Submitted by Marta Toyoda (1144061@mackenzie.br) on 2017-01-04T13:30:47Z No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Rafael Malvar Ribas.pdf: 5173927 bytes, checksum: 0ca88c67929cef8968560838940ff0c4 (MD5) / Approved for entry into archive by Paola Damato (repositorio@mackenzie.br) on 2017-01-06T18:43:05Z (GMT) No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Rafael Malvar Ribas.pdf: 5173927 bytes, checksum: 0ca88c67929cef8968560838940ff0c4 (MD5) / Made available in DSpace on 2017-01-06T18:43:06Z (GMT). No. of bitstreams: 2 license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Rafael Malvar Ribas.pdf: 5173927 bytes, checksum: 0ca88c67929cef8968560838940ff0c4 (MD5) Previous issue date: 2016-02-19 / The aim of this work is to remember the Brazilian countercultural movements that stand during the 1960s and 1970s. It is clear that in a country of continental dimensions such as Brazil there are several local cultural demonstrations that differ and intersect on one another. The main association made by the academy and the media about our counterculture is with the Tropicália movement, which brought external features adding to the local, in an hybrid art that led to its canonization. But there were other movements, although not exalted at the same level, have significant historical and cultural value. As an example there is Udigrudi in Recife, Raul Seixas from Bahia, Grã Órdem Kavernista centralized in Rio de Janeiro, the Rock and Roll and the Progressive Rock / Psychedelic especially in large urban centers, the so-called singers “Malditos” (damned), among others. / O intuito desta dissertação é o de trazer à memória os movimentos contraculturais brasileiros que vigoraram nos anos 1960 e 1970. É evidente que em um país de dimensões continentais como o Brasil se tenha diversas manifestações culturais locais que se difiram e se interseccionam. A principal associação que a academia e a mídia fazem de nossa contracultura é com o movimento tropicalista, que trouxe características externas somando-se às locais, num hibridismo artístico que levou à sua canonização. Entretanto para além da Tropicália tivemos outros movimentos, que embora não tenham sido enaltecidos ao mesmo nível, possuem expressivo valor histórico e cultural. Como exemplo podemos citar o Udigrudi em Recife, o baiano Raul Seixas, a grã ordem Kavernista centralizada no Rio de janeiro, o Rock and Roll e o Rock Progressivo/Psicodélico especialmente nos grandes centros urbanos, os cantores denominados “Malditos”, entre outros.

The trend of IP core reuse has been accelerating for years because of the increasing complexity in the System-on-Chip (SoC) designs. As a result, many IP cores of different types have been produced. Meanwhile, similar to the free software movement, an open core community has emerged because some designers choose to share their IP cores by using open source licenses. The open cores are growing fast due to their inherently attractive properties like accessible internal structure and usually no cost for license. Under this background, the master thesis was proposed by the company ENEA (Malmö/Lund branch), Sweden. It intended to evaluate the qualities of the open cores, as well as the difficulty and the feasibility of building an embedded platform by exclusively using the open cores. We contributed such an open core platform. It includes 5 open cores from the OpenCores organization: OpenRISC OR1200 processor, CONMAX WISHBONE interconnection IP core, Memory Controller IP core, UART16550, and General Purpose IOs (GPIO) IP core. More than that, we added the supports to DM9000A and WM8731 ICs for Ethernet and Audio features. On the software side, uC/OS-II RTOS and uC/TCP-IP stack have been ported to the platform. The OpenRISC toolchain for software development was tested. And a MP3 music player application has created to demonstrate the system. The open core platform is targeted to the Terasic’s DE2-70 board with ALTERA Cyclone II FPGA. It aims to have high flexibility for a wide range of embedded applications and at the same time with very low costs. The design of the thesis project are fully open and available online. We hope our work can be useful in the future as a starting point or a reference both for academic research or for commercial purposes.

