Desde o início do século XXI, vivenciamos uma explosão na produção de informações de diversos tipos, tais como fotos, áudios, vídeos, entre outros. Dentre essas informações, existem aquelas em que a informação pode ser dividida em partes menores, mas que devem ser relacionadas seguindo uma ordem total. Um exemplo deste tipo de informação é um arquivo de vídeo que foi dividido em dez segmentos identificados com números de 1 a 10. Para reproduzir o vídeo original a partir dos segmentos é necessário que seus identificadores estejam ordenados. A estrutura denominada tabela de hash distribuída (DHT) tem sido amplamente utilizada para armazenar, atualizar e recuperar esse tipo de informação de forma eficiente em diversos cenários, como monitoramento de sensores e vídeo sob demanda. Entretanto, a DHT apresenta problemas de escalabilidade quando um membro da estrutura não consegue atender as requisições recebidas, trazendo como consequência a inacessibilidade da informação. Este trabalho apresenta uma arquitetura em camadas denominada MATe, que trata o problema da escalabilidade em dois níveis: estendendo a DHT com a introdução de agentes baseados na utilidade e organizando a quantidade de requisições solicitadas. A primeira camada trata a escalabilidade ao permitir a criação de novos agentes com o objetivo de distribuir as requisições evitando que um deles tenha a escalabilidade comprometida. A segunda camada é composta por grupos de dispositivos organizados de tal forma que somente alguns deles serão escolhidos para fazer requisições. A arquitetura foi implementada para dois cenários onde os problemas de escalabilidade acontecem: (i) monitoramento de sensores; e (ii) vídeo sob demanda. Para ambos cenários, os resultados experimentais mostraram que MATe melhora a escalabilidade quando comparada com as implementações originais da DHT. / Since the beginning of the 21st century, we have experienced an explosive growth in the generation of information, such as photos, audios, videos, among others. Within this information, there are some in which the information can be divided and related following a total order. For example, a video file can be divided into ten segments identified with numbers from 1 to 10. To play the original video from these segments, their identifiers must be fully ordered. A structure called Distributed Hash Table (DHT) has been widely used to efficiently store, update, and retrieve this kind of information in several application domains, such as video on demand and sensor monitoring. However, DHT encounters scalability issues when one of its members fails to answer the requests, resulting in information loss. This work presents MATe, a layered architecture that addresses the problem of scalability on two levels: extending the DHT with the introduction of utility-based agents and organizing the volume of requests. The first layer manages the scalability by allowing the creation of new agents to distribute the requests when one of them has compromised its scalability. The second layer is composed of groups of devices, organized in such a way that only a few of them will be chosen to perform requests. The architecture was implemented in two application scenarios where scalability problems arise: (i) sensor monitoring; and (ii) video on demand. For both scenarios, the experimental results show that MATe improves scalability when compared to original DHT implementations.

A floculação é o principal estágio do tratamento da água e apesar dos significativos avanços alcançados nos últimos anos, a compreensão do mecanismo de floculação está longe de ser completa. O objetivo principal do presente trabalho foi investigar a cinética de floculação de suspensões coloidais, através da influência da dosagem de coagulante metálico e da concentração de partículas primárias nas constantes de agregação e ruptura, utilizando o modelo de Argaman e Kaufman para a cinética de floculação de um reator em batelada. A água bruta sintética foi feita com a adição de caolinita, de tal forma a obter os valores de turbidez prédeterminados de 15 UNT, 50 UNT e 100 UNT, representando águas de baixa, média e alta turbidez, respectivamente, e o coagulante utilizado foi o sulfato de alumínio. Os resultados experimentais foram obtidos em ensaios de \"jar test\" utilizando 6 dosagens de coagulantes, 6 gradientes de velocidade, 3 velocidades de sedimentação e 12 tempos de floculação para cada uma das três águas analisadas. Pode-se observar que, para água de baixa e média turbidez, com o aumento da dosagem de coagulante houve um gradativo aumento da constante de agregação, no entanto, para água de alta turbidez, para valores de dosagem de coagulante acima de 40mg.L-1, passa a ocorrer uma redução na constante de agregação, e também houve um aumento da constante de ruptura para as três águas brutas utilizadas na investigação experimental. Para todas as dosagens de coagulante empregadas, com o aumento da turbidez da água bruta, houve uma diminuição significativa da constante de ruptura ocorrida na água bruta de baixa turbidez para a água bruta de média turbidez. Embora também tenha sido observada uma variação na constante de agregação, esta foi muito pequena quando comparada numericamente com as variações na constante de ruptura. Com o aumento da turbidez da água bruta de 50 UNT para 100 UNT, a variação da constante