La qPCR est actuellement la technique de référence en matière de quantification d'ADN. Elle peut être définie comme une amplification exponentielle, cyclique et ciblée de la séquence d'ADN cible. Le caractère exponentiel de la qPCR est à la fois à l'origine de la sensibilité de la méthode mais aussi d'une potentielle variabilité inter-échantillons. Cette variabilité est compensée par le caractère cyclique de la méthode qui entraine une synchronisation de la réaction pour tous les échantillons à chaque cycle. L'amplification ciblée de la séquence choisie traduit quand à elle la spécificité de la méthode. Néanmoins, cette dernière propriété de la PCR reste la moins vérifiée. La spécificité de la qPCR est indiscutable lorsque la cible à détecter se trouve en quantité suffisante (>100 copies environ). En revanche, pour des quantités plus faibles ou en présence d'inhibiteurs, la spécificité diminue ou disparaît (limites de détection et de quantification). Cette perte de spécificité retentit de façon significative sur la précision et la reproductibilité du dosage à réaliser. Cependant, si l'hybridation non spécifique est inéluctable dans ces conditions, l'amplification non spécifique consécutive peut être limitée, voire supprimée au moyen d'amorces de PCR soit plus spécifiques, soit moins susceptibles de générer des différences inter-échantillons. La RT-qPCR, grâce à une étape initiale de transcription inverse (conversion d'un ARN en un ADN complémentaire) permet la quantification des ARN. Cependant, la transcription inverse reste moins reproductible que la PCR, générant des différences inter-échantillons délétères en diagnostic comme en recherche. Au cours de ces travaux, je propose des méthodes originales améliorant de façon significative différentes étapes de ces techniques. Premièrement, je propose une amélioration de la standardisation de l'étape de transcription inverse capable de diminuer de façon significative la variabilité inter-échantillons ; l'utilisation d'un volume constant d'extrait d'ARN pour chaque échantillon améliore considérablement la précision de la quantification d'ARN messagers. Deuxièmement, je propose une modification des amorces par incorporation de résidus d'acides nucléiques bloqués (LNA) ; cette modification permet d'augmenter la spécificité des amorces aux limites de la détection. Enfin, je propose une méthode simple et économique permettant la mesure directe de la température de fusion (Tm) dans les conditions réelles de la PCR. Ce paramètre, considéré comme capital pour la réalisation des dosages par qPCR, est généralement obtenu par des méthodes prédictives peu précises. De plus, cette méthode doit permettre de déterminer précisément les paramètres thermodynamiques des amorces (∆G, ∆H et ∆S) et ainsi avoir accès d'une part au pourcentage réel d'amorces hybridées en fonction de la température et d'autre part d'identifier la susceptibilité des amorces aux inhibiteurs. / QPCR is currently the gold standard for DNA quantification of DNA. It can be defined as an exponential, cyclic and targeted amplification of a known DNA sequence. The exponential character of qPCR is both the cause of the sensitivity of the method but also a potential variability between samples. This variability is offset by the cyclical nature of the method that causes a synchronization of the reaction for all samples in each cycle. The targeted amplification of the selected sequence translates the specificity of the method. Nevertheless, this last property of PCR remains the least tested. The specificity of qPCR is unquestionable when the target to detect is sufficient (> 100 copies). However, for smaller quantities or in the presence of inhibitors, the specificity decreases or disappears (limits of detection and quantification). This loss of specificity will have a significant effect upon the accuracy and reproducibility of the assay to be performed. However, if non-specific hybridizations are unavoidable in these conditions, consecutive nonspecific amplification may be limited or eliminated using more specific PCR primers or less likely to produce inter-sample differences. RT-qPCR allows RNA quantification because of an initial step of reverse transcription (conversion of an RNA into a complementary DNA). However, reverse transcription is less reproducible than PCR, generating harmful inter-sample differences both in diagnosis and in the research field. In this work, I propose innovative methods improving significantly different steps of these techniques. First, I propose an improved standardization of the reverse transcription step that can significantly reduce the variability between samples; using a constant volume of RNA extract for each sample substantially improves mRNA quantification accuracy. Second, I propose a primers modification by incorporating Locked Nucleic Acid residues (LNA); this modification increases the specificity of the primers to the limits of detection. Finally, I propose a simple and economical method for direct measurement of the melting temperature (Tm) in real PCR conditions. This essential parameter in the achievement of qPCR assays is generally obtained by predictive methods with low accuracy. Furthermore, this method should determine the thermodynamic parameters of the oligonucleotide sequence (∆G, ∆H and ∆S), on the one hand allowing access to the actual percentage of annealed primers according to the temperature and on the other hand, to identify the primers susceptibility in the presence of inhibitors.