During recent years, there has been a renewed interest in the study of the historical and religious values of the Old Testament. Christians everywhere seem to have become obsessed with an inherent thirst for a better understanding of this great book and the faith it teaches. This renewed desire to probe into the rich background of Semitic culture that is the very historical foundation of Christianity seems to have been brought about by two distinct and quite general conditions. The first and most obvious condition is the age in which we are living--the age of science. Today, this age is probing everywhere to discover and verify with facts the true meaning of the ever increasing amount of archeological evidence that is constantly being uncovered in the field of Old Testament research. Scientific research has far outdistanced the ability of the average man to assimilate even the wealth of knowledge that has already been uncovered. At this point, we are experiencing a cultural lag which in time should be overcome. A second motivating factor that has caused man to turn once again to a more critical study of the Old Testament has been his honest endeavor to bridge this cultural lag and also erase from posterity a blot of religious illiteracy concerning the great religious truths of this book. Many of these truths have never been brought into the full light of scientific discrimination and understanding until very recent years. One of the ideas of the Old Testament about which very little is generally known is the nature of the sacrificial system of the ancient Israelites. It is from this sacrificial system that the worship service of Christianity found the seeds of its early development. It is the purpose of this thesis to trace a view of the origin, purposes, and development of this sacrificial system from its most humble beginnings down to its completion in Jesus Christ. The fundamental aim of the thesis is to convey to the reader the idea that eternal truths were enshrined in the crude forms of early sacrificial worship and that, ultimately, these truths had their fulfillment in the person and work of Jesus Christ.

The climate impact of the construction sector, due to energy and material usage, constitutes the largest global climate burden. In Sweden, the construction sector accounts for 40% of the total energy consumption and 18% of greenhouse gas emissions. The country aims for net-zero emissions by 2045. Despite efficient new buildings, total energy usage in the sector is increasing. Enhancing energy efficiency in existing buildings is vital to reduce its climate impact. Prioritizing renovation of existing structures, instead of building new construction, is an effective strategy. This study aims to evaluate energy savings and climate impact of diverse renovation measures on 70s villas. The goal is to identify the renovation measure with highest energy savings relative to their climate impact throughout various stages of its lifecycle, productstage, transportation and construction process stage. Calculations were done using VIP-Energy and Byggsektorns miljöberäkningsverktyg. The study's calculations are based on a 1970s detached house in Ängelholm, used as a case study with an assumed remaining lifespan of 50 years. When calculating energy savings, the total savings over 50 years were included. Regarding climate impact, the analysis period implies the number of times a measure needs to be carried out during that time. The frequency depends on the technical lifespan of the renovation measure. Renovation measures included extra insulation for walls, upper level floor, crawl spaces, window replacement and installation of an exhaust air heat pump. Three insulation materials researched were: mineral wool, wood fiber and cellulose. Results for all measures were presented in terms of saved kilowatt hours per kilogram of CO2 equivalent emissions. The results show that upper-level insulation provides the most energy savings per kilogram of CO2 emissions, regardless of choice of insulation material. This is due to its longer technical lifespan, reducing overall climate impact. Installation of an exhaust air heat pump showed significant energy savings thanks to its ability to use recycled exhaust air for water heating. The results for window measures show how the choice of window frame material is directly linked to the extent of climate impact during window replacement.

