Résumé : c-Myc est un facteur de transcription (FT) dont les niveaux cellulaires sont dérégulés dans la majorité des cancers chez l’homme. En hétérodimère avec son partenaire obligatoire Max, c-Myc lie préférentiellement les séquences E-Box (CACGTG) et cause l’expression de gènes impliqués dans la biosynthèse des protéines et des ARNs, dans le métabolisme et dans la prolifération cellulaire. Il est maintenant bien connu que c-Myc exerce aussi son potentiel mitogène en liant et inhibant différents FTs impliqués dans l’expression de gènes cytostatiques. Entre autres, c-Myc est en mesure d’inhiber Miz-1, un FT comportant 13 doigts de zinc de type Cys2-His2 (ZFs) impliqué dans l’expression de plusieurs gènes régulateurs du cycle cellulaire comprenant les inhibiteurs de CDK p15[indice supérieur INK4], p21[indice supérieur CIP1] et p57[indice supérieur KIP2]. Plus récemment, il fut démontré qu’en contrepartie, Miz-1 est aussi en mesure de renverser les fonctions activatrices de c-Myc et de prévenir la prolifération de cellules cancéreuses dépendantes de c-Myc. Ces différentes observations ont mené à la suggestion de l’hypothèse intéressante que la balance des niveaux de Miz-1 et c-Myc pourrait dicter le destin de la cellule et a permis d’établir Miz-1 comme nouvelle cible potentielle pour le développement d’agents anti-cancéreux. Malgré le fait que ces deux protéines semblent centrales à la régulation du cycle cellulaire, les mécanismes moléculaires leur permettant de s’inhiber mutuellement ainsi que les déterminants moléculaires permettant leur association spécifique demeurent assez peu documentés pour le moment. De plus, la biologie structurale de Miz-1 demeure à être explorée puisque qu’aucune structure de ses 13 ZFs, essentiels à sa liaison à l’ADN, n’a été déterminée pour l’instant. Les travaux réalisés dans le cadre cette thèse visent la caractérisation structurale et biophysique de Miz-1 dans le contexte de la répression génique causée par le complexe c-Myc/Miz-1. Nous présentons des résultats d’éxpériences in vitro démontrant que Miz-1 interagit avec c-Myc via un domaine contenu entre ses ZFs 12 et 13. De plus, nous démontrons que Miz-1 et Max sont en compétition pour la liaison de c-Myc. Ces résultats suggèrent pour la permière fois que Miz-1 inhibe les activités de c-Myc en prévenant son interaction avec son partenaire obligatoire Max. De plus, ils laissent présager que que Miz-1 pourrait servir de référence pour le développement d’inhibiteurs peptidiques de c-Myc. Finalement, nous avons réalisé la caractérisation structurale et dynamique des ZFs 1 à 4 et 8 à 10 de Miz-1 et avons évalué leur potentiel de liaison à l’ADN. Les résultats obtenus, couplés à des analyses bio-informatiques, nous permettent de suggérer un modèle détaillé pour la liaison spécifique de Miz-1 à son ADN consensus récemment identifié. / Abstract : c-Myc is a transcription factor (TF) deregulated in the majority of human cancers. In heterodimer with its obligatory partner Max, c-Myc preferentially binds E-Box DNA sequences (CACGTG) and activates genes involved in protein and RNA biogenesis, metabolism and cell proliferation. It is now well established that c-Myc can also bind and inhibit some TFs involved in the expression of cytostatic genes to exert its mitogenic potential. Among those, the inhibition of Miz-1 by c-Myc is the best characterized case. Miz-1 is a TF containing 13 Cys2-His2 zinc fingers (ZFs) that is involved in the expression of many cell cycle regulators such as the CDK inhibitors p15[superscript INK4], p21[superscript CIP1] et p57[superscript KIP2]. More recently, it was shown that, on the other hand, Miz-1 is also able to reverse the transcriptional activator functions of c-Myc and to prevent the proliferation of c-Myc-dependent cancer cells. These observations led to the interesting hypothesis that the balance of c-Myc and Miz-1 levels could determine cell fate and establish Miz-1 as an interesting target for the design of novel cancer drugs. Although those proteins seem central to the regulation of the cell cycle, the molecular mechanisms allowing them to inhibit each other and the molecular determinants allowing their specific association remain poorly understood. Moreover, the structural biology of Miz-1 remains to be explored considering that none of its 13 ZF structures, essential to its DNA binding, have been determined so far. The work presented in this thesis aim at characterizing the structural biology of Miz-1 in the context of the transcriptional repression