biskra - Sahara, Sud Algérien) et capables d’hydrolyser à la fois les liaisons α-1-4 et α-1-6 de polysaccharides comme l’amidon et le pullulane, molécules d’intérêt industriel. Après isolement et caractérisation de colonies levuriennes, la méthode de plate-test-agar permet d’isoler des souches amylolytiques et de montrer que la souche L7 est la plus performante dans la production enzymatique parmi une douzaine de souches de levures productrices d’α-amylase et de pullulanase thermostables. L’identification des souches, basée sur les caractères morphologiques, les tests biochimiques et la biologie moléculaire a permis de répartir la population comme suit : 50% Clavispora lusitaniae (forme anamorph Candida lusitaniae), 25%, Pichia guilliermondii, 8% Pichia carribbicca, 8% Meyerozyma guilliermondii et 8% Rhodotorula rubra. Par sa richesse en amidon, le biotope du blé est favorable à la survie des levures amylolytiques. La majorité de ces souches dont la souche L7 est productrice de pseudo ou vrai mycélium et est tolérante à certains paramètres comme la température, la salinité, les stress osmotique et éthanolique. La souche de levure L7, Clavispora lusitaniae ABS7, semble être la plus performante dans la production d’enzymes thermostables. Son identification moléculaire a montré deux bandes avec l’endonucléase HAE III alors que les autres souches de la même espèce de Clavispora lusitaniae (L5, L9, L10, L11 et L12) présentent une seule bande. En conditions optimales (agitation 136,56 rpm, température 54,14°C, amidon 2,66g/l, extrait de levure 0,365g/l, sels 8, 75ml/l et oligo-éléments 4,3ml/l en erlenmeyers de 250 ml), la production maximale atteint les valeurs suivantes : 13456,36±300 UI pour l’ α-amylase et 12611, 6±154 UI pour la pullulanase. Ces performances sont en accord étroit avec la prédiction du modèle statistique évaluée à 13231UI pour l’α-amylase et 12825,5 UI pour la pullulanase. La production optimisée a pratiquement doublé par rapport à la production avant l’optimisation (6639,16 UI pour l’α-amylase et 6308,5 UI pour la pullulanase). En conditions optimales et en fermenteur de 2 L, la production maximale pour les deux enzymes de la levure Clavispora lusitaniaeABS7 est obtenue au bout de 28 h avec un optimum de croissance obtenu à 40 heures. La production des deux enzymes n’est donc pas associée à la croissance. La production maximale des deux enzymes s’effectue à pH 8. pic protéique. L’élution sur DEAE-cellulose confirme la présence des deux activités dans la même fraction. Les deux enzymes sont donc présentes sur la même molécule. L’α-amylase et la pullulanase sont purifiées avec un taux de purification de 50,5 et 44,6 respectivement et des rendements respectifs de 23,9% et 21,1%. L’extrait purifié montre une seule bande sur le gel de SDS-PAGE avec un poids moléculaire estimé à 75KDa et une activité amylolytique contenant à la fois les activités α-amylasique (indépendante de Ca2+) et pullulanasique (une métalloenzyme à calcium). La souche de la levure Clavispora lusitaniae ABS7 possède donc une enzyme amylolytique avec deux sites actifs. La CCM révèle une enzyme qui hydrolyse l’amidon en maltose et glucose et le pullulane en maltotriose, maltose et glucose, ce qui montre que l’enzyme est saccharifiante et correspond à une pullulanase de type II (amylopullulase). L’optimisation de l’immobilisation de l’enzyme a permis l’amélioration de l’activité: α-amylasique à 4907,75 UI (rendement 72,3 %) et celle de la pullulanase à 4491,83 UI (rendement 70,1%) avec un pH optimum de 8,5. Il ressort de notre étude que l’amylopullulanase type II libre de Clavispora lusitaniae ABS7 est thermostable puisqu’elle résiste à un traitement thermique de 75°C pendant 3 heures d’incubation et conserve 88% de son activité initiale. / This study aims to produce two amylolytic enzymes (α-amylase and