Excess production and secretion of very low-density lipoprotein (VLDL) by the liver into the circulatory system is directly related to atherosclerosis, a chronic cardiovascular disease that threatens the lives of many worldwide and continues to be a leading cause of death in the United States. The rate-limiting step in VLDL secretion is its transport from the site of biogenesis, the hepatic endoplasmic reticulum to the cis-Golgi. This step is mediated by a specialized ER- derived vesicle, the VLDL transport vesicle (VTV). Upon exit of the ER the VTV targets, fuses and delivers VLDL into the lumen of the Golgi. The targeting and fusion of the VTV with the Golgi is facilitated by specific set of soluable N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins that form a SNARE complex, which is required for the VTV-Golgi fusion and thus delivery to the Golgi. Data from our laboratory indicates that the formation of the SNARE complex requires cytosolic factors. Through the purification of liver cytosol, chromatographic steps, detailed mass spectrometry, immunodepletion and western blotting data it was identified that the protein necessary for SNARE complex formation is Hsc-70. Although Hsc-70's identification is significant, the role it plays in SNARE complex formation for VTV -Golgi fusion is a predicament and yet to be unraveled. In this study we performed a series of co-immunoprecipitation reactions to identify its role in SNARE-complex assembly. Using western blot data we confirmed binding of Hsc-70 with Sec22b, the v-SNARE on the VTV. Moreover, we confirmed the interaction of Hsc-70 with t-SNAREs, (syn5, rBet1 and GOS28) on the Golgi membrane. Removal of Hsc-70 from the liver cytosol resulted in significant reduction of SNARE-complex formation. Ultimately, the identification proteins involved in the process of VLDL delivery to the Golgi would offer therapeutic targets to control VLDL secretion into the blood by the liver.

"Mémoire présenté à la Faculté des études supérieures et postdoctorales de l'Université Laval comme exigence partielle du programme de maîtrise en Médecine Expérimentale offert à l'Université du Québec à Chicoutimi en vertu d'un protocole d'entente avec l'Université Laval pour l'obtention du grade de Maître ès sciences (M.Sc.)" / Huntingtin interacting protein-1 (HIP1) et Huntingtin interacting protein-1-related (HIP1R) ont été identifiées comme des protéines intervenant dans l’endocytose médiée par les vésicules de clathrine par leurs interactions avec d ’autres protéines endocytaires. Pour mieux comprendre les rôles attribués à HIP1/R dans cette machinerie, nous avons procédé à l’identification de nouveaux partenaires d ’interaction. Au cours de notre étude, nous avons identifié HSC70 (Heat-shock Cognate Protein70) comme un nouveau partenaire pour les domaines TALIN des deux protéines par des essais de pull-down et analyse par spectrométrie de masse. Au cours de cette étude nous avons identifié également que l’association d ’HSC70 avec le domaine TALIN d ’HIPIR, compromet sa sédimentation avec les filaments d ’actine. HIP1R est une composante du manteau de clathrine, son interaction avec HSC70 l’a impliquée dans le démantèlement des vésicules. Dans cette étude, nous avons vérifié l’intervention d ’HIPIR dans le démantèlement de la clathrine suite à son interaction avec HSC70 et la relation de ce mécanisme avec la perte d ’interaction avec l’actine.

Les déficits lymphocytaires T peuvent être corrigés par l'administration en intraveineuse (IV) de cellules souches hématopoiétiques (CSH) provenant d'un donneur. Dans un modèle d'immunodéficience lié à l'absence de la protéine kinase ZAP-70, notre équipe avait précédemment montré que l'injection intrathymique (IT) de CSH histocompatibles conduit à une reconstitution du compartiment T plus robuste et plus rapide que dans le cas où les CSH sont administrées par voie IV. Au cours de ma thèse, je me suis intéressée à l'approche IT dans un contexte non-histocompatible, où j'ai montré que l'injection de CSH semi-allogéniques directement dans le thymus permet le développement d'une thymopoièse à long-terme, même en absence de conditionnement. De plus, j'ai également montré la persistence de progéniteurs thymiques précoces (ETP) provenant du donneur dans le thymus des souris transplantées. De façon remarquable, ces ETP retiennent un potentiel de différenciation plus divers que ceux rencontrés dans le thymus d'une souris sauvage, et leur fréquence est significativement élévée après IT, ce dernier suggérant une disponibilité accrue des niches thymiques. De façon intéressante, j'ai également montré que les progéniteurs déficients en ZAP-70 pouvaient se différencier de façon importante vers le lignage CD8 lors d'une activation constante de