de ruptura ocorreu apenas de forma marginal, o que significa dizer que, embora esta seja função da turbidez da água bruta, a sua relação não deve ser simplesmente linear, conforme sugerido por Argaman e Kaufman. / Flocculation is the main stage of water treatment and in spite of significant advances in recent years, understanding of the flocculation mechanism is far from complete. The main objective of current work was to investigate the kinetics of flocculation of colloidal suspensions, throughout the influence in metallic coagulant dosage and the concentration of primary particles in aggregation and rupture constants, using the Argaman and Kaufman model for kinetics of flocculation in a batch reactor. Synthetic raw water was made with the addition of kaolin, in order to obtain predetermined turbidity values of 15 NTU, 50 NTU and 100 NTU, representing waters of low, medium and high turbidity, respectively, and the coagulant applied was aluminum sulfate. Experimental results were obtained in \"jar test\" experiments using 6 coagulant dosages, 6 velocity gradients, 3 sedimentation velocity and 12 flocculation times for each water analyzed. It can be observed that, for waters of low and medium turbidity, with the coagulant dosage increase, takes place a gradual increase in aggregation constant, however, on waters of high turbidity, with coagulant dosages above 40mg.L-1, A reduction in aggregation constant occurs, and also there was seen an increase in rupture constant for the three raw waters used in the experimental investigation. For all employed coagulant dosages, with the increase in raw water turbidity, there were a significant decrease in rupture constant occurred in raw water of low turbidity to medium turbidity raw water. Although a variation in the aggregation constant was also observed, this was very small when numerically compared with the variation in rupture constant. With the increase in raw water turbidity from 50 NTU to 100 NTU, rupture constant variation occurred only in a marginal manner, meaning to say, while this is in function of raw water turbidity, its relation might not be simply linear, as suggested by Argaman and Kaufman.

Protein solubility plays an important role whether for biophysical and structural studies, or for production and delivery of therapeutic proteins. Poor solubility could lead to protein aggregation, which is an undesired physicochemical mechanism at any stage of recombinant proteins production. To date, more than half of all recombinant therapeutic proteins are produced in mammalian cells, mainly due to the high similarity of the final product to human protein structures. However, poor secretion can occur, due to misfolded proteins or aggregates leading to cellular stress and proteolysis. Another widely-used expression system is E. coli, which can offer a cost-efficient alternative. This system has an important limitation, since proteins tends to form insoluble protein aggregates in the cytoplasm upon heterologous overexpression. Several strategies are being implemented to improved soluble expression, ranging from culture conditions to solubility enhancing tags. However, there is no universal approach or technology that solves protein aggregation. In this thesis two recently published hypotheses from our group have been applied. One stated that soluble expression of proteins was inversely correlated with the size of the largest positively-charged patch on the protein surface. The second hypothesis (of protein solubility), arose from the finding that the relative content of lysine and arginine residues separated E. coli proteins by solubility. Both hypotheses arose from a study of an extensive dataset of experimental solubilities determined for cell-free expression of E. coli proteins. In combination with other widely used strategies, such as lowering expression temperature and inducer concentration, decreasing non-charged (hydrophobic) patches and addition of helical capping for increasing stability, a rational understanding for directed alteration of solubility in a variety of recombinant proteins has been explored. This includes three protein models to test: (i) recombinant human erythropoietin (rHuEPO) (one of the top selling therapeutics) (ii) recombinant 6-Phosphofructo-2-Kinase/fructose-2,6-bisphosphatase (rPFKFB3) (a product for which over-expression has been sought for characterisation and insight into possible cancer therapy) and (iii) a set of three selected E. coli proteins containing high ratios of lysines to arginines: thioredoxin-1 (TRX), cold shock-like protein cspB (cspB), and the histidine-containing phosphocarrier protein (HPr). It was found that single or multiple point mutations (changing amino acids from positive to negative charge or vice versa; or lysines to arginines) verified the predicted effect on rHuEPO, rPFKFB3, TRX, cspB, and HPr solubility (experimentally defined as the distribution between soluble and total fractions) for expression in E. coli. In addition, the redesigned set of rHuEPO transiently expressed in HEK 293-EBNA cells, suggesting that positively-charged patch size may also influence protein secretion. Further application of these computational and experimental approaches could provide a valuable tool in the design and engineering of proteins, with enhanced solubility, stability and secretion.