pullulanase) by thermostable wheat contaminant yeast harvested in semi arid and arid zones (Biskra, Sahara, Algeria SUD) and capable of hydrolyzing both the α links 1-4 and 1-6 of polysaccharides such as starch and pullulan, molecules of industrial interest. After isolation and characterization of levuriennes colonies, the test method of agar-plate allows to isolate amylolytic strains and show, that the L7 strain is the most effective, in the enzymatic production of the 12 yeast strains producing α-amylase and pullulanase the thermostable. The identification of strains, based on morphological, biochemical tests and molecular biology has helped spread the population as follows: 50% Clavispora lusitaniae (anamorph form Candida lusitaniae), 25%, Pichia guilliermondii, 8% carribbicca Pichia, 8% Meyerozyma guilliermondii and 8% Rhodotorula rubra. By its high starch, the wheat biotope is favorable to the survival of amylolytic yeasts. Most of these strains, including the strain L7, is producer, pseudo or true mycelium and is tolerant to certain parameters such as temperature, salinity, osmotic stress and ethanolic stress. The yeast strain Clavispora lusitaniae ABS7 (L7) seems to be the most efficient in the production of thermostable enzymes. Its molecular identification showed two bands with the endonuclease HAE III while other strains of the same species Clavispora lusitaniae (L5, L9, L10, L11 and L12) have a single band. In optimal conditions (agitation 136.56 rpm, temperature 54.14 ° C, starch 2,66g / l, yeast extract 0,365g / l, salts 8 75ml / l and trace elements 4,3ml / liter Erlenmeyer flasks into 250 ml), the maximum production reached: 13456.36 ± 300 IU for the α-amylase and 12611, 6 ± 154 IU for pullulanase. This performance is in close agreement with the prediction of the statistical model 13231UI evaluated for α-amylase and 12825.5 IU for pullulanase. The optimized production almost doubled compared to production before optimization (6639.16 IU for the α-amylase and pullulanase for 6308.5 IU). In optimal conditions, and 2 L fermenter, the maximum production for the two enzymes of Clavispora lusitaniae ABS7 obtained after 28 hours, with an optimum of growth obtained at 40 hours. The production of both enzymes is thus not associated with growth. The maximum production of both enzymes is obtained at pH 8. The kinetics are characterized by an increase in carbohydrate and a substance spooning the wall of the fermenter, probably an exo-polysaccharide. The chromatographic profile on Sephacryl S200 reveals two α-amylase and pullulanase activities eluted along with the protein peak. Elution DEAE cellulose confirms the presence of both activities in the same fraction. Both enzymes are present on the same molecule. The α-amylase and pullulanase were purified with a purification rate of 50.45 and 44.59 respectively and respective yields of 23.88% and 21.11%. The purified enzyme showed a single band on SDS-PAGE gel with a molecular weight estimated at 75 KDa and an amylolytic activity containing both the α-amylase activities (independent of Ca2+) and pullulanase (a calcium metalloenzyme). The strain of the yeast Clavispora lusitaniae ABS7 therefore has an amylolytic enzyme with two active sites. TLC reveals an enzyme which hydrolyzes starch into maltose and glucose and pullulan into maltotriose, maltose and glucose, which shows that the saccharifying enzyme, and corresponds to a pullulanase type II (amylopullulase). The optimization of the immobilization of the enzyme enabled the improvement of the activity: α-amylase to 4907.75 IU (yield 72.3%) and pullulanase to 4491.83 IU (yield 70, 1%) with a pH optimum of 8.5. It appears from our study that amylopullulanase type II free is thermostable to heat treatment of 75 ° C for 3 hours of incubation, and retains 88% of its original activity.