la voie de signalisation Notch couplée à la présence d'interleukine 7 (IL-7). Après la greffe de CSH par voie IV de souris ZAP-70-/-, en absence de conditionnemt, j'ai également identifié l'accumulation d'une population de CSH présentant un phénotype particulier (Lin- Sca 1+ c-kit-), nommée LSAPT. Ces cellules LSAPT présentent un biais de différenciation vers le lignage T γδ ainsi qu'une production élevée d'IL-17, ce qui suggère que les fonctions effectrices d'une cellule T γδ sont dépendantes de leur origine progénitrices. L'ensemble de mes résultats apporte à la fois de nouveaux éléments concernant l'identification de progéniteurs T et démontrent de l'influence/coopération entre voies de signalisation et facteurs environnementaux dans la modulation de la différenciation T et de leur fonctions effectrices. / T cell deficiencies can be corrected by the intravenous (IV) injection of donor hematopoietic stem cells (HSCs). Using a murine model of ZAP-70-/- deficiency, our group previously showed that the intrathymic (IT) administration of histocompatible HSCs leads to a more robust and long-term thymopoiesis as compared to that achieved by the classical IV route. During my PhD, I found that the direct IT administration of semiallogeneic HSCs results in a sustained donor-derived thymopoiesis, overcoming histocompatibility barriers, even in the absence of conditioning. Furthermore, I found that donor-derived early thymic progenitors (ETPs) persist in the thymi of ZAP-70-/- transplanted mice, and present increased multi-lineage potential as compared to wild-type ETPs. Importantly, the frequency of donor-derived ETPs was augmented following IT transplantation, indicative of an increased progenitor niche. Interestingly, ZAP-70-deficient HSC could themselves be driven to a CD8 lineage fate in an environment where IL-7 potentiates continuous activation of the Notch pathway. Following IV transplantation of donor HSC into non-conditioned ZAP-70-/- mice, I determined that there is an accumulation of lineage-/Sca1+ donor progenitors lacking expression of the stem cell marker c-kit, termed LSAPT. These LSAPT show a biased differentiation towards the γδ T cell lineage with high IL-17-producing effector function, suggesting that progenitor origin regulates γδ T cell fate. The ensemble of my experiments provide new insights into the identity of T lineage progenitors and demonstrate how signaling pathways as well as environmental factors modulate T cell differentiation and effector function.

L’expression de ZAP-70 dans la leucémie lymphoïde chronique (LLC) est associée à une hypersignalisation du BCR et à la survenue de cytopénies auto-immunes (CAI). Les LB non tumoraux expriment aussi ZAP-70, expression corrélée à celle dans les LB tumoraux et aux CAI. Nous avons montré que ces LB non tumoraux ZAP-70+ sont polyclonaux, sans lien moléculaire entre eux ni avec le clone tumoral et qu’il n’existe pas de stéréotypie de leur BCR. Ces LB présentent par contre un enrichissement en BCR autoréactifs. Notre modèle murin knock in Zap-70+/- // Mb1-Cre+/- a montré qu’une forte expression précoce de ZAP-70 dans les LB est associée à un avantage sélectif médullaire, un enrichissement en cellules potentiellement autoréactives de type zone marginale, à un blocage partiel de la maturation et de la différentiation périphérique, ainsi qu’au développement de caractéristiques de la LLC : hypogammaglobulinémie, enrichissement en auto-anticorps circulants, hyperactivation et prolifération cellulaires augmentées. Ces résultats ouvrent de nouvelles perspectives impliquant ZAP-70 dans la compréhension du développement B et de la physiopathologie de la rupture de tolérance et de la transformation néoplasique. / ZAP-70 expression in chronic lymphocytic leukemia (CLL) is associated with BCR hypersignalling and autoimmune cytopenia (AIC) occurrence. Non tumoral B cells also express ZAP-70, which is correlated with those in tumoral B cells and AIC. We have shown that these non tumoral B cells ZAP-70+ are polyclonal, without molecular link between each other and tumoral B cells, and without BCR stereotypy. These cells are however enriched in autoreactive BCR. Our mouse model knock in Zap-70+/- // Mb1-Cre+/- revealed that a high and early ZAP-70 expression is associated with medullar selective advantage, enrichment in potential autoreactive B cells of marginal zone subtype, partial block for peripheral maturation and differentiation, along with some LLC characteristics: hypogammaglobulinemia, enrichment in circulating auto-antibodies, increase in cellular activation and proliferation. These results open new opportunities involving ZAP-70 in the understanding of B cell development and physiopathology of tolerance breakdown and neoplastic transformation.

