Aim of this diploma thesis was to prepare two protein antigens and two monoclonal antibodies, all based on VP2 minor protein of human polyomaviruses BK virus and Merkel Cell Polyomavirus. One monoclonal antibody was being prepared against unique part of VP2 protein (N-terminal epitope, not present in VP3 protein). A cell line producing such monoclonal antibody has never been established before due to low immunogenicity of the epitope. Our approach was successful in terms of mouse immunization, however, serious problems with hybridoma line stability appeared later during the preparation process. Preparation of antibody targeted to the sequence of VP2 protein of Merkel Cell Polyomavirus was another aim of this thesis. Mouse immunization and hybridoma fusion were performed successfully. After four rounds of cloning in order to purify an established clone, nine clones were cultivated in larger scale. This cultivation probably led to diminished antibody specificity and loss of production ability in most of the hybridoma cells. One more cloning should give rise to an established clone with sufficient production. Two preparations of protein antigens were performed in two expression systems. DNA encoding C-terminally truncated protein VP2 of BK virus fused with His-tag was cloned into a vector suitable for...

Les Surfaces à Haute Impédance (SHI) ont été largement étudiées pour améliorer toutes sortes de performances des antennes, comme le gain, le facteur de qualité, les formes et dimensions. L'objectif de cette thèse est de modéliser les structures de SHI et de caractériser leurs performances en vue de futures applications aux antennes.Après une brève introduction aux structures SHI et une étude de quelques modèles analytiques fréquemment traités dans la littérature, deux nouvelles méthodes numériques sont proposées pour calculer l'impédance de surface de structures SHI. Ces deux méthodes (dites « méthode du flux de Poynting » et « méthode <E>/<H> ») sont validées sur des structures symétriques, puis mises en service sur des structures de SHI asymétriques. Elles sont également validées par comparaison de résultats analytiques, numériques et expérimentaux.Nous présentons ensuite un modèle équivalent basé sur l'idée de remplacer les structures hétérogènes de SHI par une surface homogène, caractérisée par son impédance surfacique. Ce modèle nous permet d'avoir une prédiction avec un temps de calcul et une occupation de mémoire PC largement réduits. / High impedance surfaces (HIS) have proved good candidates for antenna miniaturization or antennas performance improvement. Within a certain frequency band, they can enhance the gain of an antenna while simultaneously suppressing the unwanted surface waves.In this thesis, the focus of our work is on numerical modeling of these structures by using the finite element method (FEM) based on edge elements.One of our contributions is that we propose two new numerical methods (the Poynting flux method and <E>/<H> method) to calculate the surface impedance not only for HIS structures with symmetric geometries, but also for those with asymmetric geometries. These two numerical methods have been validated by comparing analytical, numerical and experimental results.Another significant contribution of the thesis is that we introduce an equivalent model, based on the idea of replacing the heterogeneous HIS structures by a homogeneous surface, characterized by its surface impedance. Compared with the normal model, this equivalent model can save computing time and memory space.

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A presente dissertação analisa o desempenho técnico das edificações do conjunto habitacional B-10, arrolado no objeto do estudo de caso desta pesquisa, dentro da lógica da construção habitacional de interesse social e propõe uma reflexão sobre as condicionantes técnicas e gerenciais que influenciam o desenvolvimento do projeto de arquitetura orientado ao seu desempenho ideal, em relação ao ambiente construído, a partir da visão sistêmica de compreensão do edifício. A pesquisa classifica-se como exploratória e possui o caráter de aprofundamento nas questões que envolvem o objeto analisado. Optou-se pelo método Estudo de Caso, por este ser considerado uma estratégia de pesquisa comumente usada em Ciências Sociais. São feitas descrições das Patologias Construtivas detectadas nos dez subsistemas do edifício, com indicações das prováveis origens. Também são elaboradas considerações sobre como os reflexos categorizados interferem nos itens de desempenho, balizados pela ISO 6241. Os principais resultados obtidos demonstram que a partir das evidências de Patologias Construtivas diagnosticadas, relacionadas nas suas origens junto aos Responsáveis [Projeto Execução de Obra Materiais Manutenção], permitem interpretar as possíveis providências para a diminuição dos contrastes entre o Projeto de Arquitetura, o Programa de Necessidades e o Produto Obra. / This dissertation examines the technical performance of all buildings of the Housing Group \"B-10\", listed as the main subject in the study of case of this research, within the logic of housing construction of social concern and proposes a reflection on the technical and managerial conditioners that influence on the development of the architecture project oriented to its ideal performance, with regard to the built environment, from the systemic view of comprehending the building. The survey is classified as exploratory and has the characteristic of deepening on issues involving the object examined herein. The choice is for the method A Study of a Case once it is considered a commonly used strategy of search in Social Sciences. Descriptions about Constructive Pathologies detected in all ten subsystem of the building are made, with indications of probable origins. Considerations about how the categorized reflections interfere in the items of performance are also prepared, guided by ISO 6241. The main results indicate that, from the evidences of diagnosed Constructive Pathologies, related in its origins with the responsible agents [Project - Execution of Work - Materials - Maintenance], it is possible to interpret probable provisions for the contrast reduction between the Architecture Project, the Program of Needs and the Final Product - The Work.