caused by the c-Myc/Miz-1 complex. We present results from in vitro experiments showing that a domain comprised between the 12th and 13th ZFs of Miz-1 is involved in its binding to c-Myc. Moreover, we demonstrate that Miz-1 and Max compete to engage c-Myc. These results suggest for the first time that Miz-1 inhibits c-Myc by a sequestration mechanism preventing its association with its obligatory partner Max. Moreover, they argue that Miz-1 could serve as a reference for the development of c-Myc specific peptide inhibitors as a new approach for cancer drug design. Finally, we realized the structural and dynamical characterization of Miz-1 ZFs 1 to 4 and 8 to 10 and the characterization of their DNA binding potential. The results collected, coupled to bioinformatics analysis, allowed us to suggest a model for Miz-1 specific binding to its consensus DNA sequence recently unveiled.

Bacteriorhodopsin (bR) is a 7TM membrane protein expressed in Halobacterium salinarum. Due to its stability and high expression levels, bR serves as a model for other 7TM membrane proteins. Neurotensin receptor 1 (NTS1) is a member of pharmacologically relevant G protein-coupled receptor superfamily, and is the high affinity receptor for neurotensin, a 13mer peptide that can be found in the brain, gut, and central nervous system. NTS1 is a target for Parkinson’s, Schizophrenia, and drug addiction. This thesis aims to develop pulsed magnetic resonance techniques and sample preparation forms for high resolution structural studies on 7TM proteins. In this thesis, pulsed dipolar distance electron paramagnetic resonance (EPR) methods for the study of proteins in their native membrane are established. bR is spin-labeled, and a wellresolved distance distribution is measured in excellent agreement with other structural data. Preliminary distance data for a photoexcited state of bR suggests quaternary rearrangements in the native membrane that are agreement with published AFM results. A fitting method is developed to enable measurements of systems with rapid signal decay, a common feature in reconstituted systems studied by pulsed EPR methods. A physical chemical characterization of nanosized-bilayer discs termed Lipodisqs®, and the successful incorporation of bR is presented. Lipodisqs® are formed from DMPC and a polymer that is able to solubilize DMPC vesicles into discrete particles. Lipodisqs® possess a broad phase transition with increased lipid ordering compared to a DMPC dispersion. The SMA polymer interacts with the lipid tails, but does not perturb the headgroup. BR is incorporated in the monomeric form, and EPR dynamic and distance measurements confirm that Lipodisqs® preserve the native structure of bR, whilst detergent solubilisation increases the overall mobility compared to bR in its native membrane, suggesting that Lipodisqs® serve as an excellent medium for EPR studies on 7TM membrane proteins. A cysteine-depleted mutant of active, ligand binding NTS1 is constructed. Cysteines are reintroduced at positions that may be able to monitor agonist and inverse-agonist induced conformational and dynamic changes. A spin-labeling protocol is developed, and preliminary EPR measurements are discussed. Dynamic nuclear polarization (DNP) results are presented with uniformly-¹³C-labelled bR in the PM, resulting in a DNP enhancement of 16 using the biradical nitroxide polarizing agent, TOTAPOL. DNP-enhanced solid state NMR (ssNMR) is typically carried out at cryogenic temperatures, resulting in poor spectral resolution compared to ambient temperatures. Two different forms of samples are prepared that could potentially lead to better-resolved DNP spectra. BR is reverse labelled by adding natural abundance amino acids to isotopically labelled growth medium, resulting in the partial depletion of resonance signals that may obscure and crowd the NMR spectra. A crystalline sample of bR is prepared using the LCP method for crystallization, which is to date the most successful method for the crystallization of GPCRs. In summary, the first pulsed dipolar measurements of a protein in its native membrane are shown, Lipodisqs® are characterized and found to be a suitable medium for structural and functional studies of 7 TM membrane proteins, the first preliminary EPR studies on a ligand binding GPCR are presented, and novel sample preparation techniques are developed for the nitroxide-based DNP enhancement of ssNMR data. This thesis opens up several avenues for future research into 7TM membrane proteins.