Sporothrix shenckii é o agente etiológico da esporotricose, micose de carater crônico e de ampla distribuição mundial. No Brasil, vem crescendo o número de casos da doença, bem como a incidência de formas clínicas graves ou atípicas. Nosso grupo de pesquisa desenvolveu um anticorpo monoclonal contra o componente antigênico proteíco de 70 kDa, secretado pelas células leveduriformes de S. schenckii, denominado anticorpo monoclonal (AcMo) P6E7. Este AcMo apresentou atividade profilática e terapêutica na esporotricose experimental, no entanto, este anticorpo possui origem murina, o que pode gerar uma resposta imunogênica quando administrado em humanos, impossibilitando sua utilização por tempo prolongado. Visando sua possível utilização no tratamento da esporotricose humana, a nossa proposta foi a humanização do AcMo P6E7 na forma de FvFc (Fv-linker- Fc) através de engenharia genética. Inicialmente construimos duas versões uma humanizada e outra quimérica do AcMo contra a fração de 70 kDa do antigeno de S. schenckii. Os anticorpos foram expressos em vetores de expressão dicistrônicos e produzidos com eficiência e estabilidade estrutural em células de mamíferos da linhagem CHO. Posteriormente, esses anticorpos foram purificados por cromatografia de afinidade e analisados quanto a sua capacidade de ligação ao fungo e função efetora. Verificamos que os FvFcs construídos foram capazes de se ligar a porção de 70 kDa do antígeno de S. schenckii. Através de ensaios de fagocitose, constatamos que os fragmentos FvFc do P6E7 humanizado e quimérico foram capazes de opsonizar as leveduras de S. schenckii aumentando, assim, o índice fagocítico em macrófagos humanos. Esses dados em conjunto, sugerem a possível utilização do anticorpo construído no tratamento da esporotricose humana. / Sporothrix shenckii is the etiological agent of sporotrichosis, a chronical fungal infection that shows a worldwide distribution. In Brazil, there is a growing number of cases of sporotrichosis, as well as the incidence of severe or atypical clinical forms. Our research group developed a monoclonal antibody (mAb) against the fungal antigenic component a protein of 70 kDa, secreted by S. schenckii yeasts called P6E7. This mAb showed prophylactic and therapeutic activity in experimental sporotrichosis, however, this antibody has murine origin, which can generate an immune response when administered to humans, precluding their use for prolonged time. For its possible use in the treatment of human sporotrichosis, our proposal is the humanization of mAb P6E7 through genetic engineering. Initially, we built two versions of the original antibody: an humanized version and a chimeric antibody both against the 70 kDa fraction from S. schenckii antigen. The antibodies were expressed in dicistronic expression vectors and were efficiently produced in mammalian cells CHO strain, showing good structural stability. Subsequently, these antibodies were purified by affinity chromatography and assayed for their binding ability to the fungus and effector function. We found that the built os FvFcs (Fv-linker- Fc) fragments were capable of binding to the 70 kDa portion of S.schenckii antigen. Through phagocytosis assays, we found that the FvFc fragments from the humanized and chimeric P6E7 were able to opsonize S. schenckii yeasts, thus increasing the phagocytic index in human macrophages. Together, These data suggest the potential use of the antibodies constructed in the treatment of human sporotrichosis.

