Linhagem de células de inseto S2 foram cultivadas em meio TC100 suplementado em reator bubble free de 1.5 L e para estudar o seu crescimento e expressão da glicoproteína G do vírus da raiva (RVGP). Os dados permitiram propor um modelo matemático que reproduz o crescimento e produção da glicoproteína e que considera oito variáveis de estado e vinte parâmetros cinéticos. O modelo considera a velocidade específica de crescimento limitada por glicose, glutamina e glutamato e inibida por NH4+; consumo e formação de glutamina; velocidade específica de morte limitada por NH4+ e inibida por glicose; velocidade específica de produção de NH4+ e de GPV associada ao crescimento e a degradação de GPV. As concentrações de oxigênio dissolvido (pO2), glicose e glutamina foram modificadas para se avaliar a sua influência na velocidade específica de crescimento máxima e nos fatores de conversão. Os parâmetros do modelo foram ajustados usando a técnica de otimização dos poliedros flexíveis e as equações diferenciais do modelo foram integradas pelo método de Gear. / S2 cell strain from Drosophila melanogaster were cultivated in supplemented TC100 media in reactor \"bubble free\" to study their growth and expression of glycoprotein G of rabies virus (RVGP). The data allowed proposing a mathematical model that reproduces the growth and production of glycoprotein and that consider eight state variables and twenty kinetic parameters. The model considers the specific growth rate limited by glucose glutamine and glutamate and inhibited by NH4+ consumption and formation of glutamine, specific death rate limited by NH4+ and inhibited by glucose; production specific rate of NH4+ and RGPV associated with growth and degradation of RGPV. The concentrations of dissolved oxygen (pO2), glucose and glutamine were varied to evaluate their influence on maximum specific growth rate and conversion factors. The model parameters were adjusted using the flexible polyhedron optimization method and the differential equations of the model were integrated by the method of Gear.

Charles Jennens (1700-1773), the librettist of 'Saul', 'Messiah', 'Belshazzar', the final part of 'L'Allegro, il Penseroso ed il Moderato', and probably 'Israel in Egypt', amassed a huge library of music that forms the bulk of what is now known as the Aylesford collection. Jennens's collection of Handel's music was unique among those of his contemporaries, not only because it includes part-books, but also because it is unusually comprehensive. The dissertation focuses on his copies of the sacred oratorios beginning with 'Saul' (1739) because most of the collection was copied in the 1740s, the sacred oratorios were the works that Jennens was most interested in, and 'Saul' was his first collaboration with Handel. As many of these manuscripts have not been the focus of modern scholarly attention, I first establish how, when, and by whom each manuscript was copied, in order to achieve a greater understanding of how and when Jennens assembled his collection, and what his reasons were for doing so. This close study of the manuscripts reveals that Jennens made extensive alterations to the verbal text, the structure, and the music of several oratorios in his collection. His amendments to 'Saul' and 'Belshazzar' shed light on his collaboration with Handel; and his amendments to 'Samson' and 'Joseph and his Brethren' provide insights into his attitude to Handel in the mid-1740s, his approach to word-setting, his views on the adaptation of Scripture for oratorio, and his beliefs and commitments. Jennens was a highly educated man whose activities were informed by two deeply held, conflicting allegiances: to the Anglican Church and to the deposed Stuarts. An examination of how he harnessed Handel's music to deliver his religious and political messages leads to a richer and more profound understanding of the works, of the relationship between Jennens, Handel, and Handel's music, and of their place in the religious and political context of the mid-eighteenth century.