Submitted by Caroline Periotto (carol@ufscar.br) on 2016-10-10T12:55:43Z No. of bitstreams: 1 DissJMS.pdf: 4383574 bytes, checksum: 04172fa81fb0f3d2e00439bbeaf3cbd4 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:49:46Z (GMT) No. of bitstreams: 1 DissJMS.pdf: 4383574 bytes, checksum: 04172fa81fb0f3d2e00439bbeaf3cbd4 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-20T19:49:51Z (GMT) No. of bitstreams: 1 DissJMS.pdf: 4383574 bytes, checksum: 04172fa81fb0f3d2e00439bbeaf3cbd4 (MD5) / Made available in DSpace on 2016-10-20T19:49:57Z (GMT). No. of bitstreams: 1 DissJMS.pdf: 4383574 bytes, checksum: 04172fa81fb0f3d2e00439bbeaf3cbd4 (MD5) Previous issue date: 2015-12-16 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / The aim of the present work was the study of the viability of a manufacturing route of parts with the Cu-based Shape Memory Alloy (SMA) Cu- 11,3Al-3,2Ni-3Mn-0,5Zr through gas atomization followed by Selective Laser Melting (SLM) consolidation. The alloy was prepared from high purity elements in an induction furnace with a concentrate argon flow shield above the molten metal. The atomization was carried out using an induction furnace for melting and argon as atomizer gas. The atomized powder was sieved in 32-106 μm range particles sizes and consolidated by SLM 250 HL device settled at the Leibniz Institute for Solid State and Materials Research, Dresden, Germany. In the consolidation step the best combination of power (P in Watts) and velocity (V in mm/s) were selected through the visual aspect criteria. In the following step the hatching track percentage (S) guided by relative density criteria was included. The atomized powder and the consolidated samples were characterized by optical and electron microscopy, X-ray diffraction and differential scanning calorimetry. The composition and the powder morphology were suitable for the SLM processing. The parameters optimization point out that the best combinations were P310v740S40 and P310v740S50, their relative density were around 97 %. The β’ “zig-zag” martensite phase, the SMA effect cause, was prevailing in the consolidated samples microstructure, still, the microstructure although was not-uniform it was relatively grain refined, pointing out the effect of Zr addition. The consolidated samples transformation temperatures were As=172-174C, Af=194-197C, Ms=156-160C, Mf=132- 138C. The results point to a strong indicative of the viability of a manufacturing route of parts with Cu-based SMA through gas atomization followed by SLM consolidation. / O objetivo da dissertação foi o estudo da viabilidade de uma rota de fabricação de peças com liga Cu-11,3Al-3,2Ni-3Mn-0,5Zr a base de cobre com Efeito de Memória de Forma (EMF) por atomização a gás da liga, seguida pela consolidação por Fusão Seletiva por Laser (FSL). A liga foi elaborada a partir de elementos de alta pureza em forno de indução com proteção de fluxo de argônio concentrado acima do banho. As atomizações foram realizadas com fusão por indução e utilizando argônio como gás de atomização. Os pós, separados na faixa granulométrica 32-106 μm foram consolidados por FSL utilizando o equipamento SLM 250 HL do Leibniz Institute for Solid State and Materials Research, Dresden, Alemanha. Para consolidação foram selecionadas as melhores combinações de potência (P em W) e velocidade (V em mm/s) do feixe de laser pelo critério de aspecto visual das trilhas simples. Na etapa seguinte foi considerada a porcentagem de sobreposição de pistas (S) avaliada pelo critério de densidade relativa. O pó atomizado e os corpos consolidados por FSL foram caracterizados por microscopia ótica e eletrônica de varredura, difração de raios-X e por calorimetria diferencial de varredura. A composição e a morfologia do pó atomizado foram adequadas para o processo de FSL. A otimização dos parâmetros de processamento indicaram que as melhores combinações foram de P310v740S40 e P310v740S50, com densidade relativa alcançada em torno de 97%. A fase martensítica β’ “zigzag”, responsável pelo EMF, foi predominante nos corpos consolidados por FSL sendo que a microestrutura, embora não uniforme, foi relativamente refinada, indicando o efeito da adição de Zr na composição da liga. As temperaturas de transformação dos corpos consolidados foram de As=172- 174C, Af=194-197C, Ms=156-160C, Mf=132-138C. Os resultados indicam a viabilidade da rota de fabricação de peças em ligas a base de cobre com EMF por atomização a gás da liga, seguida pela consolidação por FSL.

