Esta tese tem como objetivos identificar e analisar princípios pedagógicos dos processos de trans-formação do ator que os grupos Abya Yala (Costa Rica), Yuyachkani (Peru) e Ói Nóis Aqui Traveiz (Brasil) desenvolveram ao longo de sua história e problematizar como eles poderiam ser de grande valia na formação atoral em diversas instâncias. Partiu-se principalmente de entrevistas e visitas realizadas aos grupos, assim como do estudo de material escrito e audiovisual referente ao tema. O teatro é uma construção cultural que muda de acordo com a época, o local e contexto em que se desenvolve. Do mesmo modo a formação atoral responde a essas variáveis. Atrelada às práticas cênicas dos grupos se constrói uma pedagogia para a trans-formação. Foram identificados alguns pontos em comum entre eles como o engajamento tanto no que diz respeito à vida política do país como o que se refere ao treinamento do ator. Há também nos três a busca pela autonomia do ator como criador, responsável por suas escolhas e pelo seu papel dentro do grupo. Existe neles ainda a ênfase em compartilhar sua experiência, incentiva-se os atores a serem facilitadores de processos fora ou dentro do coletivo. Esta pesquisa trouxe à tona a importância de manter contato com o fazer teatral latino-americano, revelando uma riqueza na diversidade de propostas trans-formativas e de como elas extrapolam o ambiente do grupo, podendo ser tomadas como exemplo para outros processos de formação atoral. / This thesis aims to identify and analyze the pedagogical principles of the processes of trans-formation of the actor that Abya Yala (Costa Rica), Yuyachkani (Peru) and Ói Nóis Aqui Traveiz (Brazil) developed throughout its history and discuss how they could be of great value in the formation of actors in several instances. This thesis is based mainly on interviews and visits to the groups, as well as the study of written and audio-visual material on the topic. Drama is a cultural construction that changes according to time, place and context in which it develops. Similarly the formation of actors answers to those same variables. Coupled to the group´s practices on scene it is build a pedagogy for trans-formation. We identified some common ground between them as they engage both politically, with the country´s situation, and regarding the training of the actors. All three groups demonstrate a quest for the actors independence as creators, responsible for their choices and their role within the group. They also reveal their emphasis on sharing their experience, as they encourage the actors to be facilitators of processes both inside and outside of the collective. This research has brought to light the importance of maintaining contact with the Latin American way of doing theatre, revealing a wealth of diversity of the proposed trans-formative processes that go beyond the group environment, and how they can be taken as an example to other actor-forming processes.

Os ácidos graxos (AGs) trans foram largamente utilizados pela indústria de alimentos. Entretanto, seu consumo tem sido associado a problemas de saúde, principalmente ao aumento do risco para doenças coronarianas. No Brasil, desde 2006, o conteúdo de gorduras trans obrigatoriamente precisa ser informado nos rótulos dos alimentos industrializados. Aparentemente, isso fez surgir diversos produtos que declaram não conter gorduras trans e que tradicionalmente os continham em grandes concentrações. Dessa forma, o objetivo desse trabalho foi avaliar a oferta atual desses alimentos. Para tanto, a proporção de alimentos que declaravam conter 0g de gordura trans foi avaliada, verificando variações conforme a localização do estabelecimento de venda dentro da cidade de São Paulo. Também foi realizada a comparação da composição declarada e preço dos alimentos que declaram conter 0g de AGs trans com os demais. Além disso, foram analisados alguns produtos para verificar se a informação com relação ao conteúdo de AG é confiável e se está em conformidade com a legislação. A pesquisa foi realizada nas categorias biscoito doce simples, biscoito recheado, wafer, cream cracker e biscoito de polvilho através de pesquisa em supermercados. A quantificação dos AG nas amostras foi realizada por cromatografia gasosa e os resultados foram comparados com as informações contidas nos rótulos. Dos 498 diferentes produtos encontrados nos supermercados visitados, 68,9 por cento foram de alimentos que informavam não conter gorduras trans. Não foram observadas diferenças na oferta entre as regiões da cidade, entretanto, os produtos que informavam não conter gorduras trans possuem um preço mais elevado que os demais (p<0,001). Nove produtos foram analisados e a quantidade média de gordura trans por porção encontrada foi de 0,18 (±0,29). Dentre os produtos analisados, dois deles não poderiam declarar não conter gordura trans, pois continham mais do que 0,2 g por porção, considerando-se a variação permitida pela legislação de ±20 por cento. Os resultados sugerem que a obrigatoriedade em informar a quantidade de gordura trans nos rótulos proporcionou uma oferta ampla e bem distribuída de alimentos afirmam não conter esse tipo de gordura, no entanto, tais alimentos são menos acessíveis por possuírem um preço mais elevado. Além disso, com nem sempre as informações disponíveis nos rótulos são confiáveis, existe a necessidade de maior fiscalização por parte do poder público / Trans fatty acids (FA) were extensively used by food industry. However, the consumption of this type of FA has been associated with health problems, especially with increased risk for heart diseases. Since July, 2006, Brazilian regulation has imposed that industrialized food labels must express trans fat content. This apparently contributed to several products known for having large amounts of trans fat which now declare does not contain trans fat. Thus, the purpose of this study is to examine the supply of food products that claim to be trans fat-free, checking possible variations according to the sales spots, and comparing their composition and price to those of other food products in order to verify if such claims are reliable and comply with the law. The supply of the following products was evaluated supermarkets of Sao Paulo: sweet biscuit, sandwich biscuit, wafer, cream cracker and tapioca flour biscuit. The comparison between the products was based on the