Le transport réactif des NPs en milieu poreux regroupe trois principaux processus : l’agrégation, le dépôt et le transport des NPs. Le système réel est caractérisé par la complexité et l’interdépendance de ces trois processus. En plus, ces derniers ne possèdent pas forcément la même échelle propre d’étude ou de modélisation. Cela explique la difficulté majeure pour décrire simultanément tous ces processus couplés et interdépendants dans un seul modèle aisément utilisable. Au départ, nous avons simplifié ce système en séparant les différents processus. Ensuite, nous résolvons le problème d’échelle, par l’approche d’homogénéisation ou par l’approche multi-échelles. La séparation du problème, en plusieurs processus et sur plusieurs échelles d’espace, facilite la résolution numérique et la compréhension des processus élémentaires. Dans cette étude, une approche multi-échelles a été développée pour modéliser ces trois processus (l’agrégation, le dépôt et le transport des NPs), chacun dans sa propre échelle. Nous avons considéré deux échelles de taille, l’échelle microscopique ou d’interface (nanométrique), et l’échelle mésoscopique correspondant à la taille des pores (micrométrique). A l’échelle microscopique, les processus d’agrégation et de dépôt ont été modélisés, de manière similaire, avec la théorie DLVO. Les propriétés électrostatiques de surface des NPs ou de la roche sont décrites par un modèle de complexation de surface développé sous PhreeqC. Ce modèle d’interface est testé pour deux types de matériaux, le titane pour les NPs et la silice pour la roche. A l’échelle mésoscopique, le dépôt est quantifié par la théorie classique de la filtration (CFT : Classical Filtration Theory) dans la phase initiale où le filtre est propre. Le processus de transport a été simulé par un modèle de réseau de pores (PNM : Pore Network Model) à l’échelle mésoscopique. Ce modèle de transport est couplé avec le modèle d’agrégation-dépôt et indirectement avec le modèle de complexation de surface en un seul modèle appelé PhreeqC Pore Network Transport(PPNT1.0). / Reactive transport of NPs in porous media involves three main processes: aggregation, deposition and transport of NPs. The natural system is characterized by the complexity and interdependence of these three processes. In addition, these processes do not necessarily have the same study or modeling scale. This explains the extreme difficulty to describe simultaneously all these interdependent processes in one easy-tohandle numerical model. We have simplified the system by separating the different processes. Then, we solve the problem of scale by the homogenization or the multi-scale approach. Dividing the initial problem into different processes on different scales facilitates the numerical solution and the understanding of each process separately. In this study, a multi-scale approach has been developed to model, each mechanism at its own scale. We considered two modeling scales, the microscopic or interfacial scale (nanometric scale), and the mesoscopic scale (micrometric scale). At the microscopic scale, the processes of aggregation and deposition have been modeled in a similar manner with the DLVO theory. Surface electrostatic properties of NPs and rocks are described by a surface complexation model implemented in geochemical modeling program (PHREEQC). This surface complexation model was tested for two types of materials, titanium and silica. At the mesoscopic scale, the deposition is quantified by the classic filtration theory (CFT) in the initial phase when the filter is clean. The transport of NPs in porous media was simulated by a pore network model (PNM) at the mesoscopic scale. This transport model was coupled with the deposition-aggregation model and also indirectly with the surface complexation model. These three models are coupled in a single model called: PhreeqC Pore Network Transport (PPNT1.0).