Liquid chromatography is an essential technique in manufacturing biopharmaceuticals where it is used on all scales from analytical applications in R&amp;D to full-scale production. In chromatography the target molecule, typically a protein, is separated and purified from other components and contaminants. Separation is based on different affinities of different molecules for the chromatographic medium and the physical and chemical properties of the latter determine the outcome. Controlling and designing those properties demand efficient analytical techniques. In this thesis the approach was to develop characterization methods based on nuclear magnetic resonance (NMR) spectroscopy for the assessment of various important physico-chemical properties. The rationale behind this strategy was that the versatility of NMR – with its chemical and isotopic specificity, high dynamic range, and direct proportionality between the integral intensity of the NMR signal and the concentration of spin-bearing atomic nuclei (e.g., 1H, 13C, 31P and 15N) – often renders it a very good choice for both qualitative and quantitative evaluations. These characteristics of NMR enabled us to develop two quantification methods for chromatography-media ligands, the functional groups that provide the specific interactions for the molecules being separated. Furthermore, a new method for measuring the distribution of macromolecules between the porous chromatographic beads and the surrounding liquid was established. The method, which we have named size-exclusion quantification (SEQ) NMR, utilizes the fact that it is possible to assess molecular size distribution from corresponding distribution of the molecular self-diffusion coefficient where the latter is accessible by NMR. SEQ-NMR results can also be interpreted in terms of pore-size distribution within suitable models. Finally, we studied self-diffusion of small molecules inside the pores of chromatographic beads. The results provided new insights into what affects the mass transport in such systems. The methods presented in this thesis are accurate, precise, and in many aspects better than conventional ones in terms of speed, sample consumption, and potential for automation. They are thus important tools that can assist a better understanding of the structure and function of chromatography media. In the long run, the results in this project may lead, via better chromatographic products, to better drugs and improved health. / Vätskekromatografi är en viktig teknik för tillverkning av biologiska läkemedel och används för alltifrån småskaliga analytiska applikationer till fullskalig produktion. I kromatografi separeras och renas målmolekylen (oftast ett protein), från andra komponenter och föroreningar genom att utnyttja molekylernas olika affinitet för det kromatografiska mediumet, vars fysikaliska och kemiska egenskaper har stor betydelse för hur separationen fungerar. För att kunna kontrollera och designa dessa egenskaper krävs effektiva analysmetoder. Strategin i den här avhandlingen var att utveckla metoder baserade på kärnmagnetisk resonans (NMR) spektroskopi för att karaktärisera flera viktiga fysikalisk-kemiska egenskaper. Anledningen till denna strategi är att mångsidigheten hos NMR – med dess kemiska och isotopiska specificitet, stora dynamiska omfång och direkta proportionalitet mellan NMR-signalens integralintensitet och koncentrationen av spinnbärande atomkärnor (t.ex. 1H, 13C, 31P och 15N) - ofta gör den till det bästa valet för både kvalitativa och kvantitativa tillämpningar. Dessa egenskaper hos NMR gjorde att vi kunde utveckla två kvantifieringsmetoder för kromatografimedia-ligander, dvs de funktionella grupperna som ger de specifika interaktioner som gör att molekylerna kan separeras. Dessutom har en ny metod för att mäta fördelningen av makromolekyler mellan de porösa kromatografiska pärlorna och den omgivande vätskan tagits fram. Metoden, som vi har valt att kalla size-exclusion quantification (SEQ) NMR, utnyttjar det faktum att det är möjligt att mäta