information on the labels. The quantification of trans FA in some of these foods was carried by gas chromatography and the results were compared with the information from labels. Were found 498 different products in the six supermarkets visited and 68.9 per cent of them were products that declare the absence of trans FA. There were no differences in terms of supply among regions of the city. However, the products that claim to be trans fat-free had a higher price than the others in all categories (p <0.001). No increase in the amount of saturated fat was found. In the nine products analyzed by gas chromatography the average amount of trans fat per serving was 0.18 (±0.29). Two of the products tested should not claim to be trans fat-free because they contained more than 0.2 g per serving, taking into account the acceptable variation of ±20 per cent. The results suggest that the mandatory declaration of trans fat content on labels led to a wide and well distributed supply of products that declare do not contain this kind of fat. However, these products are less accessible because they are more expensive. Furthermore, available information on the labels is not always reliable, which indicates the need to supervise such information

Made available in DSpace on 2017-07-21T14:13:04Z (GMT). No. of bitstreams: 1 Bruna Carletto.pdf: 3521002 bytes, checksum: e937254e660e6d236bdba8d92c4b247c (MD5) Previous issue date: 2015-02-23 / Fundação Araucária de Apoio ao Desenvolvimento Científico e Tecnológico do Paraná / Melanoma is a very lethal skin tumor and, when widespread, presents unfavorable prognosis. Therefore, new drugs and treatments are important to the search for more promising results. Treatment of this type of tumor surgery necessarily depend on having little effect on the supplementary chemotherapy. Studies have shown resveratrol as a drug that acts in different stages of carcinogenesis. Allied to this, modified release systems of drugs are being studied to improve unwanted features and optimize their actions. The aim of this study was to develop polymeric nanocapsules containing resveratrol and assess their potential antitumor in B16F10 melanoma strain. The nanocapsules of poly -caprolactone) (PCL) containing resveratrol were successfully prepared by interfacial deposition of preformedpolymer. All formulations showed encapsulation efficiency values suitable above 97%. Nanocapsules revealed spherical shape with smooth surface. Infrared Fourier transform spectra indicated no chemical interaction between the polymer and resveratrol after the nanoencapsulation. The analysis of X-ray diffraction showed that the process of nanoencapsulation led to an amorphisation of the system. Cell viability assays for neutral red and MTT confirmed that resveratrol-containing nanocapsules have cytotoxic effect on murine melanoma cells at concentrations of 300 and 100 micrometers. When analyzed the morphology of the cells treated with resveratrol free, it can be seen that they enter into the process of cell death by apoptosis. In the in vivo assay, the results demonstrated that treatment with resveratrol-containing nanocapsules led to a reduction in tumor volume of the mice in the groups treated with resveratrol and free controls. Treated animals also showed an increase in body mass suggesting that these animals had a better response to treatment compared to the control group. Histological analysis revealed an increase in necrotic area in the tumor and inflammation in the animals treated with the nanocapsules containing resveratrol and the treatment also led to the development of non-pulmonary hemorrhage and metastasis. These results indicate that the nanocapsules prepared from the PCL containing resveratrol have antiproliferative action front melanoma cells, with an interesting alternative in their treatment. / O melanoma é um tumor de pele muito letal e, quando disseminado, apresenta prognóstico pouco favorável. Por isso, novos fármacos e tratamentos são importantes para a busca de resultados mais promissores. O tratamento deste tipo de tumor depende necessariamente de intervenção cirúrgica tendo pouco resultado com a quimioterapia complementar. Estudos evidenciaram o resveratrol como um fármaco que atua em diversas etapas da carcinogênese. Aliado a isso, os sistemas de liberação modificada de fármacos estão sendo estudados para melhorar características indesejadas e otimizar suas ações. O principal objetivo deste trabalho foi desenvolver nanocápsulas poliméricas contendo resveratrol e avaliar o seu potencial antitumoral na linhagem de melanoma B16F10. As nanocápsulas de poli (ε-caprolactona) (PCL) contendo resveratrol foram preparadas com sucesso pelo método de deposição interfacial de polímero pré-formado. Todas as formulações apresentaram valores de eficiência de encapsulação adequados, superiores a 97%. As nanocápsulas revelaram formato esférico com superfície lisa. Os espectros de infravermelho com transformada de Fourier não indicaram nenhuma interação química entre o resveratrol e o polímero após a nanoencapsulação. As análises de difração de raios X demonstraram que o processo de nanoencapsulação levou a uma amorfização do sistema. Os ensaios de viabilidade celular por MTT e vermelho neutro confirmaram que as nanocápsulas contendo resveratrol possuem efeito citotóxico sobre as células de melanoma murino nas concentrações de 300 e 100 μM. Quando analisada a morfologia das células tratadas com resveratrol livre, pode-se observar que as mesmas entram em processo de morte celular por apoptose. No ensaio in vivo, os resultados demonstraram que o tratamento com as nanocápsulas contendo resveratrol levou a uma redução do volume tumoral dos camundongos em relação aos grupos tratados com resveratrol livre e os controles. Os animais tratados também apresentaram um aumento da massa corporal sugerindo que estes animais tiveram uma melhor resposta ao tratamento quando comparado ao grupo controle. A análise histológica revelou um aumento da área necrótica e da inflamação no tumor nos animais tratados com as nanocápsulas contendo resveratrol, e o tratamento também levou ao não desenvolvimento de hemorragia pulmonar e metástase. Esses resultados indicam que as nanocápsulas elaboradas a partir da PCL contendo resveratrol possuem ação antiproliferativa frente as células de melanoma, sendo uma alternativa interessante no seu tratamento.

