Couches minces multiferroiques d’YMnO3 (YMO) films ont été synthétisée par MOCVD sur desubstrats de Si, STO, LAO et LSAT orientées (100). L'effet de l'épaisseur des couches et de lacomposition chimique sur les propriétés structurales et magnétiques a été étudié. YMO peutcristalliser dans deux structure : hexagonale (h-YMO) et orthorhombique (o-YMO), généralementconsidérée comme les phases stables et métastables, respectivement. Les deux phases, ainsi queleur phase précurseur amorphe sont étudiées dans cette thèse. D'un côté, une croissance sélectivede la phase amorphe, h-YMO ou o-YMO est réalisé sur des substrats de Si en ajustant lesconditions de dépôt. Une étude approfondie des conditions optimales a été réalisée. Unetransformation de phase irréversible de l'état amorphe à la phase cristalline o-YMO a lieu à unetempérature à peu près constante (~ 700 ° C) et dans un court période de temps (min ~). La phaseo-YMO ainsi obtenue est stable au moins jusqu'à 900 ° C.De l'autre côté, la phase o-YMO est stabilisé par épitaxie sur des substrats de type perovskite (STO,LAO, LSAT). Les films sur STO et LSAT présentent principalement l’orientation (010) tandis queceux sur les substrats de LAO sont orientées (101). Une orientation secondaire de domaines estobservée en particulier sur des substrats de STO: rotation de 90 ° dans le plan du domaine (010).A des faibles épaisseurs les couches sont contraintes. Les mesures magnétiques montrent uncomportement de verre de spin pour chacune de phase o- ou h-YMO, indépendamment du substrat. / Multiferroic YMnO3 (YMO) films have been grown by MOCVD on (100)-oriented Si, STO, LAOand LSAT substrates. The effect of the film thickness and the chemical composition on structuraland magnetic properties has been investigated. YMO can crystallize in two structure: hexagonal(h-YMO) and orthorhombic (o-YMO), generally considered as stable and metastable phases,respectively. Both phases, together with their amorphous precursor phase, are studied in this thesis.On one side, a selective growth of the amorphous, o-YMO or h-YMO phase is achieved on Sisubstrates through the deposition conditions. An extensive study of the optimal conditions hasbeen carried out. An irreversible phase transformation from amorphous to crystalline o-YMOphase takes place at an almost constant temperature (~ 700 °C) and in a short period of time (~min). The o-YMO phase thus obtained is stable at least up to 900 °C.On the other side, the o-YMO phase is epitaxially stabilized on perovskite type substrates (STO,LAO, LSAT). The films on STO and LSAT substrates present mainly the (010) orientation whilethose on LAO substrate are (101)-oriented. Secondary domain orientation are observe in particularon STO substrates: (010) in plane with 90° rotation. Strained films are observed for smallthicknesses. The magnetic measurements show a spin glass behavior for either o- or h-YMO phase,independently of the substrate.

Depuis les années 2000, l'impression fonctionnelle connait un grand succès pour la fabrication de composants électroniques. Elle se positionne de manière complémentaire par rapport aux technologies du silicium et vise principalement les marchés de l'électronique grande surface (écrans, panneaux photovoltaïques) et de l'électronique flexible (RFID, capteurs, textiles intelligents). Dans ce travail, des couches d'argent imprimées par jet d'encre ont été caractérisées en fonction des conditions d'impression et de recuit. L'évolution de leur microstructure, de leurs propriétés électriques et mécaniques a été étudiée. Dans ce cadre, des méthodes expérimentales ont été développées et validées par la bonne corrélation entre les mesures et les modèles. La maîtrise des propriétés de ces couches et l'optimisation de leur procédé de fabrication nous ont conduits à concevoir, fabriquer et caractériser plusieurs composants flexibles: un filtre passe bande centré à 17 GHz sur un substrat polyimide, un micro-capteur de vide fonctionnant sur le principe de Pirani et un bouton-poussoir d'épaisseur inférieure à 250 µm pour des claviers souples. Enfin, des condensateurs RF ont été réalisés par la superposition de couches imprimées diélectriques (BaSrTi) et conductrices (Ag). Les performances des prototypes réalisés sont proches de l'état de l'art et ouvrent la voie à de nouvelles applications pour les technologies d'impression. Cette étude démontre le potentiel des couches d'argent imprimées par jet d'encre pour la fabrication de dispositifs flexibles. / In the last decade, functional printing has gained a large interest for the manufacturing of electronic components. It stands aside to silicon technologies and specifically targets markets of large area devices (screens, photovoltaics) and flexible electronics (RFID antennas, sensors, smart textiles). In this work, inkjet printed silver layers are characterized depending on the printing conditions and the required post-printing annealing. The evolution of their microstructure, electrical and mechanical properties is investigated as a function of the annealing temperature. The correlation of the measurements with theoretical models supports the experimental methods that were developed. The knowledge of the printed silver layers assets and the optimization of the printing process lead to the design, fabrication and characterization of flexible electronics devices: a 17 GHz band-pass filter printed on polyimide, a flexible vacuum micro-sensor working on the Pirani principle, and a 250 µm thick membrane switch for keyboards. Finally, all printed RF capacitors were realized by stacking Barium Strontium Titanate (dielectric) and silver printed layers. These prototypes exhibit performances near the state-of-the-art and suggest new opportunities for printing technologies. This thesis offers a thorough study of inkjet printed silver layers and assess their potential for the manufacturing of flexible devices.