molekylstorleksfördelningen genom att mäta motsvarande fördelning av självdiffusionskoefficienter, där den sistnämnda kan bestämmas med NMR. Resultaten från SEQ-NMR kan tolkas i termer av porstorleksfördelningar genom att använda lämpliga modeller. Slutligen studerade vi självdiffusion av små molekyler inuti porerna i kromatografiska pärlor. Resultaten gav nya insikter om vad som påverkar masstransporten i sådana system. De metoder som presenteras i denna avhandling är noggranna, precisa och på många sätt bättre än konventionella metoder när det gäller hastighet, låg provförbrukning och automatiseringspotential. De nya metoderna är därför viktiga verktyg som kan hjälpa till att ge en bättre förståelse av struktur och funktion hos kromatografimedia. I det långa loppet kan resultat från det här projektet kunna bidra till effektivare kromatografiska produkter, vilket i slutändan kan leda till bättre läkemedel och hälsa. / <p>QC 20170403</p>

Integral membrane proteins of the DP1 and reticulon families are responsible for maintaining the high membrane curvature required for both smooth ER tubules and the edges of ER sheets. Mutations in these proteins lead to motor neurone diseases such as hereditary spastic paraplegia. Reticulon/DP1 proteins contain Reticulon Homology Domains (RHD) that have unusually long (&asymp;30 aa) hydrophobic segments and are proposed to adopt intramembrane helical hairpins that stabilise membrane curvature. I have uncovered the secondary structure and dynamics of the DP1 protein Yop1p and identified a C-terminal conserved amphipathic helix that on its own interacts strongly with negatively charged membranes and is necessary for membrane tubule formation. Analyses of DP1 and reticulon family members indicate that most, if not all, contain C-terminal sequences capable of forming amphipathic helices. Together, these results indicate that amphipathic helices play a previously unrecognised role in RHD membrane curvature stabilisation. This work paves the way towards full structure determination of Yop1p by solution state NMR and marks the first high structural resolution study on an RHD protein.

A signifficant obstacle facing the healthcare industry is the phenomenon of multidrug resistance (MDR) in which a cell acquires simultaneous resistance to many unrelated drugs that it has never been exposed to. At the molecular level, MDR can be characterised by a reduction of intracellular drug levels due to their active efflux by multidrug transporters such as P-glycoprotein (Pgp). Pgp is able to efflux a phenomenally wide variety of chemically unrelated drugs and causal relationships have been established between its expression and the acquisition of MDR to numerous anticancer and central nervous system (CNS) drugs. There has thus been much effort to understand the molecular biology of Pgp and how it functions. However, many aspects of its functioning remain unclear. From a drug discovery viewpoint, we have yet to fully understand what features make some drugs susceptible to Pgp-mediated efflux (substrates) and what makes others able to inhibit Pgp function (inhibitors). From a mechanistic viewpoint, it is still uncertain what the exact nature of Pgp's binding site is, the role of ATP binding and hydrolysis in transport and how both of these interplay with ligand binding. The work presented in this thesis attempts to answer these questions from two perspectives. Firstly the mouse Pgp crystal structure [PDB 3G60] was used as a unique starting point for molecular dynamics (MD) simulations to characterise the dynamics and conformational exibility of Pgp, properties believed to be integral to its function. The simulations revealed Pgp to be a highly dynamic molecule at both its transmembrane (TM) and nucleotide binding domains (NBDs). The latter exhibited a conformational asymmetry that supports the Constant Contact model of ATPase activity. In the presence of the Pgp substrate, daunorubicin, the NBDs exhibited tighter asymmetric dimerisation leading to increased affinity for ATP. In