trans-Sialidase de Trypanosoma cruzi (TcTS) pertence à família de glicoproteínas de superfície do parasita e constitui um dos poucos exemplos naturais de glicosiltransferases superficiais encontradas em eucariotes. T. cruzi é incapaz de sintetizar ácido siálico e utiliza esta enzima para retirar este monossacarídeo de glicoconjugados do hospedeiro para sialilar moléculas aceptoras, como mucina-GPI (glicosilfosfatidilinositol), presentes na sua membrana plasmática. Esta enzima é específica em catalisar, preferencialmente, a transferência de ácido siálico para moléculas de mucina, originando ligações -2,3 com moléculas de galactose aceptoras na superfície do parasita. Considerando a heterogeneidade das moléculas de mucina de T. cruzi, é necessário que novas moléculas sejam sintetizadas a fim de que estas atuem como substratos glicopeptídicos, os quais podem levar ao melhor entendimento das interações entre enzima e substratos e permitir o planejamento racional de inibidores seletivos. Por isso, o trabalho foi divido em três rotas sintéticas: (i) preparação do doador de galactose, (ii) preparação dos aceptores-doadores e (iii) acoplamento dos dissacarídeos com aminoácidos aceptores para obtenção dos blocos de construção. Apesar dos objetivos propostos inicialmente não terem sido totalmente alcançados, o trabalho desenvolvido durante esse período permitiu a síntese do doador de galactose (3) em três etapas, aceptor de galactose (6) em cinco etapas, dissacarídeo (11) na glicosilação de 6 com 3, aminoácidos aceptores (13 e 14) e também dos blocos de construção (17 e 18) decorrente do acoplamento de 11 com os aminoácidos aceptores. Não obstante, é importante ressaltar que apesar da extensa rota planejada, porém necessária, a síntese dos blocos de construção é inédita. Portanto, pode-se concluir que o trabalho trouxe relevante contribuição no que diz respeito à química de carboidratos e à disponibilização de dados espectrométricos de compostos orgânicos para a literatura. / Trypanosoma cruzi trans-sialidase (TcTS) belongs to the family of glycoproteins expressed on the surface of the parasite and constitutes one of the few examples of natural surface glycosyltransferases found in eucariotes. T. cruzi can not synthesize sialic acid itself and uses this enzyme to scavenge this monosaccharide from host glycoconjugates to sialylate acceptors molecules, such as GPI (glycosylphosphatidylinositol) mucins, that are present in parasite plasma membrane. This enzyme is specific to catalyze, preferentially, the transference of sialic acid to mucin glycoproteins, generating -2,3-linkages with acceptor galactose molecules in the parasite surface. Considering the heterogeneity of T. cruzi mucin molecules, its necessary to synthesize new compounds that can act as glycopeptide substrates, leading to a better understanding concerning the enzyme and substrates and allow the rational design of some selective inhibitors. Thus, this work was developed in three synthetic routes: (i) the synthesis of galactose donor, (ii) synthesis of donor-acceptors and (iii) coupling between disaccharides and acceptors amino acids in order to obtain building blocks. Despite of some objectives initially proposed had not been accomplished, the developed work during this period allow the synthesis of the galactose donor (3) in three steps, donor-acceptor (6) in five steps, disaccharide (11), acceptors amino acids (13 and 14) and also the building blocks (17 and 18). However, its important highlight that the synthesis of the building blocks by this necessary, but extensive, synthetic route is unpublished. Therefore, it can be concluded that the present work brought rich contribution concerning the carbohydrate chemistry and the availability of spectrometric data of organic compounds to the literature.