La mise en forme spatiale est une problématique importante pour les lasers et ses applications. Dans de nombreux domaines (l’industrie, l’interaction laser-matière, l’injection des lasers de puissance...) il est indispensable que le faisceau laser ait une forme aplatie. Les solutions actuelles de mise en forme spatiale d'un faisceau laser quelconque en faisceau aplati (par les techniques « espace-libre » ou par les fibres fortement multimodes) ne sont pas satisfaisantes. Elles occasionnent des difficultés de maintenance, sont très sensibles aux perturbations du système et elles ne permettent pas de délivrer un faisceau spatialement cohérent. Pour cette raison nous présentons dans ce manuscrit une fibre optique microstructurée monomode délivrant un faisceau spatialement aplati. Cette fibre « mode-plat » permet de transformer n’importe quel faisceau en un faisceau aplati cohérent spatialement ce qui est un progrès par rapport aux fibres fortement multimodes utilisées jusque-là. L’intérêt des fibres optiques est également leur maintenance facilitée et leur robustesse, cette solution est donc d’un grand avantage par rapport aux solutions « espace-libres ». Grâce à cette fibre, nous avons pu réaliser une chaine laser entièrement fibrée permettant d’amplifier une impulsion d’une durée de 10 ns jusqu’à 100 µJ tout en contrôlant ses propriétés temporelles, spectrales et spatiales. En adaptant la fibre « mode-plat » à cette chaine laser, nous avons démontré la faisabilité et l’intérêt de cette fibre pour la mise en forme spatiale de faisceaux laser dans des systèmes performants et robustes. / Spatial beam shaping is an important topic for the lasers applications. For various industrial areas (marking, drilling, laser-matter interaction, high-power laser seeding…) the optical beam has to be flattened. Currently, the state of the art of the beam shaping: “free-space” solutions or highly multimode fibers, are not fully suitable. The first ones are very sensitive to any perturbations and the maintenance is challenging, the second ones cannot deliver a coherent beam. For this reason, we present in this manuscript a microstructured optical single-mode fiber delivering a spatially flattened beam. This “Top-Hat” fiber can shape any beam in a spatially coherent beam what is a progress with respect to the highly multimode fibers used in the state of the art. The optical fibers are easy to use and very robust, what is a strong benefit with respect to the “free-space” solutions. Thanks to this fiber, we could realize an all-fiber multi-stage laser chain to amplify a 10 ns pulse to 100 µJ. Moreover the temporal, spectral and spatial properties were preserved. We adapted this “Top-Hat” fiber to this multi-stage laser chain, we proved the capability and the interest of this fiber for the spatial beam shaping of the laser beams in highly performing and robust laser systems.

Fasciola cause considerable monetary loss in the agriculture industry, while parasitism of humans is an emerging disease. Fasciola cathepsin L-like proteases are believed to aid parasite invasion and survival through a range of functions including feeding, immune evasion and modulation, tissue migration, egg production and excystment. As such these proteases are considered good targets for chemotherapies and vaccine development. Fasciola cathepsins are evolutionarily divided into clades that reflect function and life stage of expression. Analysis of F. gigantica genomic DNA and mRNA identified novel cathepsin L-like sequences which are incorporated into a phylogenetic analysis of the complete Fasciola cathepsin L-like protease family. Analysis of mRNA transcripts isolated in this study also points to trans-splicing occurring amongst cathepsin transcripts, the first time this has been identified in Fasciola species. S2 subsite specificity is important in determining substrate interactions with cathepsin L-like proteases. Previous work has shown that amino acid substitutions at this site can dramatically influence substrate specificity. A number of substitutions, specifically those that have been observed, or predicted to occur during the evolution of Fasciola cathepsins L-like proteases, were introduced into the S2 subsite of FhCatL5 at aa69 to determine their influence. The introduction of L69C and L69S substitutions resulted in low overall activity indicating their expression provides no functional advantage, thus explaining the absence of such variants amongst fluke. The L69F variant showed an increase in the ability to cleave substrates with P2 proline, indicating F69 variants expressed by fluke are also likely to have this ability, similar to that shown with L69Y and FhCatL2. The introduction of a L69W substitution leads to increased cleavage of substrates with P2 proline, along with a decrease in cleavage of substrates with P2 phenylalanine. FgCatL1G transcripts were isolated from F. gigantica metacercariae. This contrasts with FhCatL5 and FhCatL2 which have been isolated in adult F. hepatica. These cathepsins differ at aa69, possessing tryptophan, leucine and tyrosine respectively. The processing and substrate specificities of each recombinant enzyme was analysed and compared. While FhCatL5 and FhCatL2 process in vitro in a manner similar to that reported for FhCatL1, FgCatL1G requires different processing conditions, including neutral pH. Combined with FgCatL1G possessing increased stability at acidic pH, this reflects the different environment into which FgCatL1G is expressed by immature compared to the adult flukes. The substrate specificity of FgCatL1G also differed from previously reported cathepsins, with a preference for P2 proline and low activity against substrates with P2 phenylalanine. This is the first time recombinant expression and purification of a cathepsin L-like protease specific to the immature life stages of Fasciola has been undertaken and had enzyme specificity analysed. This work has expanded knowledge of the repertoire of cathepsin proteases expressed at various life-stages of the liver fluke. Vaccination and/or drug inhibition studies may in the future be targeted towards cathepsins that are expressed in either the adult or immature stage, or perhaps both in a multi-targeted approach. The knowledge gained in this study may allow such targets to be chosen.