contrast, the presence of the Pgp inhibitor, QZ59-RRR led to NBD conformational changes that reduced their affinity for ATP. Thus providing an appealing mechanism for how QZ59-RRR inhibits Pgp ATPase activity. MD simulation was also used to provide atomic-detail interpretations of multiple binding stoichiometries of drug and lipid molecules observed by collaborator-led mass spectrometry experiments. This also provided opportunity to validate the Pgp simulations against novel experimental data. The second strand of the thesis explored the membrane permeation dynamics of CNS therapeutics in order to identify differences in protonation states, conformations, orientations and membrane localisation that might distinguish those that are Pgp substrates and from those that are not. These properties were studied using complementary MD simulation and nuclear magnetic resonance (NMR) techniques. The simulations revealed a novel set of criteria that in uence the likelihoodof a drug to 'flip-flop' across a membrane, a behaviour that may make drugs more susceptible to Pgp efflux. These observations were broadly consistent with the NMR experiments. However, the NMR data also highlighted limitations in the simulation approaches used in this thesis and emphasised the need to also consider the kinetics of permeation in addition to its thermodynamics.

Este trabalho de mestrado teve por objetivo a recuperação de um espectrômetro de RMN de dupla-ressonância construído na década de 1980 para o estudo de materiais no estado sólido. Esse espectrômetro foi parcialmente destruído por um raio que atingiu o prédio do IFSC-USP em 2001, tendo sua parte digital inutilizada, a qual envolvia o gerador de pulsos, e o digitalizador e promediador de sinais. Este trabalho envolveu a instalação de uma placa geradora de pulsos e um osciloscópio digital, ambos comerciais, controlados por um computador executando programas elaborados por nosso grupo. Embora o espectrômetro permita a implementação de técnicas de RMN de alta resolução de dupla-ressonância para o estudo de materiais no estado sólido, os primeiros experimentos de RMN foram realizados com materiais magnéticos, os quais permitiram sua execução sem a utilização do magneto supercondutor. No momento, esse espectrômetro encontra-se em fase de aprimoramento e testes de desempenho e já tornou possível a obtenção de resultados importantes em materiais magnéticos / The objective of this work was to repair and upgrade a double-resonance NMR spectrometer assembled in the 80s for studying solid-state materials. In 2001 this instrument was damaged by an atmospheric discharge that destroyed its digital modules, i.e., the pulse generator, digitizer, and averager. The work reported on here involved installing a new pulse-generator board and a programmable digital oscilloscope, both commercially available. These are computer-controlled by software developed by our research group. Although the spectrometer allows the use of double resonance NMR techniques for studying solid-state materials, the first experiments were done with magnetic materials, which required no superconducting magnet. Presently, the spectrometer is still being tested and improved. Even so, it is already capable of yielding important data in the study of magnetic materials

Em experimentos de Ressonância Magnética Nuclear (RMN) com Rotação da Amostra em Tomo do Ângulo Mágico (MAS), as bandas laterais são um problema comum na obtenção de espectros de alta resolução em sólidos. Em espectrômetros de alto campo magnético ou então, em sistemas que não dispõem de rotores de amostra de freqüência elevada, este problema é mais grave. Para facilitar a interpretação do espectro de alta resolução torna-se necessário suprimir estas bandas laterais. Nesta dissertação descrevemos um método simples de supressão das bandas laterais que consiste em variar a freqüência de rotação da amostra durante a aquisição do sinal, assim como, a instrumentação necessária para realizar esse experimento. Este método, que denominamos de Supressão