by Cheung Yuk Yin. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2002. / Includes bibliographical references (leaves xx-xxviii). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 摘要 --- p.iii / Table of Content --- p.iv / Abbreviations / for Amino Acids --- p.viii / for Standard Genetic Code --- p.ix / for Units --- p.x / for Prefixes --- p.xi / for Terms commonly used in the report --- p.xii / List of Figures --- p.xiii / List of Tables --- p.xiv / Chapter Chapter 1 --- Introduction / Chapter 1.1 --- Epidemiology of Hepatitis B Virus (HBV) --- p.1 / Chapter 1.2 --- Relationship between Hepatitis B Virus and Hepatocellular Carcinoma --- p.2 / Chapter 1.3 --- Brief Description of HBV Genome --- p.2 / Chapter 1.4 --- Possible Roles of HBx in Hepatocellular Carcinoma --- p.4 / Chapter 1.5 --- Novel Interacting Partner of HBx - HBx-lnteracting Protein (XIP) --- p.6 / Chapter 1.6 --- Objective --- p.6 / Chapter Chapter 2 --- Methodology / Chapter 2.1 --- Information of the HBx and XIP Clones --- p.7 / Chapter 2.2 --- "Information of the Expression Vectors (pRSETA, 6xHis-pRSETA and pET8C)" --- p.7 / Chapter 2.3 --- Sub-Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.1 --- Design of Primers for Cloning of HBx and XIP into Different Vectors --- p.9 / Chapter 2.3.2 --- Polymerase Chain Reaction (PCR) Protocol --- p.12 / Chapter 2.3.3 --- Enzyme Digestion Reaction Protocol --- p.14 / Chapter 2.3.4 --- Ligation Protocol --- p.16 / Chapter 2.3.5 --- Preparation of Competent Cells --- p.17 / Chapter 2.3.6 --- Transformation --- p.18 / Chapter 2.3.7 --- Gel Extraction Protocol --- p.19 / Chapter 2.3.7.1 --- Life Technologies CONCERT´ёØ Rapid Gel Extraction System --- p.19 / Chapter 2.3.7.2 --- QIAGEN Gel Extraction Kit --- p.20 / Chapter 2.3.8 --- Plasmid Preparation Protocol --- p.22 / Chapter 2.3.8.1 --- Life Technologies CONCERT´ёØ Rapid Plasmid Minipreps --- p.22 / Chapter 2.3.8.2 --- QIAGEN Plasmid Maxi Kit --- p.23 / Chapter 2.4 --- Expression of HBx and XIP in E. coli Strain C41 (DE3) --- p.25 / Chapter 2.4.1 --- Transformation --- p.25 / Chapter 2.4.2 --- Expression of HBx and 6xHis-HBx in E. coli Strain C41 (DE3) --- p.26 / Chapter 2.4.3 --- Expression of XIP in E. coli Strain C41 (DE3) --- p.27 / Chapter 2.5 --- Preparation of Buffers for Chromatography and Circular Dichroism Spectrum Measurement --- p.28 / Chapter 2.6 --- Purification and Refolding of HBx and His-Tagged HBx --- p.28 / Chapter 2.6.1 --- Washing of HBx and His-Tagged HBx Inclusion Bodies --- p.28 / Chapter 2.6.2 --- His-Tagged HBx Purification by Affinity Chromatography --- p.29 / Chapter 2.6.3 --- HBx Purification by Size Exclusion Chromatography --- p.30 / Chapter 2.6.4 --- Refolding of HBx and His-Tagged HBx by Oxidative Dialysis --- p.30 / Chapter 2.7 --- Purification of XIP --- p.33 / Chapter 2.7.1 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.33 / Chapter 2.7.2 --- XIP 1st Step of Purification by Hydrophobic Interaction Chromatography --- p.34 / Chapter 2.7.3 --- XIP 2nd step of Purification by Size Exclusion Chromatography --- p.34 / Chapter 2.8 --- Chemical Denaturation Experiment of HBx and XIP --- p.36 / Chapter 2.8.1 --- Preparation of Urea Buffers for the Chemical Denaturation of HBx --- p.37 / Chapter 2.8.2 --- Preparation of Different GdnHCI Buffer for the Chemical Denaturation of XIP --- p.38 / Chapter 2.8.3 --- Calculation for Chemical Denaturation Experiment --- p.39 / Chapter 2.8.3.1 --- Protein Concentration Calculation --- p.39 / Chapter 2.8.3.2 --- Residual Molar Elipticity Calculation --- p.39 / Chapter 2.8.3.3 --- Free Energy Change (ΔGu) Calculation --- p.40 / Chapter 2.9 --- Two-dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Experiment --- p.41 / Chapter 2.10 --- Interaction Confirmation between HBx and XIP --- p.42 / Chapter 2.10.1 --- "Transfection of pEGFP, pEGFP-HBx and pEGFP-XIP into HepG2" --- p.42 / Chapter 2.10.2 --- Yeast Two Hybrid System for Confirmation of HBx and XIP Interaction --- p.44 / Chapter 2.10.2.1 --- Preparation of Y187 Competent Cells --- p.44 / Chapter 2.10.2.2 --- Transformation of pGBKT7-HBx and pACT2-XIP into Y187 --- p.45 / Chapter 2.10.2.3 --- β-galactosidase Colony Lift Assay --- p.46 / Chapter Chapter 3 --- "Expression, Purification and Characterization of Hepatitis B Virus X Protein (HBx)" / Chapter 3.1 --- Introduction --- p.47 / Chapter 3.2 --- Construction of Recombinant HBx-pRSETA and 6xHis-HBx-pRSETA Plasmids --- p.48 / Chapter 3.3 --- Expression of 6xHis-HBx in E. coli C41 (DE3) using M9ZB Medium --- p.52 / Chapter 3.4 --- Expression of HBx in E. coli C41 (DE3) using M9ZB Medium --- p.54 / Chapter 3.5 --- Purification and Refolding of 6xHis-HBx Fusion Proteins --- p.56 / Chapter 3.6 --- Purification and Refolding of HBx Proteins --- p.60 / Chapter 3.7 --- Structural Characterization of Refolded HBx --- p.65 / Chapter 3.7.1 --- Introduction --- p.55 / Chapter 3.7.2 --- Experimental Analysis of HBx Secondary Structure --- p.66 / Chapter 3.7.3 --- Chemical Unfolding Experiment of HBx --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 3.8.1 --- "HBx was Expressed, Purified and Characterized instead of 6xHis-HBx" --- p.71 / Chapter 3.8.2 --- High Concentration of DTT was used to Minimize Formation of HBx Aggregates --- p.72 / Chapter 3.8.3 --- Oxidative Refolding to Ensure Proper Disulfide Bond Formation --- p.73 / Chapter 3.8.4 --- Computational Prediction and Experimental Prediction of Secondary Structure of HBx --- p.75 / Chapter 3.9 --- Concluding Remarks --- p.77 / Chapter Chapter 4 --- "Expression, Purification and Characterization of HBx-lnteracting Protein (XIP)" / Chapter 4.1 --- Introduction --- p.78 / Chapter 4.2 --- Construction of Recombinant XIP-pET8C --- p.78 / Chapter 4.3 --- Expression of XIP in E. coli C41 (DE3) using M9ZB and M9 Mediums --- p.82 / Chapter 4.4 --- Screening of Chromatographic Conditions for the Purification of XIP --- p.83 / Chapter 4.4.1 --- Introduction --- p.83 / Chapter 4.4.2 --- Purification Details --- p.83 / Chapter 4.5 --- Purification of XIP by HiTrap Phenyl HP 5-ml Column --- p.87 / Chapter 4.6 --- Purification of XIP by HiLoad 26/60 Superdex 75 Prep Grade --- p.89 / Chapter 4.7 --- Structural Characterization of XIP --- p.92 / Chapter 4.7.1 --- CD Spectrum --- p.92 / Chapter 4.7.2 --- Chemical Denaturation Experiment of XIP --- p.93 / Chapter 4.7.3 --- Two-Dimensional Heteronuclear Nuclear Magnetic Resonance (NMR) Spectrum of 15N Labeled XIP --- p.95 / Chapter 4.8 --- Discussion --- p.97 / Chapter 4.8.1 --- Purification Method Development --- p.97 / Chapter 4.8.2 --- "Do Different Protein Cosolutes, Protein Stabilizers and Detergents Help XIP to Adopt a Stable Conformation?" --- p.99 / Chapter 4.9 --- Concluding Remarks --- p.101 / Chapter Chapter 5 --- In vivo Studies of HBx and XIP Interactions / Chapter 5.1 --- Investigation of Sub-Cellular Localization of HBx and XIP in Liver Cells --- p.102 / Chapter 5.1.1 --- Introduction --- p.102 / Chapter 5.1.2 --- "Construction of Recombinant HBx-pECFP-C1, HBx-pEGFP-C1, HBx-pEYFP-C1 and XIP-pECFP-C1, XIP-pEGFP-C1, XIP-pEYFP-C1" --- p.103 / Chapter 5.1.3 --- Transfection of pEGFP-C1 HBx and pEGFP-C1 XIP into HepG2 to Find Out HBx and XIP Sub-Cellular Localization --- p.106 / Chapter 5.1.3.1 --- Introduction --- p.107 / Chapter 5.1.3.2 --- Investigation of EGFP Proteins Expression using the Confocal Microscope and the Leica TCS Software --- p.108 / Chapter 5.1.4 --- Discussion and Future Prospects --- p.111 / Chapter 5.2 --- Interaction of HBx and XIP Studied by Yeast Two-Hybrid System --- p.113 / Chapter 5.2.1 --- Introduction --- p.113 / Chapter 5.2.2 --- Construction of Recombinant HBx-pGBKT7 and XIP-pACT2 Plasmids --- p.114 / Chapter 5.2.3 --- Confirmation of HBx and XIP Interaction by Yeast Two-Hybrid System --- p.117 / Chapter 5.2.4 --- Discussion --- p.121 / Chapter Chapter 6 --- Conclusion --- p.123 / Appendix I Sequence of HBx and XIP --- p.I / Chapter II --- Vector Sequences --- p.II / Chapter III --- Vector Maps --- p.VI / Chapter IV --- Electrophoresis Markers --- p.XI / Chapter V --- Agarose Gel Electrophoresis --- p.XII / Chapter VI --- SDS-PAGE Eectrophoresis --- p.XIII / Chapter VII --- Medium for Bacterial Culture --- p.XV / Chapter VIII --- Medium for Cell Culture --- p.XVII / Chapter IX --- Medium for Yeast Culture --- p.XVIII / Chapter X --- Buffers for Yeast Transformation --- p.XIX / Reference --- p.XX