La perte de masse et de force musculaires liée à l’âge, ou sarcopénie, pourrait être partiellementexpliquée par un défaut de récupération de masse musculaire après des épisodes générateursd’atrophie musculaire. Ainsi, les périodes d’immobilisation qui augmentent avec l’âge (alitement,convalescence, fracture) pourraient être suivies d’une absence de récupération musculaire etcontribuer à la fonte musculaire au cours du vieillissement. Les causes de ce défaut de récupérationimpliquent notamment un déséquilibre du taux de renouvellement protéique et du taux derenouvellement cellulaire. L’objectif de cette thèse a donc été de mettre en évidence les mécanismesresponsables de l’atrophie musculaire chez le rat âgé au cours de l’immobilisation et ceux quiseraient défaillants afin de déceler les mécanismes à cibler pour favoriser la récupérationmusculaire.Des rats âgés ont été immobilisés pendant 8 jours par plâtrage unilatéral de la patte arrière, puislaissés en récupération pendant 40 jours après le déplâtrage. Nous avons montré que chez cesanimaux nourris avec un régime contenant 13% de caséine, l’immobilisation entraîne une atrophiedes muscles immobilisés mais, contrairement au rat adulte, le rat âgé ne récupère jamais la massemusculaire perdue. L’atrophie des muscles immobilisés peut être expliquée par 1/ une augmentationde l’apoptose et de la protéolyse ubiquitine-protéasome-dépendante musculaires, 2/ une diminutionde la régénération des cellules musculaires et 3/ une diminution de la protéosynthèse musculaire àl’état nourri. Tous ces phénomènes pourraient résulter de la présence d’un fort stress oxydant etd’une importante inflammation intramusculaire. Tous ces paramètres sont normalisés dès 10 jours derécupération, ce qui permet de stopper l’atrophie mais ne permet pas d’initier la phase derécupération musculaire. Nous avons donc testé l’effet de différentes supplémentationsnutritionnelles au cours de la période de récupération afin de favoriser un gain de masse musculairepost immobilisation. Des supplémentations en leucine (acide aminé bien connu pour stimuler laprotéosynthèse et inhiber la protéolyse) ont ainsi été réalisées. Chez les rats supplémentés, uneamélioration de la synthèse protéique et une normalisation plus précoce des activités protéolytiquesdu protéasome ont été observées. Cependant cette amélioration du métabolisme protéique ne s’estpas traduite par un gain de masse musculaire. Par contre, la modulation qualitative et quantitative desapports en protéines a pu permettre d’obtenir une récupération significative de masse musculaire :ainsi des régimes contenant 13% de lactosérum et des régimes hyper-protéinés ont permis de gagner50% de la masse perdue et ce, dès 20 jours de récupération.Nos résultats montrent que l’immobilisation chez le rat âgé aggrave la sarcopénie. Une fortealtération du métabolisme protéique permet d’expliquer la perte de muscle et la seule normalisationde la protéolyse et de la protéosynthèse permet d’expliquer l’absence de récupération musculaire.Nous avons montré que la modulation des apports en protéines au cours de la phase de récupérationpouvait permettre un gain de protéines. / Sarcopenia, the age-related muscle mass loss, might be partially explained by an impairedmuscle mass recovery of skeletal muscle mass after a catabolic state. Thus, immobilization periodswhich increase with aging could induce a muscle atrophy followed by a lack of muscle massrecovery. An imbalance of protein and cellular metabolisms are certainly involved in this absenceof recovery. The aim on this Ph.D thesis was to explore the mechanisms involved in muscle massatrophy during immobilization and their possible alteration during the recovery period in old rats.Old rats were immobilized for 8 days by unilateral hind limb casting and then allowed torecover for 40 days. Our results showed that animals fed a 13% casein diet wasted muscle mass inimmobilized muscles but, contrarily to adult animals, they never recovered the muscle mass loss.Muscle atrophy was due to 1/ an increase of apoptotic and ubiquitine-proteasome-dependentproteolytic pathways, 2/ a decrease of muscle regeneration processes and 3/ a decrease of muscleprotein synthesis at the fed state. These changes paralleled an increase of intracellular inflammationand oxidative stress. As these parameters were only normalized during the recovery period, theresultant nitrogen balance was then not enough positive as required for the muscle protein gain,hence contributing to the age-related incomplete muscle mass recovery. We tested free leucinesupplementation (an amino acid known for its stimulatory effect on protein metabolism) during therecovery period to improve muscle mass gain. This supplementation induced a greater muscleprotein synthesis in supplemented animals, but without any muscle mass gain. However, wedemonstrated here for the first time that muscle protein accretion after immobilization-inducedatrophy could be achieved with whey protein or high protein diets.In conclusion, we demonstrated that immobilization in old rats induced a muscle mass atrophyfollowed by an incomplete recovery, hence contributing to the development of sarcopenia. We alsodemonstrated that this lack of recovery cannot be overcome by a dietary free leucinesupplementation, despite a positive effect on protein metabolism, contrarily to high protein andwhey protein diets.