As porfirinas e seus derivados têm sido, ao longo dos anos, alvos de vários campos diferentes de interesse, obtendo diversas aplicações, as quais tem aumentado constantemente com o decorrer dos anos. Dentre suas aplicações, uma das mais importantes se encontra no campo da Medicina moderna, sendo a principal na área de detecção e extirpação de tecidos modificados, a partir da Terapia Fotodinâmica (do termo inglês Photodynamic Therapy – PDT), apresentando resultados promissores. Em diversos processos biológicos, as porfirinas podem existir na forma monomérica ou na forma agregada. Entretanto, a agregação de porfirinas reduz sua eficiência nas aplicações em PDT devido a redução dos tempos de vida e rendimentos quânticos de produção dos estados excitados singleto e tripleto e levando, conseqüentemente, a redução na produção de oxigênio singleto. Diversos fatores influenciam o processo de agregação das porfirinas. Dentre eles, a interação eletrostática exerce um importante papel. A modulação dessa interação pode tanto estimular a agregação, quanto diminuir a probabilidade de formação dos agregados. Deste modo, condições externas, tais como pH, força iônica e especialmente a interação com sistemas microheterogêneos podem modificar essa interação e influenciar também nas características de agregação. Neste trabalho buscamos avaliar, através de diversos métodos espectroscópicos, os efeitos da interação das porfirinas meso-tetrakis (p-sulfonatofenil) porfirina (TPPS4), aniônica, e meso-tetrakis (4-N-metilpiridil) porfirina (TMPyP), catiônica, com sistemas microheterogêneos naturais e sintéticos (células “ghost" de eritrócitos e micelas) em função da sua própria estrutura, da estrutura destes sistemas e de fatores externos (concentração, pH, força iônica). O interesse principal foi dedicado aos efeitos dessa interação na formação de agregados das porfirinas. Foram analisadas as mudanças nas características fotofísicas e fotoquímicas destes compostos, tais como espectros de absorção, rendimentos quânticos e tempos de vida dos estados excitados singleto e tripleto, produção de oxigênio singleto, etc, visando obter informações sobre o comportamento coletivo dessas porfirinas, o que é muito importante para as suas possíveis aplicações em medicina, em particular na Terapia Fotodinâmica do câncer. Foi descoberto que a interação da porfirina aniônica TPPS4 com micelas de carga oposta ou na presença de alta força iônica em pH < pKa estimula a formação seqüencial de dois tipos de agregados: inicialmente são formados agregados H que após certo período se transformam em agregados J. A formação desses agregados altera os espectros de absorção, rendimentos quânticos e tempos de vida dos estados excitados singleto e tripleto e produção de oxigênio singleto. Foi observado que a interação com porfirinas altera a c.m.c dos tensoativos, reduzindo em até três ordens de grandeza o valor da c.m.c dos tensoativos de carga oposta à das porfirinas. Associamos esta c.m.c com a formação de micelas mistas porfirina+tensoativo. Foi também observado que a adição de NaCl no sistema porfirina+micelas ou porfirina+células ghost facilita a penetração da porfirina no interior da micela ou membrana, diminuindo assim a probabilidade de contato entre as porfirinas e o oxigênio, reduzindo a constante bimolecular de supressão do estado tripleto da porfirina pelo O2. A fim de avaliar a influência da estrutura dos fotossensibilizadores (FS) na sua fototoxicidade, comparamos o efeito fototóxico de ambas porfirinas e dois corantes bisciânicos em células de linhagem neoplásica HT29. Os resultados foram analisados levando em consideração as características de ligação dos FS, sua penetração e localização nas células HT29. A fototividade de ambas porfirinas em cultura celular demonstrou ser independente do tipo de suas cargas, apresentado um perfil de toxicidade muito similar. Os BCDs apresentaram uma elevada toxicidade em comparação com a das porfirinas. Observamos que as porfirinas e os BCDs possuem uma cinética de acumulação muito similar e períodos de ligação muito próximos (2h). Entretanto, o tempo e o local de internalização parecem ser dependentes tanto da estrutura quanto da carga do FS. Assim, os BCDs se internalizaram após apenas 2 horas e se localizaram preferencialmente nas mitocôndrias. Por outro lado, a TPPS4 localiza-se principalmente nos lisossomos enquanto que a TMPyP parece estar localizada na membrana celular, ambas com um tempo de internalização de 24 horas. Estes resultados podem explicar a elevada fototoxicidade dos BCDs observada em nossos experimentos. / During several decades the porphyrins and their derivatives continue of interest in different areas including various applications, the number of which is increasing with time. One of their most important applications is in modern medicine to detect and extirpate modified tissues, the photodynamic therapy (PDT) being the principle area, which demonstrates promising results. Taking part of various biological processes, the porphyrins may exist as monomers or aggregates. However, aggregation reduces their efficacy in PDT as it shortens their lifetimes and quantum yields of excited singlet and triplet states, thus reducing the production of singlet oxygen. There exist various factors, which may modify aggregation of the porphyrins, the electrostatic interaction being very important. Modulation of this interaction can stimulate aggregation or reduce the probability of the aggregate formation. So external conditions such as pH, ionic strength and interaction with microheterogeneous systems can modify this interaction and thus