[pt] Nesta tese de doutoramento apresentamos a fabricação e a caracterização de dispositivos fotovoltaicos orgânicos (OPVs) fabricados a partir de eletrodos de grafeno e de substratos híbridos flexíveis à base de polímeros recobertos com um filme fino condutor. Para isso, inicialmente sintetizamos filmes de grafeno através da técnica de deposição química em fase de vapor (CVD), seguido de modificações no processo de transferência do grafeno para o substrato desejado. Nesta etapa, desenvolvemos uma nova metodologia utilizando uma blenda condutora de EPDM-PAni que simplifica o processo de transferência e melhora as propriedades elétricas do grafeno. Em outro momento, otimizamos diferentes substratos híbridos à base de polímeros de PVC, PVA e celulose bacteriana (BC) recobertos com um filme fino condutor de ITO. Tanto os substratos híbridos flexíveis, quanto os filmes de grafeno, foram investigados por transmitância ótica e resistência de folha a fim de avaliar os seus potenciais uso para as aplicações em OPVs. Por fim, fabricamos diversas estruturas de OPVs, tanto com o grafeno como eletrodo condutor, quanto usando os substratos híbridos flexíveis. Estes dispositivos foram caracterizados principalmente através das suas curvas características JxV, no escuro e sob iluminação. Além disso, realizamos ciclos de flexão/extensão de alguns dispositivos a fim de avaliar seu comportamento frente aos esforços mecânicos a estes submetidos. Os resultados obtidos mostraram que os filmes de grafeno fabricados são promissores para a aplicação como eletrodo condutor transparente em OPVs e que os substratos híbridos investigados podem ser utilizados em dispositivos flexíveis, visto que apresentaram comportamento semelhante aos substratos inorgânicos comumente utilizados. / [en] In this doctoral thesis we present the fabrication and characterization of organic photovoltaic devices (OPVs) assembled onto graphene electrodes and flexible hybrid polymers-based substrates coated with a conductive thin film. For this, initially the graphene films were synthesized by chemical vapor deposition (CVD) technique, followed by modifications in the transfer process of the graphene to the desired substrate. In this step, we developed a new methodology using an EPDM-PAni conductive blend that simplifies the transfer process and improves the electric properties of graphene. We also used another approach which consists in optimizing different hybrid substrates based on PVC, PVA and bacterial cellulose (BC) polymers coated with an ITO conductive thin film. The flexible hybrid substrates as well as the graphene films were investigated by optical transmittance and sheet resistance in order to evaluate their potential use for OPVs applications. Finally, we fabricate various structures of OPVs, using graphene as a conducting electrode, well as using flexible hybrid substrates. Such devices were characterized mainly through their dark and light J×V characteristic curves. In addition, we performed flexion/extension cycles in some devices in order to evaluate their behavior against the mechanical stresses submitted to them. The results showed that the graphene films are a promising material for the application as a transparent conductive electrode in OPVs and the hybrid substrates investigated can be used in flexible devices, since they presented similar behavior to the commonly used inorganic substrates.