das Bandas Laterais utilizando-se Freqüência de Rotação Baixa e Variável, baseia-se na dependência da posição das bandas laterais com a freqüência de rotação, reduzindo as coincidências entre suas posições durante a promediação, enquanto o sinal isotrópico permanece estável sempre no mesmo ponto do espectro. Para ilustrar a utilização dessa nova técnica, realizamos experimentos de RMN com o núcleo de 13C em amostras de hexametilbenzeno, tirosina, lignina e ácido húmico, e com o núcleo quadrupolar 79Br em amostras de KBr e NaBr + KBr / Sidebands in Magic-Angle-Spinning Nuclear Magnetic Resonance (MAS-NMR) experiments are a common problem for high field spectrometers or low spinning frequency systems. Although these sidebands may be used to gain information after they have been identified, it is necessary to eliminate then to simplify the interpretation of the spectra; we have recently succeeded in suppressing spinning sidebands by continuously varying the speed during the signal acquisition. This method, that we called variable low speed sideband suppression, relies on the speed dependent of the sideband positions. The purpose of this work is to describe a simple method and apparatus to suppress spinning sidebands in MAS-NMR experiments. The method allows a quick acquisition of the isotropic chemical shift spectra and works well even when the highest available spinning cannot clearly resolve the spinning sidebands. To illustrate the method, we present the 13C MAS-NMR spectra of a variety of sample acquired using cross-polarization and high power proton decoupling. Among the samples studied were: hexamethylbenzene, tyrosine, lignin and humic acid. And quadrupolar nuclei 79Br in the samples Kbr and NaBr + KBr

A alimentação humana necessita de nutrientes essenciais em sua dieta de forma balanceada. A carne é uma perfeita fonte de proteína e suplemento de ferro, zinco e outros micronutrientes como as vitaminas. O consumo de carne deve ser cada vez maior, já que a produção mundial que em 2012 era de 302 milhões de toneladas, hoje é de aproximadamente 319 milhões. Concomitantes ao aumento no consumo mundial de carne surgem dois problemas. A carne e produtos cárneos induzem ao estress oxidativo, por meio do ferro catalizado na formação de espécies reativas ou por ingestão de hidroperóxidos orgânicos durante a digestão. Tais espécies podem acarretar deterioração do produto, afetando seu tempo de exposição em prateleira e comprometendo sua qualidade, interferindo em questões econômicas. Dentro de um contexto de saúde pública, estas espécies podem gerar desequilíbrio celular, mutações genéticas e danos cromossômicos, desencadeando doenças degenerativas no organismo humano. Uma dieta balanceada deve ser inspirada na natureza. Por meio da evolução as plantas desenvolveram, devido ao estress oxidativo, a capacidade de produzir compostos bioativos como xantinas e compostos fenólicos, como mecanismo de proteção. A Erva-mate, Ilex paraguariensis contém alta quantidade relativa destes compostos, que atuam como antioxidantes, os quais podem ser usados como aditivos na alimentação, com o objetivo de modificar o estado redox do organismo animal. Isto pode induzir mudanças no metabolismo, afetando a composição do tecido muscular e, consequentemente, a qualidade da carne. O presente trabalho se propôs a investigar as mudanças no perfil de composição dos metabólitos polares e seu impacto na estabilidade redox da carne de frangos de corte que tiveram suas dietas suplementadas com diferentes níveis de extrato de erva-mate na ração. A adição de tais extratos claramente promoveu uma mudança quantitativa na composição de metabólitos polares da carne como mostrado a partir de análises de 1H RMN e impactou positivamente na estabilidade redox segundo análises por spin-trapping RPE. Por fim, a adição de 0,075% m/m de extrato de erva-mate na alimentação de frangos de corte mostrou-se como o tratamento mais indicado, promovendo mudanças bioquímicas no metabolismo do animal, melhorando a estabilidade oxidativa da carne, seu