Submitted by Alison Vanceto (alison-vanceto@hotmail.com) on 2017-02-21T12:43:27Z No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-24T11:24:18Z (GMT) No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Approved for entry into archive by Ronildo Prado (ronisp@ufscar.br) on 2017-04-24T11:24:33Z (GMT) No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) / Made available in DSpace on 2017-04-24T11:31:33Z (GMT). No. of bitstreams: 1 DissMCD.pdf: 3338735 bytes, checksum: a38bd4887b4213867a0c295a634f94c1 (MD5) Previous issue date: 2016-08-08 / Não recebi financiamento / Although effective, current vaccinations against rabies, one of the most lethal infectious diseases in the world, present security issues of administration and production costs. In this scenario, modern biotechnology has become a source of new alternatives of great interest for vaccine production. The main antigen capable of conferring neutralizing immune response against infection by rabies virus is the glycoprotein of rabies virus (RVGP), which the production by recombinant DNA technology has been developed by researchers at the Viral Immunology Laboratory (LIV) of the Butantan Institute of São Paulo using various expression systems in Drosophila melanogaster S2 cells. One of the latest developments is S2MtRVGP-H-His cell line, obtained by stable transfection with plasmids containing cDNA from other components of RVGP and histidine tag to facilitate purification, both under control of the inducicle metallothionein promoter. This work aims to study the kinetic characteristics of cell growth and production of recombinant glycoprotein rRVGP rabies virus strain of Drosophila melanogaster S2MtRVGP-H-His, in order to evaluate the potential of a bioreactor with agitation induced by waves (Wave) for the scale-up production of rRVGP. The first stage of the study, involving batch cultures in 20 mL Schott bottle with commercial culture medium Sf900-III, allowed us to determine the optimal temperature of cultivation (28ºC), time of induction of expression (72 h), the specific growth rate ranging from 0.022 to 0,034 h-1; maximum cell density 1.82×107 cel.mL-¹ and rRVGP produced from 0.07 to 0.99 μg.mL-1. Based on these results, was started the second part of the study performed in the Single-use Wave bioreactor, involving batch cultures with 650 mL of Sf900-III, with 60% of dissolved oxygen and pH ranging without control from 6.2 to 7.0. The culture in the bioreactor showed maximum specific growth rate of 0,035 h-1, maximum cell density was 1.1×107cel.mL-¹ and RVGP produced 0.85 μg.mL-1. The production of large scale rRVGP with S2MtRVGP-H-His cells using the Wave bioreactor has shown to be viable, reproducible and with high potential to scale-up. / Embora eficazes, as vacinas atuais contra a raiva, uma das doenças infecciosas mais letais do mundo, apresentam problemas relacionados com a segurança de administração e o custo de produção. Nesse contexto, a biotecnologia moderna se torna uma fonte de alternativas inovadoras de grande interesse para produção de vacinas. O principal antígeno capaz de conferir uma resposta imunológica neutralizante contra o vírus rábico é a glicoproteína do vírus da raiva (RVGP), cuja produção por tecnologia de DNA recombinante vem sendo desenvolvida por pesquisadores do Laboratório de Imunologia Viral (LIV) do Instituto Butantan de São Paulo, utilizando vários sistemas de expressão em células de Drosophila melanogaster S2. Um dos mais recentes desenvolvimentos é a linhagem S2MtRVGP-H-His, obtida mediante transfecção estável com plasmídeos contendo entre outros componentes o cDNA da RVGP e a cauda de histidina para facilitar a purificação, ambos sob controle do promotor indutível da metalotioneína. O presente trabalho tem como objetivo o estudo de características cinéticas de crescimento celular e de produção de glicoproteína recombinante do vírus da raiva rRVGP da linhagem de Drosophila melanogaster S2MtRVGP-H-His com vistas a avaliação do potencial de um biorreator com agitação induzida por ondas (waves) para escalonamento da produção de rRVGP. A primeira etapa dos trabalhos, envolvendo cultivos em batelada em frasco Schott com 20 mL de meio de cultura comercial Sf900-III, permitiu a determinação da temperatura ideal (28ºC), o tempo apropriado de indução da expressão (72 h) e das velocidades específicas de crescimento de 0,022-0,034 h-1, densidade celular máxima de 1,82×107 cel.mL-¹ e rRVGP produzida de 0,07-0,99 μg.mL-1. Com base nesses resultados, iniciou-se a segunda parte dos trabalhos com cultivos em biorreator Wave utilizando 650 mL de meio Sf900-III, com concentração média de oxigênio dissolvido de 60% da saturação com ar e pH variando sem controle de 6,2-7,0. O cultivo no biorreator apresentou velocidade específica máxima de crescimento de 0,035 h-1, densidade celular máxima de 1,1×107 cel.mL-¹ e rRVGP produzida de 0,85 μg.mL-1. A produção da rRVGP em larga escala com células S2MtRVGP-H-His utilizando o biorreator Wave mostrou ser uma alternativa viável, reprodutível e com grande potencial de escalonamento.