En ville, les systèmes de récupération et d'infiltration des eaux pluviales dans le sous-sol ont pour conséquence d'augmenter la connectivité hydrologique entre la surface et la nappe phréatique. Ces pratiques d'infiltration produisent de nombreuses perturbations physico-chimiques au niveau de la nappe (ex. augmentation des variations thermiques, baisse des concentrations en oxygène dissous, enrichissement de la nappe en matière organique dissoute) mais les conséquences sur le compartiment microbien restent peu connues. L'objectif principal de la thèse est de déterminer les effets de l'infiltration des eaux pluviales sur les communautés microbiennes des nappes phréatiques, aussi bien en termes d'abondance, d'activités que de diversité génétique bactérienne. En se basant sur les changements environnementaux associés à l'infiltration des eaux pluviales et l'analyse des communautés bactériennes, un objectif fondamental est d'évaluer l'importance des phénomènes de dispersion (ex. transferts) et de sélection par des facteurs abiotiques (ex. disponibilité des ressources nutritives) sur les assemblages bactériens au sein des nappes phréatiques. Ces travaux ont été axés sur des expérimentations de terrain utilisant deux approches d'échantillonnage : une méthode active (prélèvements d'eau) et une méthode passive (incubation de substrats artificiels). La description des communautés a été effectuée par une méthode de séquençage de nouvelle génération (i.e. Illumina MiSeq) en se basant sur le gène rrs. Les résultats de ce travail mettent en avant une influence significative des pratiques d'infiltration sur les bactériomes d'un aquifère. En effet, le développement, les activités et la diversité des micro-organismes retrouvés dans la nappe ont été stimulés significativement par l'enrichissement en carbone organique dissous biodégradable engendré par ces pratiques. Néanmoins, cet impact est fortement réduit dans les systèmes étudiés où la zone non saturée est épaisse (> 10 m) et agit comme un filtre physique, chimique et biologique efficace entre le bassin d'infiltration et l'aquifère. Les faibles similarités entre les structures génétiques des bactériomes des eaux d'infiltration et dans la nappe indiquent que la zone non saturée joue un rôle efficace sur la rétention des bactéries dans les systèmes étudiés. En conclusion, cette thèse constitue la première étude d'envergure visant à quantifier la réponse du compartiment microbien des aquifères à des perturbations engendrées par l'infiltration des eaux pluviales en milieu urbain. Elle ouvre aussi de nouvelles perspectives sur les méthodes et outils d'évaluation de la qualité des nappes phréatiques / In urban area, managed aquifer recharge (MAR) systems raises hydrological connectivity between surface and groundwater. These infiltration practices are the cause of many disturbances in groundwaters (e.g. increase of thermal variations, decrease of dissolved oxygen or enrichment in organic matter) but associated consequences on microbial compartment remains unclear. The main aim of the thesis is to determine the effects of stormwater runoff infiltration on microbial communities of groundwater, in terms of abundance, activities and bacterial diversity. Based on environmental changes associated to MAR practices and bacterial community analyses, a fundamental question is to assess the importance of dispersal (e.g. transfers) and selection by abiotic factors (e.g. nutrients availability) on groundwater communities assemblage. This study is based on field experiments with two complementary strategies of sampling: an active one (i.e. groundwater sampling) and a passive one (incubation of artificial substrate). Communities’ description was made by next-generation sequencing (i.e. Illumina MiSeq) of rrs gene. The results showed a significant influence of MAR practices on microbial communities. Growth, activities and diversity of groundwater micro-organisms were mainly stimulated by biodegradable dissolved organic carbon enrichment associated to MAR practices. Nonetheless, this impact was reduced in systems where the vadose zone is thick (> 10 m) and acts as a physical, chemical and biological filter between the infiltration basin and the aquifer. Low similarities between bacterial communities of infiltration waters and bacterial communities of groundwaters reveal that vadose zone is effective on the retention of bacteria in studied systems. To conclude, this thesis constitutes the first major study that aimed to quantify microbial compartment response to disturbances caused by MAR practices in urban area. It also opens new perspectives on assessment tool for groundwater quality

La bronchopneumopathie chronique obstructive ou BPCO est une atteinte des voies respiratoires causée par le tabagisme. Cette maladie pulmonaire chronique et non réversible pour laquelle il n’y pas de traitement curatif se caractérise par une inflammation permanente du tractus respiratoire. Celle-ci est due à l’afflux massif de cellules de l’inflammation, principalement des neutrophiles et macrophages, qui libèrent après activation, de nombreuses protéases actives. Ces protéases vont alors dégrader les protéines de structure comme l’élastine, ce qui va entraîner la dégradation progressive des alvéoles pulmonaires et au final une altération de plus en plus marquée de la fonction respiratoire. Parmi les différentes protéases présentes dans le poumon, la MMP-12 de macrophage, joue un rôle clé dans la physiopathologie de la maladie. / Chronic obstructive pulmonary disease or COPD is a lung disease caused by tobacco smoking. This is a chronic and non reversible disease for which no curative treatment is available yet. Permanent inflammation of the airways is a hallmark of COPD because immune cells such as neutrophils and macrophages are continuously recruited. Once activated, these cells release numerous active proteases which participate to the degradation of structural proteins of the lungs such as elastin, leading to lung emphysema as a consequence of lung alveoli degradation. Among the different proteases found in the lungs, macrophage MMP-12 has been reported to play a key pathogenic role in COPD development.

