The activity of protein kinases is heavily dependent on the phosphorylation state of the protein. Kinase phosphorylation states have been prepared through biological or enzymatic means for biochemical evaluation, but the use of protein chemical modification as an investigative tool has not been addressed. By chemically reacting a genetically encoded cysteine, phosphocysteine was installed via dehydroalanine as a reactive intermediate. The installed phosphocysteine was intended as a surrogate to the naturally occurring phosphothreonine or phosphoserine of a phosphorylated protein kinase. Two model protein kinases were investigated on: MEK1 and p38α. The development of suitable protein variants and suitable reaction conditions on these two proteins is discussed in turn and in detail, resulting in p38α-pCys180 and MEK1-pCys222. Designed to be mimics of the naturally occurring p38α-pThr180 and MEK1-pSer222, these two chemically modified proteins were studied for their biological function. The core biological studies entailed the determination of enzymatic activity of both modified proteins, and included the necessary controls against their active counterparts. In addition, the studies on p38α-pCys180 also included a more detailed quantification of enzymatic activity, and the behaviour of this modified protein against known inhibitors of p38α was also investigated. Both modified proteins were shown to be enzymatically active and behave similarly to corresponding active species. The adaptation of mass spectrometry methods to handle the majority of project's analytical requirements, from monitoring chemical transformations to following enzyme kinetics was instrumental in making these studies feasible. The details of these technical developments are interwoven into the scientific discussion. Also included in this thesis is an introduction to the mechanism and function of protein kinases, and on the protein chemistry methods employed. The work is concluded with a projection of implications that this protein chemical modification technique has on kinase biomedical research.

O complexo Sporothrix schenckii reúne espécies etiologicamente relacionadas à esporotricose, uma micose que pode acometer seres humanos e animais. Foi realizada a identificação fenotípica e molecular de 85 isolados provenientes de quatro estados brasileiros (Minas Gerais, Rio de Janeiro, Rio Grande do Sul e São Paulo). Foram pesquisadas a produção de enzimas, o perfil de inibição por leveduras killer, a suscetibilidade aos antifúngicos comerciais e aos extratos de plantas. Os isolados foram identificados como S. schenckii, S. brasiliensis e S. globosa, com predomínio de S. schenckii. Houve discordância de 37,7% entre a identificação das espécies do complexo S. schenckii utilizando metodologias fenotípicas e genotípicas. Entre os antifúngicos testados, a terbinafina foi o fármaco mais ativo; seguido por cetoconazol e itraconazol, enquanto fluconazol e voriconazol foram os menos ativos. Cinco isolados fúngicos foram detectados como resistentes ao itraconazol, sendo um S. globosa e quatro S. schenckii. Não houve diferença nos perfis de suscetibilidade aos antifúngicos entre as espécies do complexo Sporothrix schenckii. Entre os extratos de origem vegetal, o mais ativo foi o proveniente de Pterocaulon polystachyum, mostrando que o uso popular das plantas reforça a importância de pesquisas etnofarmacológicas, abrindo a possibilidade de encontrar novos agentes antifúngicos clinicamente eficazes. Doze das 18 leveduras killer avaliadas apresentaram atividade frente a todos os isolados do complexo S. schenckii estudados. No entanto, não houve diferença na suscetibilidade as toxinas entre as espécies de Sporothrix. Todos os isolados produziram desoxiribonuclease, urease e proteinase. Atividade fosfolipase e esterase foi detectada em 83 (97,6%) e 80 (94,1%), respectivamente, dos isolados testados. Todas as amostras do complexo S. schenckii produziram, pelo menos, quatro das enzimas avaliadas, e 78 (91,8%) dos isolados produziram todas as enzimas analisadas no estudo. No entanto, não foi possível diferenciar as espécies de Sporothrix baseado no perfil enzimático. Entre as enzimas extracelulares avaliadas nos isolados do complexo S. schenckii, desoxiribonuclease e esterase foram produzidas em maior quantidade, podendo vir a ser um fator de virulência. Além disso, o caldo