As Heat Shock Proteins (HSPs) ou proteínas do choque térmico são encontradas em todas as células e são classificadas de acordo com seu peso molecular. Dentre elas encontram-se as de 27, 60, 70, 90 e 110 kDa, sendo as mais estudadas no contexto da reprodução as da família 60 e 70. Essas proteínas são ditas como chaperoninas, em razão do seu importante papel no dobramento e desdobramento de outras proteínas celulares sem alterar sua conformação final, e são expressas frente a qualquer tipo de estresse como calor, vírus, bactéria, hormônios, diferenciação celular, etc, e influenciam nas respostas imune inata e adquirida. A placenta também expressa essas proteínas, uma vez que é um órgão de intenso estresse e diferenciação celular durante toda a gestação. Nesse estudo, busca-se avaliar a expressão ou não dessas proteínas na placenta bovina e para isso foram utilizadas 30 amostras de diferentes animais em estágios distintos de gestação, fixadas em formol tamponado a 10% e processadas pela técnica de imuno-istoquímica. O mesmo numero de amostras foi também processado para a análise de imuno-microscopia eletrônica de transmissão pelas técnicas de \"freeze-substitution\" e marcação por pós-embebição. Na imuno-istoquímica, as HSPs 60 e 70 foram localizadas nos trofoblastos, epitélio materno e células binucleadas. A expressão da HSP 60 foi maior no início declinando no segundo e terceiro terço. Já a expressão da HSP 70 manteve-se praticamente constante, evidenciando a forte expressão dessa proteína durante todo o período. Na análise de imuno-microscopia eletrônica de transmissão, ambas as famílias foram localizadas nas células binucleadas (núcleo, citoplasma e vesículas) e epitélio materno (núcleo e citoplasma) em todos os terços gestacionais. O perfil das proteínas estudadas na placenta bovina foi diferente quando comparada à placenta humana, pois nesta última, a intensidade da expressão para a HSP 70 diminuiu com o decorrer da gestação enquanto para a HSP 60 foi constante durante todo a gestação. Provavelmente essas diferenças podem estar relacionadas ao fato dessas amostras terem sido coletadas de mulheres com gravidez interrompidas e também pelo tipo de placentação distinta. A bovinocultura de corte é de extrema importância para a econômica para o Brasil e se faz necessário o conhecimento de fatores que possam melhorar suas características reprodutivas. Dessa forma os resultados obtidos nesse estudo contribuirão certamente de subsídio para experimentos futuros sobre o papel das Heat Shock Proteins na placenta bovina. / Heat Shock Proteins (HSP) can be found in any kind of cell. These proteins are classified according to their molecular weight and their known families include the HSP 27, 60, 70, 90 and 110 kDa. Among these, HSP 60 and 70 are the ones of interest in reproduction. They were known as chaperonines because of their capacity to fold and unfold other proteins into the cell, without changing their own conformation. They are expressed during several stress conditions likes virus and bacteria infections, hormones, heat, cellular differentiation, etc, and also take part signalizing for innate and acquired immune responses. Heat shock proteins are expressed in several tissues and organs, including the placenta. In this study we have evaluated the expression of these proteins in the bovine placenta, using thirty samples from different animais with distinct gestational periods, fixed in 10% formalin and processed for immunohistochemistry. The same numbers of samples were processed for immunoelectron microscopy using freeze-substitution and post embedding labeling techniques. The immunohistochemistry results show the expression of HSP 60 and 70 in trophoblasts, maternal epithelia and binucleated cells. The HSP 60 expression was higher in the beginning of gestation, becoming lower during the second and third trimester. Heat shock protein 70 expression were practically constant throughout the gestation. The immunoelectron microscopy analysis revealed that both HSP 60 and 70 were located in the cytoplasm and nucleio binucleated cells and maternal epithelia from the beginning to the end of pregnancy. The immunolocalization of HSP 60 and 70 in the bovine placenta were distinct from the ones found in studies on women, probably due to the differences of the placentation type and to the fact that those samples were collected from abnormal or discontinuous pregnancy. Beef production in Brazil is an important economical activity and studies to improve the bovine reproductive characteristics are necessary and must be expended, therefore our results certainly contributes for further studies on HSP function during pregnancy in this species.