Deposition of dispersed phase from flowing dispersions onto a substrate surface is of utmost relevance for various industrial processes like fouling of sparingly soluble salts, such as CaCO3 and CaSO4 in heat exchangers and desalination evaporators which tend to form deposits on flow surfaces of tubes or pipelines conveying hard water and in water filtration and purification processes since concentration of CaCO3 in many natural water resources is equal to or greater than the saturation level. The study of deposition is also of intrinsic interest for biophysics and colloid science where vascular calcification i.e. precipitation and deposition of calcium phosphates (hydroxyapatites) in the muscular layer of the blood reduces arterial compliance and promotes congestive heart failure. Experiments were conducted on straight, circular cross section silica tubing of inner di-ameter (ranging from 0.88 mm to3.40 mm) to study the effect of electrostatic interaction and hydrodynamics on the deposition behavior of CaCO3 on silica surface when streams of aqueous solutions of Ca(NO3)2 and Na2CO3 with a concentration of 40.0 g l−1 and 25.9gl−1 respectively flowing at a volumetric flow rate of 1 l h−1 each is mixed to form CaCO3 dispersion which was then transported through silica tubing at a steady volumetric flow rate of 2lh−1. The in situ phenomenology of CaCO3 particles transport, deposition, and evolution of spatial and temporal patterns of the CaCO3 deposition on the silica surface along with the dendritic growth of CaCO3 during the flow was visually documented with the aid of a 100X optical microscope. The study discussed the deposition behavior of dispersed phase of CaCO3 from its aqueous dispersion on the silica tubing during flow and attributed charge inversion from negative to positive of silica surface, due to the adsorption of Na+ formed during precipitation reaction of CaCO3, as a plausible reason for the reversal of electrostatic interaction from attraction between initially negatively charged silica surface and positively charged CaCO3 particles which promoted deposition and subsequent evolution of patterns of CaCO3 deposition on the silica surface during the early stage of experiments to repulsion between finally net positively charged silica surface and positively charged CaCO3 particles which resulted in retarded deposition and subsequent emergence of sparsely adhered CaCO3 agglomerates as localized, limited patches of CaCO3 deposits on the silica surface during the later stage of the experiments

The effect of substrate surface treatment (substrate sputter cleaning) in a cathodic arc plasma prior to unbalanced magnetron deposition of transition metal nitride coatings on the performance of the coated components has been investigated. In particular the influence of parameters such as ion species, ion energy and exposure time on the changes in substrate surface topography, microstructure and micro-chemistry were studied employing transmission electron microscopy, energy dispersive X-ray analysis, electron energy loss spectroscopy, X-ray diffraction, atomic force microscopy and optical microscopy. The consequences for both the microstructure of subsequently grown transition metal nitride coatings and their adhesion were elucidated. The relevance for practical applications was demonstrated using the example of dry high-speed milling tests, which showed that an appropriate choice of substrate surface pre-treatment parameters can double the life time of the coated tools. This was found to be due to an improved adhesion as a result of a combina-tion of reduced oxygen incorporation at the interface between coating and substrate and local epitaxial growth of the coating. The latter is promoted by certain sub-strate surface pre-treatment procedures, which provide clean surfaces with preserved crystallographic order.

Orientadora: Profa. Dra. Lúcia Helena Gomes Coelho / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Ciência e Tecnologia Ambiental, 2016. / Considerando a situação atual dos recursos hídricos, cuja qualidade vem sendo comprometida pela poluição causada, principalmente, pelo descarte inadequado de efluentes não tratados ou ineficientemente tratados, evidencia-se a necessidade de se criar técnicas e processos que permitam tratar o efluente de modo eficiente, de forma mais econômica e sustentável, cujos resíduos finais sejam de mais fácil disposição ou reutilização, visando a minimização dos impactos ambientais do despejo de efluentes e dos processos de tratamento de águas em si. O presente trabalho avaliou o potencial da utilização de conchas de moluscos (Venerupis pulastra) como substrato natural na remoção de fósforo inorgânico dissolvido (ortofosfato) em meio aquoso com vistas ao desenvolvimento de tecnologia de tratamento terciário de efluentes de baixo custo e baixo impacto ambiental. Foram realizados ensaios em batelada que consistiram da adição de 0,5 g a 2,0 g do substrato natural (previamente preparado) em 15 mL de solução de concentração conhecida de