Sabouraud dextrose mostrou potencial para ser usado na avaliação in vitro da atividade antifúngica frente ao complexo S. schenckii. / The Sporothrix schenckii complex combines species etiologically related to sporotrichosis, a mycosis which can affect humans and animals. The phenotypic and genotypic identification of 85 strains from four Brazilian States (Minas Gerais, Rio de Janeiro, Rio Grande do Sul and São Paulo) was performed. The enzymatic production, profile of inhibition by killer yeasts, susceptibility to marketed antifungal and to plant extracts were surveyed. The isolates were identified as S. schenckii, S. brasiliensis and S. globosa, with the predominance of S. schenckii. There was 37.7% disagreement regarding the species classification using phenotypic and genotypic methodologies. Among the tested antifungals, terbinafine was the most active drug, followed by ketoconazole and itraconazole, while fluconazole and voriconazole were the least active ones. Five isolates - one S. globosa and four S. schenckii - were resistent to itraconazole. There was no difference as to the profiles of the susceptibility to the antifungal agents among the Sporothrix species. The most active vegetal extract was from Pterocaulon polystachyum, showing that the popular use of these plants reinforces the importance of ethnopharmacological researches, with the possibility of finding new clinically effective antifungal agents. Twelve out of the 18 evaluated killer yeasts showed activity against all the tested strains of the S. schenckii complex. However, there was no difference in susceptibility to the toxins among the Sporothrix species complex. All the isolates were desoxiribonuclease, urease and proteinase positive. Phospholipase and esterase activities were detected in 83 (97.6%) and 80 (94.1%), respectively, among the isolates evaluated. All the S. schenckii complex strains produced at least four of the evaluated enzymes, and 78 (91.8%) of the isolates produced all the enzymes analyzed in the study. However, it is not possible to differentiate the Sporothrix species based on their enzymatic profile. Among the extracellular enzymes evaluated in the S. schenckii complex isolates, desoxiribonuclease and esterase were the most prominent ones, and their production may be a virulence factor. Furthermore, the Sabouraud dextrose broth showed potential to be used in the in vitro evaluation of the antifungal activity against the S. schenckii complex.

O complexo Sporothrix schenckii reúne espécies etiologicamente relacionadas à esporotricose, uma micose que pode acometer seres humanos e animais. Foi realizada a identificação fenotípica e molecular de 85 isolados provenientes de quatro estados brasileiros (Minas Gerais, Rio de Janeiro, Rio Grande do Sul e São Paulo). Foram pesquisadas a produção de enzimas, o perfil de inibição por leveduras killer, a suscetibilidade aos antifúngicos comerciais e aos extratos de plantas. Os isolados foram identificados como S. schenckii, S. brasiliensis e S. globosa, com predomínio de S. schenckii. Houve discordância de 37,7% entre a identificação das espécies do complexo S. schenckii utilizando metodologias fenotípicas e genotípicas. Entre os antifúngicos testados, a terbinafina foi o fármaco mais ativo; seguido por cetoconazol e itraconazol, enquanto fluconazol e voriconazol foram os menos ativos. Cinco isolados fúngicos foram detectados como resistentes ao itraconazol, sendo um S. globosa e quatro S. schenckii. Não houve diferença nos perfis de suscetibilidade aos antifúngicos entre as espécies do complexo Sporothrix schenckii. Entre os extratos de origem vegetal, o mais ativo foi o proveniente de Pterocaulon polystachyum, mostrando que o uso popular das plantas reforça a importância de pesquisas etnofarmacológicas, abrindo a possibilidade de encontrar novos agentes antifúngicos clinicamente eficazes. Doze das 18 leveduras killer avaliadas apresentaram atividade frente a todos os isolados do complexo S. schenckii estudados. No entanto, não houve diferença na suscetibilidade as toxinas entre as espécies de Sporothrix. Todos os isolados produziram desoxiribonuclease, urease e proteinase. Atividade fosfolipase e esterase foi detectada em 83 (97,6%) e 80 (94,1%), respectivamente, dos isolados testados. Todas as amostras do complexo S. schenckii produziram, pelo menos, quatro das enzimas avaliadas, e 78 (91,8%) dos