tempo de exposição em prateleira e um produto de maior qualidade e segurança para o consumidor. / Human food needs to supply essential nutrients in balance. Meat is a perfect protein source and supplies iron and zinc and many micronutrients like vitamins. The consumption of meat would increase in the next years because the global production in 2012 was 302 millions of tons, and actually is 319 millions of tons, approximately. Concomitantly with the increasing of global consumption of meat, two problems arise. Meat and meat products also hold the risk of inducing oxidative stress through iron catalyzed formation of reactive species or by ingestion of organic hydro peroxides during digestion. These species can cause the deterioration of the product, affecting its shelf-life, compromising its quality and influencing economic aspects. In the context of the public health, these species can generate cellular imbalance, genetic mutations, and chromosome damages, resulting in degenerative diseases of the human organism. A balanced diet should be inspired by Nature. Through evolution, oxidative stress made plants to produce bioactive compounds like xanthines and phenolic compounds to protect against oxidative stress. Yerba-mate, Ilex paraguariensis, contains relative high amounts of these compounds that act as antioxidants, which may be use as a feeding additive in order to modify the redox status of the animal organism. This may induce change in the animal metabolism, affecting the muscle tissue composition and thus the meat quality. The present investigation aims to screen changes in the polar metabolites composition and its impact on the redox stability of meat from broilers fed with different levels of yerba-mate extract as feed additive. The addition of mate extract to broilers feed clearly provided a quantitative change in the polar metabolites of meat as shown by 1H NMR analysis and impacted positively in the redox stability of meat as probed by spin-trapping EPR analysis. In conclusion, addition of 0.075 % w/w of yerba-mate extract to the broilers feed shown up like the best treatment because it produced biochemical changes in the animal metabolism improving the oxidative stability meat which may positively increase the product shelf-life and a safer and higher quality product to the consumer.

Neste trabalho, é investigado o efeito exercido por grupos alquiltio, sobre a basicidade de nitrilas alifáticas. Com esta finalidade, foi estudada uma extensa série de nitrilas alquiltio-substituídas em posição &#945;, &#946;, &#947; e &#948;, juntamente com nitrilas não substituídas, escolhidas para efeito de comparação. O conjunto de compostos investigados foi o seguinte: acetonitrila, propionitrila, butironitrila, valeronitrila, hexanonitrila, heptanonitrila, octanonitrila, isobutironitrila, 2-metilvaleronitrila, pivalonitrila, 2,2\'-dimetilvaleronitrila, metiltioacetonitrila, &#945;-metiltiopropionitrila, etiltioacetonitrila, &#945;-etiltiopropionitrila, &#945;-etiltioisobutironitrila, terc-butiltioacetonitrila, &#946;-etiltiopropionitrila, &#946;-etiltiobutironitrila, &#947;-etiltiobutironitrila, &#948;-etiltiovaleronitrila e etilselenoacetonitrila. É apresentada inicialmente, uma revisão bibliográfica sobre vários aspectos eletrônicos e estruturais do ciano grupo, incluindo potenciais de ionização e participação de orbitais moleculares na formação de pontes de hidrogênio. são também apresentados métodos utilizados para a determinação de constantes de associação aceptor-doador pela ressonância magnética nuclear. São fornecidos os resultados de basicidade relativa das nitrilas não substituídas e &#945;-alquiltio-nitrilas. Tais dados foram obtidos através de medidas dos deslocamentos químicos, na r.m.n., dos protons de doadores, fenol e p-clorofenol, em sistemas ternários nitrila-doador fenólico-tetracloreto de carbono. São apresentados também, valores de constantes de associação para os mesmos sistemas, determinados pelo emprego do método de Benesi e Hildebrand. São apresentados os dados de