Introdução: A imobilização prolongada no leito pode desencadear alterações em diferentes sistemas do corpo e na capacidade funcional. Durante a internação hospitalar, o repouso acontece com frequência e alterações na funcionalidade instaladas neste período podem perdurar mesmo após a alta. Este ambiente pode induzir o paciente à inatividade física o que pode afetar principalmente a força muscular. Objetivos: Avaliar o impacto da internação hospitalar na capacidade funcional e força muscular de pacientes hospitalizados e identificar possíveis preditores de ocorrência de eventos pós alta. Métodos: Trata-se de um estudo coorte prospectivo que inclui 250 pacientes internados em enfermarias do Instituto Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. Foram incluídos pacientes de ambos os gêneros, com idade >=50 anos. Foram excluídos pacientes com déficits cognitivos, dellírium, aqueles que não conseguiram realizar os testes propostos e/ou apresentarem > 48 horas de admissão na enfermaria. Dados clínicos e sociodemográficos foram avaliados a partir da análise dos prontuários. A força de preensão palmar (FPP) foi avaliada pelo dinamomêtro palmar e a capacidade funcional (CF) avaliada pelo índice de Barthel. Para verificar a ocorrência de quedas e re-internações, foi realizado um acompanhamento por telefone de 1, 3 e 6 meses pós-alta. Análise estatística: As variáveis numéricas foram representadas em mediana e intervalo interquartil (5% a 95%) e média e desvio padrão (DP). As variáveis categóricas foram descritas em valores absolutos e porcentagem. A comparação entre os sujeitos adultos e idosos no momento da admissão, foi realizada através do teste de Mann-Whitney Rank Sum Test e teste de Qui-Quadrado. A comparação entre as pontuações da admissão e alta hospitalar da FPP e CF foi realizada através do teste t de Student e Wilcoxon Signed Rank Test. Um modelo de Regressão Logística Múltipla foi utilizado para identificar possíveis preditores de perda de FPP durante a hospitalização e a ocorrência de quedas e re-internação no acompanhamento pós-alta. Resultados: A CF avaliada pelo Índice de Barthel não sofreu alterações (p=0,83), no entanto, foi verificada uma redução significativa de FPP durante o período de hospitalização (p=0,03), sendo a FPP no momento admissão o principal preditor de perda de força. A ocorrência de quedas e re-internações aumentam progressivamente com o acompanhamento pós-alta, sendo os principais preditores de ocorrência de quedas o auto-relato de queda prévia e a idade e, para re-internação o tempo de hospitalização. Conclusão: A hospitalização pode gerar perda de força de preensão palmar, no entanto, a capacidade funcional avaliada pelo Indice de Barthel não se altera. A ocorrência de queda e re-internação aumentam com o período de acompanhamento pós-alta e foi possível identificar preditores para a ocorrência destes eventos / Introduction: Prolonged bed rest can trigger changes in different body systems and functional capacity. During hospitalization, the rest happens frequently and changes in functionality installed in this period can last even after hospital discharge. This environment can induce the patient to physical inactivity which can affect the musculoskeletal system, especially the muscular strength. Objectives: Evaluated the impact of hospitalization on functional capacity and muscle strength of hospitalized patients. Methods: This was a prospective cohort study including 250 patients in wards of the Hospital Central Institute of the Faculty of Medicine, University of São Paulo. They included patients of both genders, aged >= 50 years. Patients with cognitive deficits, dellírium, those who cannot perform the tests proposed and / or submit > 48 hours of admission to the ward. Clinical and sociodemographic data were evaluated from the analysis of medical records. The hand grip strength (HGS) was evaluated by the hand dynamometer and the functional capacity (FC) evaluated by the Barthel Index. To verify the occurrence of falls and re-hospitalizations, a telephone follow-up of 1, 3 and 6 months post-discharge was performed. Statistical analysis: Numerical variables were represented in median and interquartile range (5% to 95%) and mean and standard deviation (SD). Categorical variables were described in absolute values and percentage. The comparison between adult and elderly subjects at admission was performed using the Mann-Whitney Rank Sum Test and Chi-Square test. The comparison between admission and discharge scores of FPP and CF was performed using Student\'s t-test and Wilcoxon Signed Rank Test. A Multiple Logistic Regression model was used to identify possible predictors of FPP loss during hospitalization and the occurrence of falls and re-admission at post-discharge follow-up. Results: The FC assessed by the Barthel Index did not change (p = 0.83); however, there was a significant reduction in FPP during the hospitalization period (p = 0.03). The occurrence of falls and re-hospitalizations increase progressively with post-discharge follow-up, the main predictors of occurrence of falls being the self-reported prior fall and the age, and for re-hospitalization, hospitalization time (p < 0,001). Conclusion: Hospitalization may lead to loss of hand grip strength, however, the functional capacity assessed by the Barthel Index does not change. The occurrence of fall and re-hospitalization increased with the post-discharge period and it was possible to identify predictors for the occurrence of these events