The aim of this thesis is introduce the mobile mapping system IP-S2, which is using by company GEODIS BRNO, and perform data collection by this system. Measure the identical points, determine their coordinates and compare with coordinates obtained by mobile mapping system. The conclusion of this thesis is focused on testing the accuracy of the mobile mapping system.

No description available.

La fibrose kystique (FK) est caractérisée par une inflammation pulmonaire chronique. Cette dernière est définie entre autres par une augmentation importante de la production de mucus. Celui-ci s'accumule dans les bronches empêchant ainsi l'éventuel passage de l'air. De plus, le mucus forme une barrière protectrice pour les bactéries qui va causer l'inflammation des poumons. La protéase human airway trypsin-like (HAT) a été retrouvée dans les expectorations de patients atteints d'asthme et bronchite chronique, pathologies similaires à la FK. Elle semble jouer un rôle dans l'inflammation car elle augmente entre autres la production de MUC5AC, protéine composant majoritairement le mucus. Le but de cette étude était de synthétiser la HAT dans une conformation active dans un système eucaryote pour étudier ses propriétés biochimiques, enzymatiques ainsi que son potentiel pro-inflammatoire dans la lignée cellulaire pulmonaire Calu-3. L'ADNc codant pour la partie soluble de la HAT a été inséré dans le vecteur pMT-BiP V5-His. Cet ADN recombinant fut ensuite transfecté dans les cellules de Drosophile Schneider 2 (S2). La HAT recombinante a par la suite été purifiée et son activité protéolytique testée avec un substrat fluorogénique. Ses propriétés biochimiques et enzymatiques ont été étudiées ainsi que son effet sur la production d'IL-8 et sa détection dans les expectorations de patients. La HAT purifiée était «enzymatiquement» pure, donc l'effet protéolytique observé avec le substrat fluorogénique est dû à la protéase. Les analyses de ses propriétés biochimiques montrent que l'activité protéolytique de la HAT est inhibée par un pH acide (<6.5), certains inhibiteurs synthétiques (l'aprotinine, la leupeptine et le PEFABLOC) et endogène (i.e: alpha-2 anti-plasmine (a2-AP)) de protéases à sérine. D'un autre côté, les essais enzymatiques révèlent que la HAT a une préférence pour les substrats ayant une arginine en P4, P3 et Pl plutôt que ceux ayant un glutamate en P4, P2 et P1'. Finalement, l'étude sur l'implication physiologique de l'enzyme démontre que la protéase ne semble pas avoir d'effet ou au plus peu d'effet sur la production d' IL-8. [Symboles non conformes]