Enquanto as células humanas sintetizam purinas pela via de novo e pela via de recuperação, protozoários parasitas as sintetizam somente pela via de recuperação. Por essa razão, as enzimas que compõem essa via são importantes alvos para o desenvolvimento de novas drogas antiparasitárias. A enzima APRT converte adenina e &#945-D-5-fosforibosil 1-pirofosfato (PRPP) a AMP na via de recuperação de purinas. Nesse trabalho, a APRT e a APRT-His recombinantes foram caracterizadas por métodos bioquímicos e espectroscópicos. As expressões do gene aprt contidos nos vetares pET29+ (Novagen) e pQE30 (Qiagen) renderam 5 e 10 mg.mL-1 de APRT e APRT-His, respectivamente, na forma solúvel. A APRT permaneceu estável e homogênea in vitro em Tris pH 7,5 contendo 5 mM de MgSO4 e 150 mM de KCl mas a APRT-His mostrou-se instável e insolúvel nesse pH e acima de 0,5 mg.mL-1. O estudo de solubilidade revelou que a APRT-His é parcialmente estabilizada em Tris pH 8,5 contendo 150 mM de KCl devendo ser purificada e mantida nesse tampão durante os ensaios espectroscópicos e a adição de 50 mM de histidina mostrou-se eficiente para a concentração da enzima até 8mg.mL-1. A caracterização bioquímica da APRT e da APRT-His revelou que elas são diméricas nos seus tampões e têm PI igual a 6,45 &#177 0,20 e 7,7 &#177 0,16, respectivamente. Os ensaios de atividade enzimática indicaram que a APRT é duas vezes mais ativa do que a APRT-His. Os espectros de CD da APRT-His foram mais intensos do que os espectros da APRT e mostraram perfil de hélice &#945 . Os resultados da desconvolução revelaram que a APRT-His tem cerca de 10% mais hélice-&#945 do que a APRT. O valor de teor de estrutura secundária da APRT equivale aos valores extraídos dos dados cristalográficos da APRT de L donovani e de L tarentolae. Os espectros de emissão de fluorescência mostraram que a APRT-His e a APRT possuem máximos de emissão em 342 e 332 nm, respectivamente. Além disso, eles indicaram que o PRRP e o AMP suprimem a fluorescência do Trp presente na APRT. A supressão foi relacionada à posição dos ligantes localizados no sítio ativo da enzima e a ausência de supressão nas amostras de APRT-His foi relacionada à presença de Mg2+. Os resultados indicam que a presença dos resíduos de histidina na região N-terminal da APRT-His induziu a modificação estrutural da enzima levando a precipitação contínua. Nesse sentido, a ausência dos resíduos de histidina incorporados à enzima favoreceu a estabilidade da proteína in vitro. / Human cells synthesize purine nucleotide by again and salvage pathways, while parasitic protozoa use only salvage pathways. For this reason, the enzymes that compound the salvage pathway are important targets to development of new antiparasitic drugs. The enzyme adenine phosphoribosyltransferase (APRT) converts adenine and &#945-D-5-phosphoribosyll-pyraphosphate (PRPP) to adenosine monophosphate (AMP) at salvage pathway. In this work, the APRT and APRT-His recombinants had been characterized by biochemical and spectroscopic methods. The expression of the aprt genes from L. tarentolae inserted into pET29+ (Novagen) and pQ30 (Qiagen) vectors yielded 5 and 10 mg.mL-1 of the APRT and APRT-His, on soluble form, respectively. The APRT remained stable and homogeneous in vitro at Tris pH 7.5 containing 5 mM MgSO4 and 150 mM KCl, but APRT-His was instable and insoluble above 0.5 mg.mL-1 at the same pH. The solubility study showed that histidine increased the APRT-His solubility and it is partially stabilized at Tris pH 8.5 containing 150 mM KCl. The addition of the histidine 50 mM was efficient for concentrations up to 8 mg.mL-1.Then, the APRT-His was purified and storage in that buffer for spectroscopic assays. The biochemical characterization of the APRT and APRT-His indicated that a both are dimercs in its buffers, and they have isoelectric points at pH 6.45 &#177 0.20 and 7.7 &#177 0.16, respectively. By enzymatic activity assays, the APRT is twice activer than APRT-His. The CD spectra of the APRT-His were more intense than the APRT spectra. and showed helix &#945 profile. The fluorescence spectra marked a maximum emission fluorescence at 342 nm for the APRT-His and 332 um for the APRT. In addition, the spectra revealed that PRPP and AMP quenched the fluorescence of the tryptophan (Trp) into APRT. The quench was related to position of the ligands inside active site of the enzyme and the absent of fluorescence of the Trp, inside APRT-His, was related to absent of the Mg2+. The results has demonstrated that the presence of the histidine residues at N-terminal region of the APRT-His induced to conformational changes of the enzyme following to continuos precipitation. In the same sense, the absent of histidine residues associated to enzyme favored to stability of the protein in vitro.