affect the aggregation characteristics. In this work we present the results received with the help of various spectroscopic techniques on interaction of anionic meso-tetrakis (p-sulfonatophenyl) (TPPS4) and cationic meso-tetrakis (4-N-methyl-pyrydiumil) (TMPyP) porphyrins with natural and synthetic microheterogeneous systems (“ghost" erythrocyte membranes and micelles) in function of their proper structure, the structure of these systems and the external factors (concentration, pH, ionic strength). The principle attention was paid to the effects of this interaction on aggregation of the porphyrins. We analyzed variations in the porphyrin photophysical and photochemical characteristics such as absorption spectra, quantum yields and lifetimes of excited singlet and triplet states, singlet oxygen production, etc., to look for the regularities in their collective behavior which is very important at their possible application in medicine, in PDT of cancer, in particular. We found that the interaction of the anionic porphyrin TPPS4 with micelles of the opposite charge or in the presence of high ionic strength at pH<pKa stimulated sequential formation of two types of aggregates: at the beginning H aggregates were formed which transformed with time in J type. Formation of the aggregates modified the porphyrin absorption spectra, their lifetimes and quantum yields of excited singlet and triplet states and the production of singlet oxygen, as well. We observed that the interaction with porphyrins reduced up to three orders of magnitude the c.m.c. values for surfactants with the charge opposite to that of the porphyrin. We attribute these reduced c.m.c. values to formation of mixed micelles (surfactant + porphyrin). We observed that in the presence of NaCl in the systems (porphyrin + micelle) or (porphyrin + “ghost" cells) the dye penetrated more easily the interior of the micelle or the membrane, reducing the probability of its contact with oxygen, and, hence, reducing the bimolecular constant of triplet suppression by O2. To evaluate the influence of the structure of photosensitizers (PS) on their phototoxicity we compared phototoxic effects of both porphyrins and two biscyanine dyes upon the neoplasic cells line HT29. The results were analyzed taking into account the characteristics of photosensitizer binding, penetration and localization in cells. The photoefficacy of both porphyrins was demonstrated independent of the type of their charge, their phototoxicities being very close. The BCDs demonstrated the elevated phototoxicity as compared with that of porphyrins. We observed that both porphyrins and BCDs possessed very close accumulation kinetics and their binding periods were similar (2 h). Nevertheless, the time of the PS entrance to the cell and their intracellular localization were shown to depend on their structure and charge. So, the BCDs entered the cell in two hours and were localized preferably in mitochondrias. The TPPS4 localized in lysosomes, while TMPyP seemed to prefer the cell membrane, the time of entrance for both being close to 24 hours. These results may explain higher BCDs phototoxicity observed in the experiments.

Submitted by LUDMILA MAGALHÃES null (mag.ludmila@gmail.com) on 2017-03-15T01:30:12Z No. of bitstreams: 1 Dissertação_Ludmila_Magalhães.pdf: 611574 bytes, checksum: efdd25c5174f4d490dccf0f24fb99f43 (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2017-03-20T23:04:21Z (GMT) No. of bitstreams: 1 magalhaes_l_me_jabo.pdf: 611574 bytes, checksum: efdd25c5174f4d490dccf0f24fb99f43 (MD5) / Made available in DSpace on 2017-03-20T23:04:21Z (GMT). No. of bitstreams: 1 magalhaes_l_me_jabo.pdf: 611574 bytes, checksum: efdd25c5174f4d490dccf0f24fb99f43 (MD5) Previous issue date: 2017-02-17 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O esterco bovino contém elevados teores de matéria orgânica, que pode elevar os teores de carbono orgânico no solo e proporcionar melhorias nos atributos físicos do solo. Isso ocorre devido o carbono orgânico ser um agente cimentante das partículas minerais do solo. Objetivou-se com esse trabalho avaliar o efeito da adubação orgânica (esterco bovino) nos atributos do solo: carbono orgânico, carbono orgânico particulado, carbono orgânico associado aos minerais, distribuição de agregados do solo por classes de tamanho, índice de estabilidade de agregados do solo, condutividade hidráulica do solo saturado, macroporosidade, microporosidade, porosidade total, resistência do solo à penetração e densidade do solo. O experimento foi realizado na Fazenda de Ensino, Pesquisa e Extensão da UNESP, Câmpus de Jaboticabal, em um Latossolo Vermelho eutroférrico, de textura argilosa. O delineamento experimental foi em blocos casualizados, constando de cinco doses de esterco bovino (0, 15, 30, 45 e 60 Mg ha-1) com quatro repetições. Foram coletadas amostras de solo indeformadas, na camada de 0-0,10 m, para a determinação da condutividade hidráulica do solo saturado (K0), macroporosidade (Ma), microporosidade (Mi), porosidade total (PT), resistência do solo à penetração (RP) e densidade do solo (Ds). Na mesma camada, foram coletadas amostras de solo deformadas para a determinação da distribuição de agregados do solo por classes de tamanho, índice de estabilidade de agregados do solo (IEA), carbono orgânico (CO), e fracionamento físico do carbono orgânico, em que foi determinado o carbono orgânico particulado (COP). O carbono orgânico associado aos minerais (COAM) foi obtido por diferença entre o CO e o COP. Foi realizada a análise de variância para a regressão polinomial, para os atributos do solo avaliados