fósforo (0,5 mg L-1) sob agitação em mesa agitadora por períodos de tempo estabelecidos. Foi avaliada a influência dos parâmetros: tempo de detenção hidráulica substrato/solução, pH da solução, quantidade de substrato, granulometria do substrato e velocidade de rotação da mesa agitadora orbital na eficiência de remoção de ortofosfato em amostras sintéticas de concentração conhecida. Todos os parâmetros foram variados no sistema utilizando abordagem univariada e multivariada. Para os testes com abordagem univariada, os resultados de eficiência de remoção de fósforo variaram de 14% a 68%, evidenciando o potencial de adsorção do substrato e a importância dos parâmetros estudados no desempenho do processo. Com a análise destes dados, foi possível fazer um melhor delineamento dos parâmetros e níveis dos parâmetros a serem utilizados nas análises com abordagem multivariada. Os resultados de eficiência de remoção para as análises com abordagem multivariada alcançaram valores entre 50% e 98%, apontando que as condições utilizadas nesta etapa do experimento (parâmetros e níveis) mostraram-se muito mais favoráveis ao processo de adsorção de fósforo pelo substrato. Os testes realizados com materiais de diferentes conchas (lotes e espécies diferentes) apontaram resultados semelhantes nas três amostras estudadas, mantendo eficiência acima de 96%. Por fim, foram realizados ensaios aplicando a técnica em duas diferentes amostras de efluentes provenientes de tratamento secundário (que possui como objetivo a remoção de matéria orgânica do efluente), sendo eles: biorreator de membranas submersas (BRM), com concentração inicial de fósforo (Ci) de 3,9 mg L-1) e lagoas de estabilização (Ci = 0,5 mg L-1). Obteve-se 91% de eficiência de remoção de fósforo para a amostra do BRM e 86% de eficiência de remoção para a amostra de lagoas de estabilização. Verificou-se eficiência elevada para remoção de fósforo nestas amostras, entretanto, com resultados abaixo dos encontrados em amostras sintéticas na mesma condição, apontando possíveis interferências das substâncias existentes na matriz do efluente no processo de adsorção. / Considering the current situation of water resources, whose quality has been compromised by pollution caused mainly by improper disposal of untreated effluents or ineffectively treated, highlights the need to create techniques and processes able to treat effluent, in an efficiently, economically and sustainably way, and its final waste able to be easier disposal or reuse, in order to minimize the environmental impact of the discharge of effluents and water treatment process itself. This present research proposes to evaluate the potential of shellfish shell (Venerupis pulastra) as a natural substrate in the removal of inorganic phosphorus (orthophosphate) from water, aiming the development of a low-cost and environmentally friendly tertiary wastewater treatment technology. The first step was the acquisition and preparation of the shellfish shell substrate. After that, laboratory tests were conducted batchwise, using 15ml of aqueous solutions containing 0.5mg/L of phosphorus with addition of the natural substrate (varying mass from 0.5g to 2g) under shaken for a set time. It was evaluated the influence of the parameters: contact time with the substrate and the solution, pH of the solution, amount of substrate mass, the substrate particle size and the rotational speed of the orbital shaker in the efficiency of orthophosphate removal from the synthetic sample of known concentration. All parameters were varied in the system using univariate and multivariate (design of experiments - DOE) approach. For the univariate approach tests, the phosphorus removal efficiency results ranged from 14% to 68%, showing the adsorption of the substrate potential and the importance of the parameters studied on the process performance. The results provided by univariate approach were used to organize a more specific delineation of the parameters and levels to be used in the analysis with multivariate approach. The removal efficiency results for analysis varying parameters with multivariate approach reached values between 50% and 98%, showing that the conditions used in the design of experiment (parameters and levels) proved to be more favorable to the phosphorus adsorption process. The tests with different materials (different lots and species of shellfish) showed similar results in the three samples studied, maintaining the removal efficiency of orthophosphate above 96%. Finally it was held experiments with two samples of effluents from different secondary treatment systems. The efficiency results for removal were 91% for the effluent from the bioreactor of submerged membranes (BRM) and 86% for effluent from secondary treatment from stabilization lagoons. There was high efficiency for removing phosphorus in these samples, however, with results below those found in synthetic samples in the same condition, pointing out possible interference of substances in the effluent matrix in the adsorption process.