isolados produziram todas as enzimas analisadas no estudo. No entanto, não foi possível diferenciar as espécies de Sporothrix baseado no perfil enzimático. Entre as enzimas extracelulares avaliadas nos isolados do complexo S. schenckii, desoxiribonuclease e esterase foram produzidas em maior quantidade, podendo vir a ser um fator de virulência. Além disso, o caldo Sabouraud dextrose mostrou potencial para ser usado na avaliação in vitro da atividade antifúngica frente ao complexo S. schenckii. / The Sporothrix schenckii complex combines species etiologically related to sporotrichosis, a mycosis which can affect humans and animals. The phenotypic and genotypic identification of 85 strains from four Brazilian States (Minas Gerais, Rio de Janeiro, Rio Grande do Sul and São Paulo) was performed. The enzymatic production, profile of inhibition by killer yeasts, susceptibility to marketed antifungal and to plant extracts were surveyed. The isolates were identified as S. schenckii, S. brasiliensis and S. globosa, with the predominance of S. schenckii. There was 37.7% disagreement regarding the species classification using phenotypic and genotypic methodologies. Among the tested antifungals, terbinafine was the most active drug, followed by ketoconazole and itraconazole, while fluconazole and voriconazole were the least active ones. Five isolates - one S. globosa and four S. schenckii - were resistent to itraconazole. There was no difference as to the profiles of the susceptibility to the antifungal agents among the Sporothrix species. The most active vegetal extract was from Pterocaulon polystachyum, showing that the popular use of these plants reinforces the importance of ethnopharmacological researches, with the possibility of finding new clinically effective antifungal agents. Twelve out of the 18 evaluated killer yeasts showed activity against all the tested strains of the S. schenckii complex. However, there was no difference in susceptibility to the toxins among the Sporothrix species complex. All the isolates were desoxiribonuclease, urease and proteinase positive. Phospholipase and esterase activities were detected in 83 (97.6%) and 80 (94.1%), respectively, among the isolates evaluated. All the S. schenckii complex strains produced at least four of the evaluated enzymes, and 78 (91.8%) of the isolates produced all the enzymes analyzed in the study. However, it is not possible to differentiate the Sporothrix species based on their enzymatic profile. Among the extracellular enzymes evaluated in the S. schenckii complex isolates, desoxiribonuclease and esterase were the most prominent ones, and their production may be a virulence factor. Furthermore, the Sabouraud dextrose broth showed potential to be used in the in vitro evaluation of the antifungal activity against the S. schenckii complex.

Made available in DSpace on 2017-07-10T19:23:56Z (GMT). No. of bitstreams: 1 TESE_ FRANCIELI .pdf: 3210488 bytes, checksum: 139233665a2fb9191412af6ecba0f292 (MD5) Previous issue date: 2015-02-06 / The composting process has been used as the main way to stabilize agro-industrial waste from the broiler production chain. These wastes are generated in the run-fattening stage and during the slaughtering and processing of meat, originally inadequate to composting process due to its high levels of nitrogen. The objective of this research was to evaluate different lignocellulosic materials available regionally as a carbon source to be added to the composting process. The lignocellulosic materials were carding cotton waste, pruning of urban trees, sawdust, crushed sugarcane bagasse and crushed napier grass, which mixed with other wastes (reproductive poultry bedding, hatchery waste, flotation sludge, sausage skins and coal from boilers), constituted the treatments. Five windrows were set up and monitored, with C:N ratio of around 30. The windrows were turned twice a week in the first month and once a week in the following months until stabilization, confirmed by the decline of the windrow temperature until it reaches values of room temperature. At every turning, moisture was adjusted to 60%. The time of composting was evaluated, as well as mass reductions and volume (parameters related to optimization of the composting area); losses of N, P and K; concentration of N, P and K and the ratio of humic to fulvic acid - HA / FA that to characterize the