em função das doses de esterco bovino, utilizando o teste F com nível de significância de 5% de probabilidade. Os valores de CO, COP, IEA, PT, Ma, RP, Ds e K0 foram influenciados pelo esterco bovino, aplicado ao solo. Os valores de COAM e Mi não foram influenciados pelo esterco bovino aplicado ao solo. A aplicação de esterco bovino proporcionou no Latossolo Vermelho eutroférrico, o aumento do carbono orgânico, do carbono orgânico particulado, da porosidade total, da macroporosidade e do índice de estabilidade de agregados do solo em água, e a redução da densidade do solo e da resistência do solo à penetração. Já o carbono orgânico associado aos minerais e a microporosidade não foram influenciados. / Cattle manure contains high levels of organic matter, wich can increase the organic carbon content in the soil and provide improvements in the physical attributes of this. This occurs because the organic carbon it is a cementing agent for the soil mineral particles. From this, the objective of this paper was to evaluate the effect of organic fertilization (cattle manure) on the soil attributes: organic carbon, particulate organic carbon, organic carbon associated to minerals, soil aggregates distribution by sizes classes, soil aggregates stability index, saturated soil hydraulic conductivity, macroporosity, microporosity, total porosity, soil resistance to penetration, and soil bulk density. The experiment was carried out at the Teaching, Research and Extension Farm of UNESP, Jaboticabal Campus, in a clayey Oxisol. The experimental design was a randomized complete block, consisting of five rates of cattle manure (0, 15, 30, 45 e 60 Mg ha-1), with four replications. Undisturbed soil cores were collected at the 0-0.10 m deep layer to determine the hydraulic conductivity of saturated soil (K0), macroporosity (Ma), microporosity (Mi), total porosity (PT), soil resistance to penetration (RP) and soil bulk density (Ds). At the same layer, deformed soil cores were collected to determine the soil aggregates distribution by sizes classes, aggregate stability index of soil (IEA), organic carbon (CO), and physical fractionation of organic carbon, in with was determined the particulate organic carbon (COP). The organic carbon associated to minerals (COAM) was obtained by difference between CO and COP. An Analysis of Variance was performed to polynomial regression, to the evaluated soil attributes in function of cattle manure rates, using the F test with 5% probability. The values of CO, COP, IEA, PT, Ma, RP, Ds and K0 were influenced by the cattle manure applied to the soil. The values of COAM e Mi were not influenced by the cattle manure applied to the soil. The cattle manure addition provided on the Oxisol the increase of organic carbon, particulate organic carbon, total porosity, macroporosity and soil aggregates stability in water index, and the decrease of soil bulk density and soil resistance to penetration. The organic carbon associated to minerals and microporosity were note influenced.

La réponse au choc thermique est un mécanisme de défense largement décrit au cours duquel l’expression préférentielle des protéines de choc thermique Hsp aide la cellule à récupérer des dommages causés par l’hyperthermie, comme la dénaturation/agrégation des protéines. Une des conséquences du choc thermique mise en évidence au laboratoire, est l’activation du facteur de transcription NFκB. Cette activation a lieu pendant la période de récupération suivant ce stress. Par comparaison de la réponse au choc thermique de cellules témoins ou déficientes en NFκB, nous avons cherché à étudier les conséquences de l’activation de NFκB par le choc thermique. Nous avons montré que NFκB active un mécanisme augmentant la survie des cellules soumises à une hyperthermie : l’autophagie. L’absence d’induction de ce mécanisme conduit à la mort par nécrose des cellules déplétées en NFκB. Dans ces cellules, l’induction artificielle de l’autophagie restaure une survie normale au stress thermique. Nous avons montré que les principaux régulateurs de l’autophagie (complexes mTOR et PI3Kinase de Classe III) ne sont pas des cibles modulées par NFκB, en réponse à une hyperthermie. En revanche, l’accumulation de protéines dénaturées voire agrégées est un élément primordial pour l’activation de l’autophagie-dépendante de NFκB. En effet dans les cellules déficientes pour NFκB, contrairement aux cellules témoins, l’accumulation de protéines agrégées induite par le traitement hyperthermique, mais aussi par l’expression de formes mutées d’HspB5, n’est pas résorbée ; ceci indique que le contrôle qualité des protéines est altéré dans ces cellules. Cette altération pourrait provenir d’un défaut de formation du complexe BAG3-HspB8 en absence de NFκB. En effet, nous avons montré que la forte expression des gènes bag3 et hspb8, induite suite au stress thermique, est dépendante de NFκB et que l’accumulation du complexe BAG3-HspB8, observé dans les cellules témoins soumises au choc thermique, est inhibée dans les cellules déficientes pour NFκB. Nos résultats démontrent que NFκB induit un processus autophagique en réponse à l’agrégation protéique induite par l’hyperthermie. Ce mécanisme, nécessitant la formation du complexe BAG3-HspB8, augmente la survie des cellules probablement par l’élimination des protéines agrégées générées au cours du stress thermique / The heat shock response is a widely described defense mechanism during which the preferential expression of heat shock proteins (Hsps) helps the cell to recover from thermal damages such as protein denaturation/aggregation. We have previously reported