In the New Testament and in the early church fathers’ writings, the Son is understood to be ontologically equal to the Father and subordinate to him. Whether understood as ingenerate-generate, sender-sent, commanded-obedient, subordination shows the distinction between the Father and Son. As seen in church history, minimizing these distinctions can lead to modalism and pressing them too far leads to Arianism. In the Bible, obedience or subordination does not mean ontologically inferior. Rather, obedience results from faith and love. Although some fathers connected obedience to Christ’s humanity, they were doing so while rejecting the Arian argument that the Son’s obedience meant he was ontologically inferior. They affirmed the voluntary obedience of the Son as an expression of his love for the Father and rejected any sense of coercion or determinism. The doctrine of the eternal generation of the Son from the Father’s ousia held together the equality and subordination of the Son to the Father. Beginning with Christ’s atoning work rather than metaphysics, P. T. Forsyth and Jürgen Moltmann believe that the Son’s obedience is crucial for the atonement to be the free act of grace of the Sovereign God. Because of this, the Son’s obedience must be divine, and thus eternal. Otherwise, the obedience would be from Christ’s humanity, and humanity would contribute in inappropriate ways to the atonement. They also believe that subordination, obedience, humility, and servanthood complete the understanding of divine love. The unity provided by the same divine love is expressed according to the particularity of the Person. In the Trinitarian relationship, the Son’s eternal obedience is his free response to the Father. Here subordination is not oppression, but perfect love freely given to the perfect Lover. This fuller conception of divine love that a proper emphasis on obedience affords has great potential to help Trinitarain theology contribute to the elimination of oppression and the improvement of human relationships and to do so in a manner consistent with the biblical witness.

This thesis makes a historically grounded theoretical contribution to an emerging “critical” approach to refugee studies. Utilising the insights of Michel Foucault and Hannah Arendt, it seeks to reconceptualise academic and policy understandings of what has come to be known as “the refugee problem" through an examination and critique of its (implicit) conceptual foundations. The thesis proceeds through a series of historically-informed moves oriented by the relationship between power, subjectivity, and agency, and argues that the key to reconceptualising the refugee problem lies in understanding how these three concepts rely upon and reinforce one another in a particular historically contingent configuration. The objectives of this thesis are threefold and connected. First, it unpacks a deceptively unproblematic term, “the refugee problem” to reveal the complicity of understanding the “refugee (as) problem” in perpetuating the plight of increasing numbers of the world's population, despite the alleviation of the difficulties these people face being the professed goal of the refugee regime. Second, in so doing it contributes to a growing body of literature seeking to counter the voicelessness and abjection into which refugees and asylum seekers are cast. And third, on the basis of this, to begin a conversation about rethinking the nature of the “solutions” we seek to a reframed “refugee problem.” Engaging in a (Foucaultian) genealogical analysis of “the refugee problem”, the first half of the thesis charts the historically-contingent development of a distinct “refugee problem discourse”, revealing that the construction of refugees as passive victims of political forces is the effect both of such discourse and of the international refugee regime as a classificatory regime of truth and subjectivity, rather than an expression of any essential nature of “the refugee.” The thesis then turns to Hannah Arendt's work as a theoretical lens through which to reframe our understanding of the “refugee problem” and to investigate how to identify and open up creative forces for re-subjectification processes and “solutions” not tied to the classificatory and subjectivising logic of the refugee regime or sovereign state system.  Practices of rights claiming, and the City of Sanctuary movement in the UK are examined as two such processes, with the potential of posing “counter-narratives” of problems and solutions which challenge the technocratic, or population-management, approach of the refugee regime.