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Selective laser melting (SLM) of metallic powders is an additive manufacturing technique that is widely employed to produce 3D components, and is fast becoming an important method for manufacturing near-net shape and complex metallic parts. In this thesis, a comprehensive investigation on the effect of SLM on the mechanical and corrosion properties of the Al-12Si (AS), 316L stainless steel (SS), and 18(Ni)-300 grade managing steel (MS) is investigated, with particular emphasis on the developing (micro- as well as mesa-)structure -property correlations. Detailed microstructural characterization combined with quasi-static tensile, fracture toughness, fatigue crack growth, and unmatched fatigue tests were conducted. The effect of post-SLM heat treatment as well as the scanning strategy (linear vs. checker board hatch style) was examined and the results are compared with those of conventionally manufactured (CM) counterparts. The SLM alloys exhibit a mesostructured, in addition to the fine cellular structure along the boundaries. In a case of SLM-AS, the fine cellular structure imparts higher strength at the cost of ductility, while the mesostructured, which arises due to the laser track hatching, causes the crack path to be tortuous, and in turn leads to substantial increase in fracture toughness. This imparts significant anisotropy to the toughness while tensile properties are nearly-isotropic. The experimental results of SLM-SS also show that higher tensile strengths properties with a marked reduction ductility. In spite of these, the fracture toughness, which ranges between 63 and 87 MPa.m0.5, of the SLM-SS is good, which is a result of the mesostructured induced crack tortuousity.Both tensile and toughness properties of SLM-SS were found to be anisotropic in nature. Upon aging SLM-MS, nanoscale precipitation of intermetallic compounds occurs within the cells that, in turn, lead in marked improvements in tensile strengths properties, but substantial reductions in ductility and fracture toughness. Overall, the mechanical performance, except ductility, of the SLM-MS after aging is found to be similar to that of CM-MS. Importantly, the lack of ductility does not lead to a reduction in toughness. Although the SLM-MS alloy possesses a mesostructured, no significant anisotropy in the mechanical behaviour is observed. The unnoticed studies on SLM-AS, -SS, and -MS reveal that the tensile residual stresses, gas-pores, and unmelted powder particles, can degrade the unmatched highest fatigue properties considerably and hence need be eliminated for high fatigue strength. Room temperature, electrochemical corrosion resistances (CRs) of SLM-AS, -SS and -MS in 0.1M NaCl solution were also evaluated and compared with those CM counterparts. While SLM improves CRs of AS and SS, it degrades that of MS. The results are discussed in terms of microstructural refinement and porosity that are common in SLM alloys.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Diques de basaltos toleíticos de baixo-TiO2 ocorrem na porção oriental do Enxame de Diques da Serra do Mar, na Região dos Lagos, entre Niterói e Búzios. Eles têm direções, preferencialmente, NE-SW, subordinadamente, NNE-SSW e mais raramente, NW-SE, e formas intrusivas variáveis. Os basaltos são holocristalinos a hipocristalinos, inequigranulares a equigranulares, intergranulares e intersertais. Eles são constituídos essencialmente de plagioclásio, augita e/ou pigeonita, com olivina corroída, minerais opacos e apatita como minerais acessórios e biotita, bowlingita, idingisita, uralita e saussurita como minerais secundários. A assembléia de fenocristais