Submitted by RONIVALDO RODRIGUES DA SILVA (rds.roni@yahoo.com.br) on 2016-03-01T13:46:53Z No. of bitstreams: 1 Tese Doutorado RONIVALDO R. SILVA.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-03-01T18:27:48Z (GMT) No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) / Made available in DSpace on 2016-03-01T18:27:48Z (GMT). No. of bitstreams: 1 silva_rr_dr_sjrp.pdf: 3318357 bytes, checksum: 82fadd527a2ede34e2a0a237a881e8f8 (MD5) Previous issue date: 2016-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Aspártico (E.C. 3.4.23), cisteíno (E.C. 3.4.22) e serino peptidases (E.C. 3.4.21) são endopeptidases, cujos modos de ação são dependentes de resíduos de ácido aspártico, cisteína e serina presentes no sítio catalítico, respectivamente. Atualmente, vários estudos são realizados na busca por novas enzimas com relevantes propriedades bioquímicas para aplicação industrial. Neste contexto, nós propomos a produção de enzimas em bioprocesso submerso, purificação, estudo das propriedades bioquímicas e determinação da especificidade catalítica das peptidases secretadas pelos fungos filamentosos Rhizomucor miehei, Phanerochaete chrysosporium e Leptosphaeria sp. Inicialmente, após produção por bioprocesso submerso, estas enzimas foram purificadas utilizando cromatografias de exclusão molecular e troca iônica. Em ensaios de inibidores na atividade enzimática, notamos inibição das peptidases por pepstatina A (R. miehei), ácido iodoacético/N-Etilmaleimida (P. chrysosporium) e fluoreto de fenil metil sulfonila (Leptosphaeria sp), sendo então definidas como aspártico, cisteíno e serino peptidases, respectivamente. Por SDS-PAGE (12%), as massas moleculares foram estimadas em 37 kDa (aspártico), 23 kDa (cisteíno) e 35 kDa (serino). O máximo de atividade proteolítica foi alcançado em pH 5,5 e 55 ºC para peptidase aspártica secretada por R. miehei; pH 7 e faixa de temperatura 45-55 ºC para cisteíno peptidase secretada por P. chrysosporium, e pH 7 e 45 ºC para serino peptidase secretada por Leptosphaeria sp. Sob efeito de incubação a diferentes pH, a peptidase aspártica mostrou-se estável em condições ácidas (pH 3-5); cisteíno peptidase foi estável em ampla faixa de pH (pH 4-9), e serino peptidase mostrou-se mais estável em condições com tendências alcalinas e pH ligeiramente ácido (pH 5-9). Em todas estas faixas de pH citadas, as peptidases apresentaram atividade proteolítica acima de 80% por 1 hora de incubação. Quanto à estabilidade térmica, a cisteíno peptidase mostrou-se mais termoestável dentre as três enzimas e serino peptidase descreveu a menor tolerância à temperatura. Em incubação com agentes desnaturantes, observamos redução na atividade proteolítica sob efeito de surfactantes iônicos (0,02-1%): dodecil sulfato de sódio (SDS) e brometo de cetil-trimetil amônio (CTAB); íon cobre II (5 mM); Ditiotreitol (DTT) e guanidina (ambos na faixa de 10-200 mM) para todas as peptidases. Por último, em estudo de especificidade catalítica destas enzimas, observamos a preferência por aminoácidos aromáticos (F e W), básicos (K e R) e apolares (em particular, resíduo de metionina) para peptidase aspártica. Alta especificidade descrita por cisteíno peptidase, cuja preferência catalítica é notória por aminoácidos básicos (K, H e R), especialmente na posição P3 e lisina-dependência para catálise na posição P'3. Em serino peptidase, notamos maior aceitação por aminoácidos apolares (G, I, L, M e V), básicos (H e R) e polares neutros (N e Q) para as diferentes posições avaliadas no substrato. / Aspartic (EC 3.4.23), cysteine (EC 3.4.22) and serine peptidases (EC 3.4.21) are endopeptidases whose modes of action are dependent on aspartic acid, cysteine and serine residues present in the catalytic site, respectively. Currently, several studies are conducted in the search for new enzymes with relevant biochemical properties for industrial application. In this context, we propose the production of enzymes in submerged bioprocess, purification, the study of biochemical properties and determining the catalytic specificity peptidases secreted by the filamentous fungus Rhizomucor miehei, Phanerochaete chrysosporium and Leptosphaeria sp. Initially, after production submerged bioprocess, these enzymes have been purified using size-exclusion and ion exchange chromatographies. In the effect of inhibitors on enzyme activity, we note peptidase inhibition by pepstatin A (R. miehei), iodoacetic acid/ N-Ethylmaleimide (P. chrysosporium) and phenyl methyl sulfonyl fluoride (Leptosphaeria sp), suggesting that these enzymes are aspartic, cysteine and serine peptidases, respectively. For SDS-PAGE (12%), molecular weights were estimated at 37 kDa (aspartic), 23 kDa (cysteine) and 35 kDa (serine). Maximum proteolytic activity was achieved at pH 5.5 and 55 °C for aspartic peptidase secreted by R. miehei; pH 7 and temperature range 45-55 °C for cysteine peptidase secreted by P. chrysosporium and pH 7 and 45 °C for serine peptidase secreted by Leptosphaeria sp. Under incubation at different pH effect, aspartic peptidase was stable under acidic conditions (pH 3-5); cysteine peptidase was stable in wide pH range (pH 4-9), and serine peptidase was more stable under alkaline conditions and pH slightly acidic (pH 5-9). In all these pH ranges mentioned, peptidases showed proteolytic activity above 80% by 1 hour incubation. As regards the thermal stability, cysteine peptidase was more thermostable enzyme and serine peptidase described the lowest temperature tolerance. In incubation with denaturing agents, we observed a decrease in proteolytic activity under the effect of ionic surfactant (0.02-1%) sodium dodecyl sulfate (SDS) bromide and cetyl-trimethyl ammonium bromide (CTAB); copper (II) ion (5 mM); Dithiothreitol (DTT) and guanidine (both in the range of 10-200 mM) for all peptidases. Finally, the study of catalytic specificity of these enzymes, we found a preference for aromatic amino acids (F and W), basic (K and R) and nonpolar (in particular, methionine residue) to aspartic peptidase. High specificity described by cysteine peptidase, which a catalytic preference is notorious for basic amino acids (K, R and H), especially in position P3 and lysine-dependence for catalysis at position P'3. In serine peptidase, for different evaluated positions, we noticed greater acceptance by nonpolar amino acids (G, I, L, M and V), basic (M and R) and neutral polar (N and Q).