that NFκB transcription factor is activated during the recovery period after heat shock. Thus, we aimed to analyze the consequences of NFκB activation during heat shock recovery, by comparing the heat shock response of NFκB competent and incompetent cells. We demonstrated that NFκB plays a major and crucial role during the heat shock response by activating autophagy, which increases the survival of heat-treated cells. Indeed, we observed that autophagy is not activated during heat shock recovery leading to an increased level of necrotic cell death in NFκB incompetent cells. Moreover, when autophagy is artificially induced in these cells, the heat shock cytotoxicity is turned back to normal. We showed that the key regulators of autophagy (mTOR complex, and class III PI3Kinase complex) are not regulated by NFκB after heat shock. In contrast, we observed that aberrantly folded/aggregated proteins accumulation is a prime event in the activation of NFκB -mediated autophagy. Moreover, NFκB -depleted cells accumulate higher levels of protein aggregates induced by either heat shock treatment or mutated form of HspB5, indicating that the protein quality control process seem to be altered in these cells. This alteration could be caused by a defect in BAG3-HspB8 complex formation in NFκB -depleted cells. We demonstrated that heat shock treatment induces a NFB-dependent overexpression of the bag3 and hspb8 genes. Moreover, the accumulation of BAG3-HspB8 complex in heat shocked NFκB -competent cells is inhibited by NFκB depletion. Our findings how / prove / highlight revealed that NFκB -induced autophagy during heat shock recovery is an additional response to protein denaturation/aggregation induced by heat shock. This process depends on the BAG3-HspB8 complex formation and increases cell survival, probably through clearance of aggregated proteins

In last few years Value-at-Risk (Var) is a very popular and frequently used risk measure. Risk measure VaR is used in most of the financial institutions. VaR is popular thanks to its simple interpretation and simple valuation. Valuation of VaR is a problem if we assume a few dependent risks. So VaR is estimated in a practice. In presented thesis we study theory of stochastic bounding. Using this theory we obtain bounds for VaR of sum a few dependent risks. In next part of presented thesis we show how we can generalize obtained bounds by theory of copulae. Then we show numerical algorithm, which we can use to evaluate bounds, when exact analytical evaluate isn't possible. In a final part of presented thesis we show our results on practical examples.

Tendo em vista o fato de que o sistema radicular das culturas modifica as propriedades do solo ao seu entorno e da praticamente ausência de trabalhos científicos para avaliação das diferenças estruturais entre solo e rizosfera, o objetivo deste estudo foi aprofundar o conhecimento destas diferenças, avaliando a agregação do solo junto às raízes e fora da sua influência, em diferentes rotações culturais. A amostragem foi realizada na camada superficial do solo (0,00 - 0,05 m) em cada sistema de rotação, considerando como rizosfera o solo aderido às raízes e como solo sem influência das raízes, aquele não aderido a elas. Para avaliar a estabilidade estrutural do solo, foram feitas as análises da argila prontamente dispersa em água, da argila mecanicamente dispersa em água e da estabilidade de percolação. As análises granulométrica e química também foram realizadas. Os dados obtidos foram submetidos à análise de variância e, quando significativo, ao teste de médias Tukey (p<=0,05). Posteriormente, foram feitas correlações entre os atributos físicos utilizados como indicadores da estabilidade dos agregados e os demais atributos avaliados. A rizosfera apresentou agregados mais estáveis em água do que o solo fora dela, sendo a argila mecanicamente dispersa em água o indicador mais sensível na detecção desse resultado. Foi evidênciado que o sistema radicular de diferentes culturas influenciaram de forma diferente a agregação das partículas do solo. As correlações mais significativas foram para a argila mecanicamente dispersa em água. / Considering the fact that the crops root system modifies the soil properties around it and in the almost absence of scientific works to evaluate the structural differences between soil and rhizosphere, the objective of this study was to increase the knowledge of these differences by evaluating soil aggregation close to the root and out of its influence, at different crop rotations. Sampling was performed in the soil surface layer (0.00 - 0.05 m) in each rotation system, considering as rhizosphere the soil adhered to the roots and as soil without influence of the roots, that not adhered to them. To evaluate the structural stability of the soil, analyzes of clay readily and mechanically dispersed in water and of the percolation stability were made. The granulometric and chemical analyzes were also made. Data obtained were analysed by the analysis of variance and, when significant, by Tukey test (p<=0.05). Then, the correlations were made between the physical attributes used as indicators of the aggregates stability and the other attributes evaluated. Rhizosphere presented more stable aggregates in water than the soil outside it, being the mechanically clay dispersed in water the most sensitive indicator in the detection of this outcome. It was evidenced that the root system of different crops influenced of different way the soil particles aggregation. The more significant correlations were those for clay mechanically dispersed in water.