destas rochas foi estimada em 15% de olivina, 40% de augita e 45% de plagioclásio. Os rochas inserem-se numa série basáltica subalcalina de afinidade toleítica de baixo-TiO2 pouco expandida (MgO= 8,13-5,91%peso), não tendo sido encontradas amostras representativas de líquidos basálticos primários. Esta suíte predominante foi denominada Suíte Costa Azul. No entanto, os dados litogeoquímicos apontam para a existência de mais que uma suíte de baixo-TiO2 na área de estudo que, segundo modelos de fusão parcial em equilíbrio modal, poderiam ter sido geradas por diferentes quantidades de fusão a partir de uma mesma fonte com granada residual. Em termos regionais, a suíte Costa Azul pode ser relacionada com a Suíte Esmeralda que ocorre na subprovíncia Sul de Paraná-Etendeka, muito embora abrangendo um espectro mais amplo de razões Ti/Y. As rochas da suíte Costa Azul evoluíram por 42% de cristalização fracionada envolvendo uma assembléia de fenocristais composta por 15% de olivina, 40% de augita e 45% de plagioclásio, sem mudança de assembléia fracionante. Misturas de componentes mantélicos empobrecidos (mínimo de 72% de D-MORB) e enriquecidos (máximo de 28% de manto litosférico subcontinental) estão associadas à petrogênese desta suíte. Misturas entre componentes do tipo pluma e litosféricos não conseguem explicar as composições parentais da suíte. Os modelos petrogenéticos permitiram a proposição de um modelo geodinâmico envolvendo delaminação do manto litosférico subcontinental englobado por células convectivas ascendentes do manto sublifosférico subjacente em níveis astenosféricos pouco profundos durante um estágio avançado de rifteamento do supercontinente Gondwana. / Low-TiO2, tholeiitic basalt dykes occur eastwards the Serra do Mar Dyke Swarm within the Região dos Lagos from Niterói up to Búzios city. The dykes strike preferentially NE-SW bearing variable intrusive structures. The basalts are holo- to hypocrystaline, equigranular to inequigranular rocks with mostly intergranular and intersertal textures. They are composed mostly by plagioclase, augite and/or pigeonite and have corroded olivine, opaque minerals and apatite as accessory phases. Secondary mineralas are represented by biotite, bowlingite, iddingisite, uralite and saussurite. The phenocrysts assemblage comprise 15% olivine, 40% augite and 45% plagioclase. The rocks comprise a low-TiO2, subalkaline, tholeiitic basaltic suite hereafter called the Costa Azul suite. Nevertheless, lithogeochemical data point to the existence of more than one low-TiO2 suite in the study area. Geochemical modelling has shown that these suites can be related by different degrees of partial melting from a similar mantle source with residual garnet. The Costa Azul suite can be related with the Esmeralda low-TiO2 basaltic suite within the Paraná-Etendeka CFB province due south Brazil although comprising a wider Ti/Y ratio range. The rocks of the Costa Azul suite evolved by 42% of fractional crystallisation involving a phenocryst assemblage with 15% de olivine, 40% augite and 45% plagioclase. Binary mixing modelling point to at least 72% of a D-MORB component and 28% of a subcontinental lithospheric mantle (SCLM) component in the petrogeneses of the Costa Azul basalts. Models involving mixing between plume-like asthenospheric and SCLM components cannot explain the geochemistry of parental liquids in the Costa Azul suite. A geodynamic model involving continental lithosphere delamination and entrainment by ascending convection cells from the underlying asthenospheric depleted mantle during an advanced stage of Gondwana rifting is depicted on the basis of the petrogenetic models proposed for the Costa Azul suite.