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Peltophorum dubium (Sprengel) Taubert is a native forest species that shows fast development with environmental and economic importance. However, studies related to the production of quality seedlings by vegetative propagation are still incipient. As a result, this study investigated methods for multiplication and in vitro root formation of Peltophorum dubium. For multiplication was evaluated the use, alone or combined, of the cytokinins 6-Benzylaminopurine (BAP), Kinetin (KIN), isopentenyladenine (2iP) and Thidiazuron (TDZ) in different concentrations and its association with the auxin alpha-naphthalene acetic acid (ANA) or activated charcoal. In the formation of roots were evaluated the auxins NAA, indole-3-butyric acid (IBA)and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations and the exposure time of shoots to these growth regulators and their cultivation in nutrient media MS and WPM. We evaluated also alternative substrates (vermiculite, Plantmax ® or fine sand) associated with different volumes of MS nutrient medium containing agar or not, in the in vitro root formation, with the aim of providing a more porous medium to the development of root system. There wasn t emission of adventitious shoots in the absence of cytokinins in epicotyls of Peltophorum dubium. TDZ and 2iP, at 5 or 10 μM, associated with 0.015 μM NAA in MS medium promoted the highest percentage of buds. However, the shoots showed little development, intensive formation of callus at the base and leaf chlorosis, which didn t permit its use. In the absence of cytokinin was not observed leaf chlorosis. The inclusion of 1 g L-1 of activated charcoal to the nutrient medium MS provided more vigorous shoots and the use of epicotyl containing cotyledonary nodes allowed the formation of new shoots in all treatments. When BAP was used alone was not observed the emission of adventitious buds on epicotyl; these only started to occur when we used combinations of BAP with KIN or 2iP. In MS nutrient medium, the highest callus formation was observed in the presence of 10 μM IBA, after 30 days. In nutrient medium WPM, the highest callus formation occurred in the absence of IBA. Reduced percentage of root formation in Peltophorum dubium shoots were both obtained in MS medium as in WPM, regardless of the absence or presence of IBA. In MS nutrient medium, regardless of the presence or absence of auxin and independent of the concentration of this class of growth regulators, callus formation occurred at the base of the shoots of Peltophorum dubium to 30 days in vitro culture. Both the nutrient media MS and WPM possibilite the formation of roots on shoots of Peltophorum dubium with the use of "pulse" treatments of IBA at concentrations between 0 and 20 μM for six days in nutrient medium. However, the root formation was not satisfactory. There was intense callus formation at the base of the shoots at 60 days of culture, when the vermiculite was used as substrate in the process of inducing rooting. At 60 days it was possible to obtain root formation on all substrates. The combination of MS medium supplemented with 10 μM IBA, vermiculite and agar provided 36.8% of root formation in shoots, as well as improved the quality of roots of Peltophorum dubium. The use of epicotyl containing cotyledonary nodes on MS nutrient medium, supplemented with activated charcoal reduces the callus at the base of the explants and raises the emission of adventitious shoots for 75% and provides the obtaining of more vigorous shoots and leaves without chlorosis. The auxins NAA, IBA and 2,4-D in concentrations of up to 20 μM during the whole period of culture are not efficient in promoting the in vitro formation of roots on shoots of Peltophorum dubium. The use of pulse treatment with IBA for six days and the reduction the concentration of salts from the nutrient medium after this period allows the formation of roots, independent of IBA concentration and use of MS or WPM media. Most promising results are obtained using the combination of vermiculite, nutrient medium and agar, both in percentage of root formation, and the quality of root system formed. / Peltophorum dubium (Sprengel) Taubert é uma espécie