La polarité cellulaire est une caractéristique commune à tous les organismes. Jusqu’à récemment, il était assumé que la sécrétion de protéines vers des domaines polaires de la cellule végétale se faisait de façon non polarisée, mais ce point de vue a été re-étudié, la sécrétion est polarisée mais la dynamique, les voies de traficempruntées et les mécanismes sont toujours inconnus. Précédemment, mon laboratoire d’accueil a caractérisé un enrichissement en sphingolipides contenant des acides gras à très longues chaines (VLCFAs) au niveau d’un sous-domaine du trans-Golgi Network (TGN) appelé Vésicules de Sécrétions (SVs). Plus précisément, il a été montré que la longueur des acides gras des sphingolipides jouait un rôle critique dans la sécrétion du transporteur d’auxine PIN2 des SVs vers des domaines polaires de la membrane plasmique. Pendant ma thèse, je me suis intéressé à la question suivante : comment les sphingolipides agissent-t-ils au TGN? En identifiant le protéome des SVs, ainsi qu'en utilisant des outils génétiques et pharmacologiques en combinaison avec la visualisation de marqueurs lipidiques, j'ai pu identifier que les sphingolipides agissent sur l’homéostasie des phosphoinositides en mettant en avant un lien fonctionnel entre ces deux classes de lipides au sein de la cellule végétale. En utilisant un set de marqueurs des phosphoinositides (PIPs), j’ai pu montrer que les sphingolipides ciblent principalement le phosphatidyl-inositol-3-phosphate, PI(3)P et le phosphatidylinositol- 4-phosphate, PI(4)P. De plus, mon analyse protéomique a montré que la localisation d'un ensemble de protéines liées aux PIPs était diminuée dans les SVs/TGN immunopurifiées quand la composition des sphingolipides est altérée. Mes résultats nous forcent à revoir notre vision de la dynamique des lipides au niveau des membranes, et suggère l’idée que la dynamique de remodelage de la composition d’une classe de lipide, les phosphoinositides, peut être modulée par une autre classe de lipide, les sphingolipides. / Cell polarity is a defining feature of all organisms. Until very recently, it was thought that delivery of proteins to polar domains of root epidermal cells plasma membrane was non-polar, but this view has been re-examined, the delivery is polar but the dynamics, the paths taken, and the mechanisms are unknown. My host team previously characterised an enrichment of Very-Long-Chain-Fatty-Acids (VLCFAs)-containing sphingolipids at the site of secretory vesicles (SVs) sub-domain of the trans-Golgi Network (TGN). Moreover, the length of sphingolipids acyl-chain was found to play a critical role in secretory sorting of the auxin carrier PIN2 from SVsassociated TGN to apical polar domain of the plasma membrane (PM). During my PhD, I addressed the following question: how sphingolipids act at SVs/TGN? Using proteomics of SVs, genetics and pharmacological tools in combination with visualisation of lipid probes we could identify that sphingolipids act on phosphoinositides (PIPs) homeostasis establishing a new functional link between these two lipids in plant cells. Using a set of multi-affinity fluorescent PIPs probes I could show that sphingolipids target phosphatidylinositol-3-phosphate (PI3P) and phosphatidylinositol-4-phosphate (PI4P). Moreover, my proteomic analyses show that several PIPs-related proteins are downregulated in immuno-purified TGN-associated SVs when the sphingolipid composition is altered pharmacologically. My results force the reassessment of our view of lipid membranes dynamics and highlight the idea that dynamic remodelling of the composition of one lipid class, the phosphoinositides, can be modulated by another lipid class, the sphingolipids.