Fundação Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro / Nesta dissertação foram estudas rochas máficas dos complexos alcalinos de Morro de São João, Rio Bonito, Tanguá, Gericinó-Mendanha, Morro Redondo, Itatiaia e Passa Quatro. Essas rochas ocorrem na forma de diques e/ou sills. As amostras coletadas foram classificadas como lamprófiros, fonolitos, gabros e diabásios alcalinos. A análise geoquímica permitiu identificar um trend fortemente insaturado e um trend moderadamente alcalino para os complexos estudados. O primeiro é caracterizado por foiditos e fonolitos como membros parentais e mais evoluídos, respectivamente, enquanto o segundo tem basaltos alcalinos como membros parentais e traquitos como os mais evoluídos. Todas as amostras plotam no campo da série alcalina, sendo majoritariamente miaskíticas, sódicas ou potássicas. Adicionalmente, o estudo geoquímico indicou que os complexos alcalinos representam câmaras magmáticas distintas, onde diferentes processos evolutivos tiveram lugar. As modelagens apontaram dois processos de diferenciação distintos nos complexos estudados. Os complexos alcalinos de Morro de São João, Morro Redondo, Gericinó-Mendanha e Itatiaia estariam relacionados a processos de cristalização fracionada. Por outro lado, o Complexo Alcalino de Passa Quatro teria sido diferenciado por processos de cristalização fracionada com esvaziamento e posterior reabastecimento da câmara magmática (RTF). De um modo geral, esses modelos indicaram a presença de mais do que uma série magmática nos complexos estudados e a não cogeneticidade entre as séries agpaíticas e miaskíticas. A discriminação de fontes foi feita com base na análise dos elementos terras raras das amostras parentais de cada um dos complexos (gabro em Morro de São João e lamprófiro nos demais). No entanto, este procedimento não foi aplicado para o Complexo Alcalino de Morro Redondo, uma vez que todas as suas amostras apresentaram valores de MgO muito abaixo do típico para líquidos parentais. O líquido parental do Complexo Alcalino do Gericinó-Mendanha apresentou razões de La/Yb e La/Nb, maior e menor que a unidade, respectivamente, típicas de derivação a partir fontes férteis. Os líquidos parentais dos outros complexos alcalinos tiveram suas razões La/Yb e La/Nb maiores que a unidade, típicas de derivação a partir de fontes enriquecidas. Os modelos desenvolvidos revelaram que os líquidos parentais de cada um dos complexos estudados estariam relacionados a fontes lherzolíticas com granada residual. Além disso, a fusão parcial destas fontes teria ocorrido num intervalo de 1 a 7% dentro da zona da granada. Finalmente, as modelagens petrogenéticas elaboradas permitiram a proposição de um cenário geodinâmico, envolvendo a descompressão adiabática do manto litosférico e sublitosférico anomalamente aquecidos. As características geoquímicas dos líquidos parentais parecem ter sido controladas essencialmente pela mistura desses dois tipos de fontes. / Mafic rocks from the Morro de São João, Rio Bonito, Tanguá, Gericinó-Mendanha, Morro Redondo, Itatiaia and Passa Quatro alkaline complexes were studied based on field, petrographic and lithogeochemical data. The mafic rocks are mostly alkaline lamprophyres and alkaline gabbro (in Morro de São João). The complexes show both strongly undersaturated and mildly alkaline evolutionary trends, having foidites and alkaline basalts as parental compositions. Trachytes and phonolites are the most common evolved rocks. Geochemical modelling has show that miaskitic and agpaitic series are unlikely to be related by fractional crystallization. Enriched mantle sources predominate although a fertile mantle source seems to be related with the Gericinó-Mendanha complex. Parental compositions are related to small amounts of partial melting (1-7%) of garnet lherzolite within the garnet stability field in the mantle. Simple geodynamic models indicate that the alkaline complexes are related to the adiabatic decompression of anomalously hot (~1570C) mantle although unrelated to lithospheric translation over a fixed hotspot or mantle plume. Parental compositions are likely to be strongly controled by mixing of distinctive lithospheric mantle sources and a more homogeneous sublithospheric mantle. The lithospheric components seem to be related with the accreted terranes during the Gondwana amalgamation in Early Proterozoic times.