agronomic value of the final compost, in addition to monitoring of microbiological parameters such as basal respiration and activity enzymatic β-glucosidase, cellulase, acid and alkaline phosphatase. With the aid of techniques of Multivariate Analysis (Cluster Analysis and Principal Component), it was concluded that the treatment which used the carding cotton waste as a carbon source allowed optimizing the use of composting area and provided the production of a organic compost with greater agronomic value. Regarding the microbiological parameters, these were most intense in the thermophilic phase, being the cellulase activity most accentuated. / O processo de compostagem é utilizado como principal forma de estabilizar resíduos agroindustriais provenientes da cadeia produtiva do frango de corte. Trata-se dos resíduos gerados no período que antecede a fase de engorda e durante o abate e industrialização da carne, originalmente inadequados ao processo de compostagem por apresentarem altos teores de nitrogênio. Objetivou-se avaliar diferentes materiais lignocelulósicos disponíveis regionalmente como fonte de carbono a ser adicionado na compostagem destes resíduos. Os materiais lignocelulósicos avaliados foram: resíduos da desfibrilação do algodão, podas de árvores urbanas, serragem, bagaço de cana moído e capim napier triturado, que em mistura com os demais resíduos (cama de matrizeiro, resíduos de incubatório, lodo de flotador, tripa celulósica e carvão), constituíram os tratamentos. Foram montadas e monitoradas cinco leiras, com relação C:N em torno de 30. As leiras foram revolvidas duas vezes por semana no primeiro mês e uma vez por semana nos meses seguintes até a estabilização, confirmada pelo declínio da temperatura da leira até atingir os valores da temperatura ambiente. A cada revolvimento, a umidade foi corrigida para 60%. Os parâmetros avaliados foram o tempo de compostagem e as reduções de massa e volume (parâmetros relacionados à otimização do pátio de compostagem); perdas de N, P e K, concentração de N, P e K e relação ácidos húmicos:ácidos/fúlvicos AH/AF que permitiram caracterizar o valor agronômico do composto final. Monitorou-se os parâmetros microbiológicos, como a respiração basal e a atividade enzimática de β-glucosidase, celulase, fosfatase ácida e alcalina. Com auxílio de técnicas da Análise Multivariada (Análise de Agrupamento e de Componentes Principais), concluiu-se que o tratamento em que se utilizou o resíduo da desfibrilação de algodão como fonte de carbono permite otimizar a utilização do pátio de compostagem e proporciona a produção de um composto orgânico com maior valor agronômico. Com relação aos parâmetros microbiológicos, estes foram mais intensos na fase termofílica, sendo a atividade de celulase a mais pronunciada

Ce travail fait l’objet d’une avancée scientifique dans le développement de la technique d’IRM rehaussée par l’effet Overhauser dans la souris à 0,2 T. Cette dernière repose sur le transfert de polarisation des spins électroniques saturés d’un radical libre vers les spins des protons (généralement de l’eau) voisins pour rehausser le signal RMN du proton. Notre équipe a développé cette technique pour détecter une activité protéolytique au travers de deux stratégies. La première partie de la thèse a été de détecter pour la première fois une activité protéolytique in situ dans des souris saines et in vitro sur cellules vivantes. L’efficacité du rehaussement par effet Overhauser repose sur le temps de corrélation des spins des électrons non-appariés. Un radical nitroxyde greffé à l’élastine a été utilisé comme substrat. La protéolyse de ce dernier par des élastases pancréatiques a conduit l’observation en 3D d’un rehaussement du signal RMN de plus de 10 fois dans le tube digestif de souris vivantes. De plus, des développements méthodologiques, tels que l’implémentation de la séquence TrueFISP, le sous-échantillonnage par la méthode “Keyhole”, et la reconstruction des données en 3D ont été faits. La deuxième stratégie repose sur des molécules de nitroxyde ayant l’unique propriété de pouvoir décaler leurs pics de résonance après hydrolyse. Un nitroxyde phosphorylé en position Béta pouvant être détecté à deux fréquences spécifiques différentes avant et après hydrolyse d’un groupement chimique a été synthétisé par des chimistes à Marseille. L’hydrolyse de cette macromolécule a été observée