Esta tese tem como objetivos identificar e analisar princípios pedagógicos dos processos de trans-formação do ator que os grupos Abya Yala (Costa Rica), Yuyachkani (Peru) e Ói Nóis Aqui Traveiz (Brasil) desenvolveram ao longo de sua história e problematizar como eles poderiam ser de grande valia na formação atoral em diversas instâncias. Partiu-se principalmente de entrevistas e visitas realizadas aos grupos, assim como do estudo de material escrito e audiovisual referente ao tema. O teatro é uma construção cultural que muda de acordo com a época, o local e contexto em que se desenvolve. Do mesmo modo a formação atoral responde a essas variáveis. Atrelada às práticas cênicas dos grupos se constrói uma pedagogia para a trans-formação. Foram identificados alguns pontos em comum entre eles como o engajamento tanto no que diz respeito à vida política do país como o que se refere ao treinamento do ator. Há também nos três a busca pela autonomia do ator como criador, responsável por suas escolhas e pelo seu papel dentro do grupo. Existe neles ainda a ênfase em compartilhar sua experiência, incentiva-se os atores a serem facilitadores de processos fora ou dentro do coletivo. Esta pesquisa trouxe à tona a importância de manter contato com o fazer teatral latino-americano, revelando uma riqueza na diversidade de propostas trans-formativas e de como elas extrapolam o ambiente do grupo, podendo ser tomadas como exemplo para outros processos de formação atoral. / This thesis aims to identify and analyze the pedagogical principles of the processes of trans-formation of the actor that Abya Yala (Costa Rica), Yuyachkani (Peru) and Ói Nóis Aqui Traveiz (Brazil) developed throughout its history and discuss how they could be of great value in the formation of actors in several instances. This thesis is based mainly on interviews and visits to the groups, as well as the study of written and audio-visual material on the topic. Drama is a cultural construction that changes according to time, place and context in which it develops. Similarly the formation of actors answers to those same variables. Coupled to the group´s practices on scene it is build a pedagogy for trans-formation. We identified some common ground between them as they engage both politically, with the country´s situation, and regarding the training of the actors. All three groups demonstrate a quest for the actors independence as creators, responsible for their choices and their role within the group. They also reveal their emphasis on sharing their experience, as they encourage the actors to be facilitators of processes both inside and outside of the collective. This research has brought to light the importance of maintaining contact with the Latin American way of doing theatre, revealing a wealth of diversity of the proposed trans-formative processes that go beyond the group environment, and how they can be taken as an example to other actor-forming processes.

O trabalho teve por objetivo o desenvolvimento de bases gordurosas para margarinas utilizando o método de modificação por interesterificação, como alternativa ao processo de hidrogenação parcial, que forma isômeros trans. / The aim of the study was to develop soft margarines by using intertesterification, as an alternative of hydrogenation, which forms trans isomers.

Os ácidos graxos trans competem com os ácidos graxos essenciais inibindo as enzimas envolvidas na síntese dos ácidos graxos polinsaturados de cadeia longa. Quantidades adequadas de ácidos graxos essenciais na dieta minimizam este efeito. No entanto, pouco se conhece da ação dos isômeros trans, sob condições de restrição ou deficiência dietética dos ácidos graxos essenciais, sobre o percentual de deposição tanto dos próprios isômeros trans, como dos ácidos graxos polinsaturados nos lipídios dos tecidos. Foram avaliadas dietas ricas em ácidos graxos trans e com diferentes concentrações de ácidos graxos essenciais em ratos. A incorporação de ambos foi proporcional à concentração na dieta, mas variou entre os tecidos estudados. Verificou-se que as quantidades de ácidos graxos trans incorporadas pelo tecido adiposo foi a maior, enquanto que o cérebro incorporou a menor quantidade. A despeito da incorporação dos ácidos graxos trans, o perfil de ácidos graxos do cérebro manteve-se estável em relação às variações dietéticas. Não se observou acúmulo dos ácidos graxos trans em função do prolongamento do consumo da dieta. Os ácidos graxos essenciais incorporados aos tecidos também foram modulados pela dieta, e em concentrações adequadas influenciaram na menor deposição dos isômeros trans no tecido adiposo, plasma e tecido cardíaco. Avaliando-se a composição dos ácidos graxos nos fosfolipídios do tecido cardíaco, observou-se que a fosfatidiletanolamina incorporou a maior porcentagem dos isômeros trans seguida da fosfatidilcolina e da cardiolipina, respectivamente. Embora tenha incorporado quantidades intermediárias dos ácidos graxos trans a fosfatidilcolina teve uma maior alteração no perfil de ácidos graxos em comparação aos demais. A reduzida concentração de ácidos graxos trans incorporados na cardiolipina das mitocôndrias e no cérebro sugere a existência de um mecanismo protetor para a manutenção da composição lipídica necessária às atividades funcionais. / Trans fatty acids compete with essential fatty acids inhibiting the enzymes of the long chain polyunsaturated fatty acids synthesis. Appropriate amounts of dietary essential fatty acids minimize this effect. However, little is known about the action of trans isomers on the deposition percentage of these own trans isomers itself or as polyunsaturated fatty acids in tissue lipids, when under dietary restriction or deficiency conditions of essential fatty acids. Diets high in trans fatty acids and with different concentrations of essential fatty acids were assessed. The incorporation of trans isomers and of essential fatty acids was proportional to their dietary concentration, but varied among the tissues studied. It was verified that the adipose tissue incorporated the largest amount of trans fatty acids while the brain incorporated the smallest. In spite of the trans fatty acids incorporation, the profile of brain fatty acids was maintained stable in relation to dietary variations. No accumulative deposition of trans fatty acids was observed in relation to extended length of time of dietary consumption Essential fatty acids incorporated in tissues were also modulated by diet and in moderate concentrations influenced the lower deposition of trans isomers in adipose tissue, blood plasma and heart. By assessing fatty acids composition in phospholipids of heart, it was verified that phosphatidylethanolamine incorporated the largest percentage of trans isomers followed by phosphatidylcholine and cardiolipin, respectively. Phosphatidylcholine presented the greatest change in fatty acids profile when compared to the others phospholipids, although incorporating intermediate amounts of trans isomers. The reduced concentration of trans fatty acids incorporated in cardiolipin and brain suggests that there is a protector mechanism for maintenance of the lipids composition required for functional activities.