florestal nativa de rápido crescimento e dotada de importância ambiental e econômica. No entanto, os estudos relacionados à produção de mudas de qualidade por meio da propagação vegetativa são, ainda, incipientes. Em virtude disso, este trabalho objetivou estudar metodologias para a multiplicação e para a formação de raízes in vitro de Peltophorum dubium. Para a multiplicação foram avaliados o emprego isolado ou combinado das citocininas 6-Benzilaminopurina (BAP), Cinetina (CIN), Isopenteniladenina (2iP) e Thidiazuron (TDZ) em diferentes concentrações, bem como sua associação com a auxina Ácido alfa-Naftaleno Acético (ANA) ou carvão ativado. Na formação de raízes foram avaliadas as auxinas ANA, Ácido 3-Indol Butírico (AIB) e Ácido 2,4-Diclorofenoxiacético (2,4-D) em diferentes concentrações, bem como o período de exposição das brotações a esses fitorreguladores e seu cultivo nos meios nutritivos MS e WPM. Foram avaliados, ainda, substratos alternativos (vermiculita, Plantmax® ou areia fina) associados a diferentes volumes de meio nutritivo MS, contendo ou não ágar, na formação in vitro de raízes, com o intuito de oferecer um meio mais poroso para o desenvolvimento do sistema radicular. Não ocorreu emissão de brotações adventícias na ausência de citocininas em epicótilos de Peltophorum dubium. TDZ e 2iP, a 5 ou 10 μM, associados a 0,015 μM de ANA, em meio MS, promoveram as maiores porcentagens de brotações adventícias. No entanto, as brotações apresentaram pequeno desenvolvimento, intensa formação de calos na base e clorose foliar, o que inviabilizou a sua utilização. Na ausência de citocininas não ocorreu clorose foliar. A inclusão de 1 g L- 1 de carvão ativado ao meio nutritivo MS proporcionou a obtenção de brotações mais vigorosas e a utilização de epicótilos contendo nós cotiledonares permitiu a formação de novas brotações em todos os tratamentos testados. Quando BAP foi utilizado isoladamente não foi observada a emissão de brotações adventícias em epicótilos; estas só passaram a ocorrer quando foram utilizadas combinações de BAP com CIN ou 2iP. Em meio nutritivo MS, a maior formação de calos foi observada na presença de 10 μM de AIB, aos 30 dias. Em meio nutritivo WPM, a maior formação de calos ocorreu na ausência de AIB. Reduzidas porcentagens de formação de raízes em brotações de Peltophorum dubium foram obtidas tanto em meio MS como em WPM, independentemente da ausência ou da presença de AIB. Em meio nutritivo MS, independente da presença ou não de auxinas e, independente da concentração utilizada, ocorreu calogênese na base das brotações de Peltophorum dubium aos 30 dias de cultivo in vitro.Tanto o meio nutritivo MS quanto o WPM possibilitaram a formação de raízes em brotações de Peltophorum dubium com a utilização de tratamentos pulse de AIB, em concentrações entre 0 e 20 μM, durante seis dias no meio nutritivo. No entanto, a formação de raízes não foi satisfatória. Ocorreu intensa formação de calos na base das brotações, aos 60 dias, quando a vermiculita foi utilizada como substrato no processo de indução ao enraizamento. Aos 60 dias foi possível obter formação de raízes em todos os substratos testados. A combinação de meio MS acrescido de 10 μM de AIB + vermiculita + ágar proporcionou 36,8% de formação de raízes nas brotações, bem como melhorou a qualidade do sistema radicular de Peltophorum dubium. A utilização de epicótilos contendo nós cotiledonares, em meio nutritivo MS, acrescido de carvão ativado, reduz a calogênese na base dos explantes e eleva a emissão de brotações adventícias para 75%, bem como proporciona a obtenção de brotações mais vigorosas e sem clorose foliar. As auxinas ANA, AIB e 2,4-D, em concentrações de até 20 μM durante todo o período de cultivo não são eficientes em promover a formação in vitro de raízes em brotações de Peltophorum dubium. O emprego de tratamento pulse com AIB por seis dias e a redução na concentração de sais do meio nutritivo após esse período permite a formação de raízes, independente da concentração de AIB e da utilização dos meios MS ou WPM. Resultados mais promissores são obtidos com a utilização da combinação de vermiculita, meio nutritivo e ágar, tanto na porcentagem de formação de raízes, como na qualidade do sistema radicular formado.