in vivo dans l’estomac de souris saines avec des rehaussements de plus de 400% et imagée en 3D avec une bonne résolution spatio-temporelle. Ainsi, une prochaine étape serait de poursuivre ce travail sur un modèle pathologique et développer cette technique à un champ magnétique plus bas. / This work relates the continuity and advances in the implementation of the Overhauser-enhanced Magnetic Resonance Imaging technique on a 0.2 T scanner. Briefly, OMRI technique is based on polarization transfer of saturated electronic spins from free nitroxide radicals to proton spins of surrounding water molecules in the aim to drastically enhance proton NMR signal. To this technique, our research team has merged specific strategies for proteolytic activity detection. The first strategy relies on a 3D visualization of proteolytic activity happening in intact living cells or in vivo in healthy mice. With an Overhauser switch based upon changes in molecular tumbling time, high Overhauser enhancements of 10-fold were observed in the intestinal tract of mice after that elastolytic activity of our probe: the nitroxide-labeled elastin macromolecule took place. In addition, MRI developments - TrueFISP sequence implementation, undersampling Keyhole method and data reconstruction were carried out for imaging these rapid biological processes. A second exquisite strategy is also described using nitroxides with shifting resonant peaks. Here, a Beta-phosphorylated nitroxide molecule was specifically detected at two distinct frequencies: one for its substrate and the other for its product once hydrolysis took place. This hydrolysis was imaged in 3D in the stomach of living mice with Overhauser enhancements of more than 400% and with a good spatiotemporal resolution. The perspectives of this work lie on a future detection of a pathological proteolytic activity in vivo and eventually and development of very low magnetic field OMRI.

In biosensors, printing involves the transfer of materials, proteins or cells to a substrate. It offers many capabilities thatcan be utilized in many applications, including rapid deposition and patterning of proteins or other biomolecules.However, issues such as stability when using biomaterials are very common. Using proteins, enzymes, as biomaterialink require immobilizations and modifications due to changing in the structural conformation of the enzymes, whichleads to changes in the properties of the enzyme such as enzymatic activity, during the printing procedures andrequirements such as solvent solutions. In this project, an innovative approach for the fabrication of proteinmicroparticles based on cross-linking interchange reaction is presented to increase the stability in different solvents.The idea is to decrease the contact area between the enzymes and the surrounding environment and also preventconformation changes by using protein microparticles as an immobilization technique for the enzymes. The theory isbased on using a cross-linking reagent trigging the formation of intermolecular bonds between adjacent proteinmolecules leading to assembly of protein molecules within a CaCO3 template into a microparticle structure. TheCaCO3 template is removed by changing the solution pH to 5.0, leaving behind pure highly homogenous proteinmicroparticles with a size of 2.4 ± 0.2 μm, according to SEM images, regardless of the incubation solvents. Theenzyme model used is Horse Radish Peroxidase (HRP) with Bovine Serum Albumin (BSA) and Glutaraldehyde (GL)as a cross-linking reagent. Furthermore, a comparison between the enzymatic activity of the free HRP and the BSAHRPprotein microparticles in buffer and different solvents are obtained using Michaelis-Menten Kinetics bymeasuring the absorption of the blue product produced by the enzyme-substrate interaction using a multichannelspectrophotometer with a wavelength of 355 nm. 3,3’,5,5’-tetramethylbenzidine (TMB) was used as substrate. As aresult, the free HRP show an enzymatic activity variation up to ± 50 % after the incubation in the different solventswhile the protein microparticles show much less variation which indicate a stability improvement. Moreover, printingthe microparticles require high microparticle concentration due to contact area decreasing. However, usingmicroparticles as a bioink material prevent leakage/diffusion problem that occurs when using free protein instead.