A infecção por HIV-1 leva a uma séria imunodeficiência causada principalmente pela depleção de linfócitos T auxiliadores, a principal célula-alvo do vírus. Além dos linfócitos T CD4, o HIV-1 também pode interagir e infectar macrófagos e células dendríticas (DCs). As DCs são resistentes à infecção pelo HIV-1, mas podem internalizar vírions em compartimentos e transferi-los para linfócitos T CD4&#43, em um processo chamado trans-infecção. Para promover sua infecção, o HIV-1 subverte o citoesqueleto da actina da célula hospedeira em várias etapas de seu ciclo. Em DCs o citoesqueleto também é essencial para internalização do HIV-1 e formação dos compartimentos. Miosinas são proteínas motoras que interagem com filamentos de actina e estão envolvidas em diversos processos celulares, incluindo migração, transporte de moléculas, endocitose e reciclagem de componentes de lipid rafts. Apesar de existirem mais de 40 tipos de miosinas em humanos, apenas a miosina 2a foi estudada no contexto da trans-infecção. Por isso, nosso objetivo nesse trabalho foi estudar o papel das miosinas 1c e 1e na maturação de células dendríticas derivadas de monócitos (MDDCs) e na internalização de HIV-1 por estas células. Confirmamos por Real Time PCR a expressão de 10 miosinas em MDDCs de doadores saudáveis, depois verificamos que há regulação negativa da expressão do gene da miosina (myo1c) em MDDCs de pacientes HIV&#43. Analisamos a ativação das células em resposta ao lipopolissacarídeo (LPS) por meio da expressão de CD86 e HLA-DR em MDDCs silenciadas para myo1c e 1e. Não houve diferença na expressão dos marcadores de ativação em células silenciadas para miosina 1e (myo1e) em relação ao controle. No entanto, na maioria dos doadores testados, o silenciamento da myo1c interferiu com o aumento de expressão desses marcadores, indicando que a myo1c possa ter um papel na ativação celular por LPS. Ademais, a localização subcelular do HIV-1 em MDDCs silenciadas para myo1c e ativadas com LPS ficou mais próxima ao fenótipo de células imaturas. Contudo, não houve diferença na quantidade HIV-1 internalizado por MDDCs silenciadas para as miosinas 1c e 1e ou tratadas com um inibidor específico de miosinas do tipo 1. Estes resultados sugerem que a myo1c pode estar envolvida na ativação de células dendríticas e consequentemente alterar o mecanismo de internalização do HIV-1 por MDDCs. / Infection by human immunodeficiency virus (HIV) leads to severe immunodeficiency caused by depletion of T helper cells, the main targets of the virus. Besides T CD4&#43 cells, HIV-1 can infect and interact with other immune cells, including dendritic cells and macrophages. Dendritic cells are resistant to HIV infection, however, they can bind and internalize HIV in compartments and then transfer the virus to CD4&#43 T cells in a process called trans-infection. To promote infection, HIV-1 subverts actin cytoskeleton of host cell at several points of its cycle. In DCs, cytoskeleton is also essential to HIV-1 internalization and compartment assembly. Myosins are motor proteins that can interact with actin and take part in several cellular processes, including migration, molecular trafficking, endocytosis and lipid raft recycling. Even though there are about 40 myosin types, only myosin 2a has been investigated in trans-infection. Thus, our aim was to evaluate the role of myosins 1c and 1e in monocyte derived dendritic cell (MDDC) activation and HIV-1 internalization. We have validated the expression of 10 myosins in MDDCs by real-time PCR, and observed a down regulation of myosin 1c gene in HIV&#43 patients. We have evaluated cell activation in response to lipopolysaccharide (LPS) through CD86 and HLA-DR expression in myosin 1c and 1e knocked down MDDCs. There was no change in expression of activation markers in myosin 1e knocked down MDDCs compared with control cells. However, in most donors, myosin 1c knock down impaired the increase of activation markers following LPS treatment, suggesting that myosin 1c may play a role in cell activation by LPS. In addition, subcellular location of HIV-1 in MDDCs knocked down for myosin 1c and activated with LPS, was similar to immature cell phenotype. Nevertheless, we have not observed changes in the amount of HIV-1 internalized by myosin 1c or 1e knocked down MDDCs or in MDDCs treated with myosin I inhibitor. These data suggest that myosin 1c may play a role in MDDC activation and therefore alter the mechanism of HIV-1 internalization by MDDCs.