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.

A presente dissertação analisa o desempenho técnico das edificações do conjunto habitacional B-10, arrolado no objeto do estudo de caso desta pesquisa, dentro da lógica da construção habitacional de interesse social e propõe uma reflexão sobre as condicionantes técnicas e gerenciais que influenciam o desenvolvimento do projeto de arquitetura orientado ao seu desempenho ideal, em relação ao ambiente construído, a partir da visão sistêmica de compreensão do edifício. A pesquisa classifica-se como exploratória e possui o caráter de aprofundamento nas questões que envolvem o objeto analisado. Optou-se pelo método Estudo de Caso, por este ser considerado uma estratégia de pesquisa comumente usada em Ciências Sociais. São feitas descrições das Patologias Construtivas detectadas nos dez subsistemas do edifício, com indicações das prováveis origens. Também são elaboradas considerações sobre como os reflexos categorizados interferem nos itens de desempenho, balizados pela ISO 6241. Os principais resultados obtidos demonstram que a partir das evidências de Patologias Construtivas diagnosticadas, relacionadas nas suas origens junto aos Responsáveis [Projeto Execução de Obra Materiais Manutenção], permitem interpretar as possíveis providências para a diminuição dos contrastes entre o Projeto de Arquitetura, o Programa de Necessidades e o Produto Obra. / This dissertation examines the technical performance of all buildings of the Housing Group \"B-10\", listed as the main subject in the study of case of this research, within the logic of housing construction of social concern and proposes a reflection on the technical and managerial conditioners that influence on the development of the architecture project oriented to its ideal performance, with regard to the built environment, from the systemic view of comprehending the building. The survey is classified as exploratory and has the characteristic of deepening on issues involving the object examined herein. The choice is for the method A Study of a Case once it is considered a commonly used strategy of search in Social Sciences. Descriptions about Constructive Pathologies detected in all ten subsystem of the building are made, with indications of probable origins. Considerations about how the categorized reflections interfere in the items of performance are also prepared, guided by ISO 6241. The main results indicate that, from the evidences of diagnosed Constructive Pathologies, related in its origins with the responsible agents [Project - Execution of Work - Materials - Maintenance], it is possible to interpret probable provisions for the contrast reduction between the Architecture Project, the Program of Needs and the Final Product - The Work.

<p>Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.</p>

<p>Abstract</p><p>Crimean-Congo Hemorrhagic Fever virus (CCHF) and Lassa virus are giving sources illness to humans. In addition to zoonotic transmission, CCHF and Lassa virus can spread from person to person. After a short incubation period, CCHF and Lassa virus infections are characterized by a sudden onset of high fever, chills, headache and cough just like flu. Even some people are vomiting and have diarrhoea. After a few days of illness hemorrhagic manifestations occur. Treatment options for CCHF and Lassa viruses are limited, and there is no vaccine available for use in humans. The purpose of the present study was to produce recombinant nucleocapsid protein of Lassavirus and CCHF virus including an aminoterminal His-tag by a Semliki Forest Virus Replicon (pSFV 4.2). The recombinant proteins are planned to be used in future development of diagnostic methods.</p>

Several plant defense systems consist of enzymes that act on glucosides and produce a toxic compound. In the intact plant tissue the substrate and enzyme are kept apart. The system studied here consists of the substrate 2-O-β-D-glucopyranosyl-4-dihydroxy-1,4-benzoxazin-3-one and the enzyme glucan 1,3-β-glucosidase in rye. The aim was to determine the properties of a cell wall β-glucosidase. Two different systems for expression and purification of β-glucosidase fused to a tag were used: a 6xHistidine tag system and a thioredoxin tag system. The sequence of the β-glucosidase had previously been determined so now the gene was subcloned into E.coli. A direct PCR on colonies, a test expression, a restriction digestion of plasmids and sequencing was made to analyze the transformation, which all turned out successful. Then the β-glucosidase solubility was determined. Finally a purification of the β-glucosidase from E.coli under native conditions and a pNPG assay was carried out. For the (His)6-tagged protein, the recombinant β-glucosidase tended to end up in the insoluble pelleted fraction which indicated formation of inclusion bodies. The cell wall 1,3-β-glucosidase was soluble with the thioredoxin system, but the percentage of soluble protein fraction was around 5% only of the total protein. In eluates from a nickel-nitrilotriacetic acid column the presence of recombinant protein was confirmed with Western blot, but contaminating bands were also present. Purified elauted fractions did not exhibit detectable β-glucosidase activity. It was not possible to purify active enzyme. From a BLAST search it was clear that the most similar enzymes all had putative glycosylation sites and lack of glycosylation could be a reason for the protein not to fold properly.