α-synuclein is a small presynaptic protein whose misfolding and aggregation are considered drivers of the neurological disorders Parkinson’s disease, multiple system atrophy, dementia with Lewy bodies and related synucleopathies. α-synuclein exists in a dynamic state that changes from an α-helical conformation when bound to liposomes to natively unfolded in solution, the majority being in the latter state. The disease process by which native healthy α-synuclein undergoes a change in conformation to form β-sheet oligomers and fibrils is still unresolved. The fibrillation process has been widely studied by several different techniques and the structure of the fibrils has been determined by NMR, scanning transmission electron microscopy and X-ray diffraction. The early stages of aggregation into β-sheet rich oligomers, despite having been widely studied, has proven difficult to follow due to the heterogeneity of the species formed and the unpredictability of the process. The goal of the work reported here was to design and develop a novel, reproducible and quantitative assay to study the early stages of α-synuclein aggregation and to establish a platform for discovery of novel compounds that inhibit this process. These compounds could then be taken as a starting point for the development of new drugs for the treatment of synucleopathies. The assay developed herein has been designed, established and demonstrated to be suitable for the screening of α-synuclein aggregation inhibitors. The assay quantitatively measures aggregation using α- synuclein site-specifically labelled with green and red fluorescent dyes. Proteins labelled with the green dye are bound to microbeads. α-synuclein labelled with the red dye aggregates on the bead-linked green α-synuclein. The first part of the thesis describes the development of the tools required for the assay. α-synuclein single cysteine mutants were produced to introduce a specific attachment point to the protein. Single isomer carboxytetramethylrhodamine was synthesised in large scale for the label. Two different trifunctional tags that allow both the fluorescent labelling of the protein and the addition of a group for bead attachment in a single step were synthesised. Optimisation of the attachment of the functionalised proteins to beads of differing materials was accomplished enabling further development of the bead-based aggregation assay. With all tools established, the second part of the work comprised the development of the bead-based α-synuclein aggregation assay. Solid supports made of two different materials, TentaGel and Agarose, with two different types of bead surface attachment chemistry for α-synuclein were investigated, Ni-NTA on bead with His6-tag on the target or dibenzylcyclooctyne on bead and azide conjugation for the target. Only the combination of Ni-NTA agarose beads linking to His6-tag functionalised α-synuclein was found to be suitable for quantitative measurement of the aggregation process. Using 20 % EtOH, α-synuclein on-bead aggregation was reproducible within a 5 h time-frame with a linear dependence of aggregation rate as function of protein concentration on-bead. The third part of the thesis describes the research into novel starting points for the discovery of inhibitors of α-synuclein aggregation. In the peptides field, the most active peptides in the literature were selected and synthesised for study under the same conditions to find the most active ones. The most active peptide could be modified with non-natural amino acids to increase affinity and stability. While peptides and peptidomimetics would be applied in mechanistic studies, small molecular inhibitors of aggregation might represent lead compounds. One known inhibitor of α-synuclein aggregation was selected, NPT200-5, and an on-bead synthesis was developed so a diversity library could be generated around its four different building blocks. Finally the peptides, the NPT200-5 amide derivative and some known small molecule inhibitors of α-synuclein aggregation, such as curcumin, baicalein and EGCG amongst others, were screened on the bead-based α-synuclein aggregation assay. Strong inhibitory effects of curcumin and baicalein demonstrated the efficacy of the newly developed assay. In summary, the tools for the development of a novel micro-bead-based α-synuclein aggregation assay have been successfully produced. A novel bead-based α-synuclein early stage aggregation assay has been developed and optimised. Validation of this new technique was achieved with known small molecules inhibitors of α-synuclein aggregation.