deslocamentos químicos e de constante de associação para as &#946;-, &#947;- e &#948;-etiltionitrilas e nitrilas não substituídas correspondentes pelo estudo de sistemas ternários nitrila-doador fenólico-tetracloreto de carbono. São fornecidos os deslocamentos químicos do próton etinílico do fenilacetileno associado às nitrilas &#946;-, &#947;- e &#948;-etiltio-substituídas e nitrilas não substituídas correspondentes, em sistema binário. São descritas as preparações de nove nitrilas a saber: butironitrila, octanonitrila, isobutironitrila, metiltioacetonitrila, &#945;-etiltiopropionitrila, &#946;-etiltiopropionitrila, &#946;-etiltiobutironitrila, &#948;-etiltiovaleronitrila e etilselenoacetonitrila, das quais, três são compostos novos. Os resultados obtidos permitem concluir que: a) a associação com doador de protons, em &#945;-alquiltionitrilas, ocorre no ciano grupo e não no enxofre; b) as nitrilas &#945;-alquiltio-substituídas apresentam uma diminuição de basicidade com relação aos análogos não substituídos; c) as nitrilas &#946;-etiltio-substituídas, estudadas nos sistemas ternários, apresentam basicidade ligeiramente menor do que as correspondentes não substituídas; d) a substituição de um grupo metilena em posições afastadas (&#947; e &#948;), em relação ao ciano grupo, por enxofre, não conduz a alteração de basicidade quando as medidas são efetuadas em sistemas ternários e provoca um aumento da basicidade quando se trabalha em sistemas binários. São discutidas as causas da diminuição de basicidade em &#945;-alquiltio-nitrilas. A influência dos efeitos estérico e indutivo, como únicos determinantes desta diminuição, é excluída devido à diminuição de frequência de estiramento do ciano grupo nestes compostos e ao resultado de basicidade e frequência da etilselenoacetonitrila. Discute-se a divergência de resultados na série de nitrilas &#946;-, &#947;- e &#948;-etiltio-substituídas, nos sistemas ternários e binários. É sugerida a hipótese da transferência de carga intra ou intermolecular, envolvendo o enxofre e o ciano grupo, em tais compostos. Finalmente, é proposta a interação entre enxofre e ciano grupo, como causa preponderante para a diminuição de basicidade em &#945;-alquiltio-nitrilas. É também sugerido, que a pequena diminuição de basicidade, observada para as &#946;-etiltio-nitrilas, em sistema ternário, seja devida ao efeito indutivo do enxofre. / The effects of alkylthio groups on the basicities of aliphatic nitriles have been investigated. A large number of unsubstituted and alkylthio-nitriles have been studied, including the following compounds: acetonitrile, propionitrile, butyronitrile, valeronitrile, hexanenitrile, heptanenitrile, octanenitrile, isobutyronitrile, 2-methylvaleronitrile, trimethylacetonitrile, 2,2\'-dimethylvaleronitrile, methylthioacetonitrile, &#945;-methylthiopropionitrile, ethylthioacetonitrile, &#945;-ethylthiopropionitrile, &#945;-ethylthioisobutyronitrile, tert-butylthioacetonitrile, &#946;-ethylthiopropionitrile, &#946;-ethyl thiobutyronitrile, &#948;-ethylthiovaleronitrile and ethylselenoacetonitrile. A literature review on several electronic and structural aspects of the cyano-group, in aliphatic nitriles, like its ionization potentials and its hydrogen bonding ability as measured by n.m.r. spectroscopy, is presented, as well as the methods of determination of association constants by n.m.r. spectroscopy. The relative basicities data measured from hydrogen chernical shifts of phenol and p-chlorofenol associated to the above nitriles, in ternary systems, using carbon tetrachloride as solvent, are reported. The association constants for the same systems, determined by Benesi-Hildebrand method are given. The relative basicities data of &#946;-, &#947;- and &#948;-alkylthio-nitriles measured from chemical shifts of phenylacetylene, in binary systems, are presented. The syntheses of the following nitriles: butyro nitrile, octanenitrile, isobutyronitrile, methylthioacetonitrile, &#945;-ethylthiopropionitrile, &#946;-ethylthiopropionitrile, &#946;-ethylthiobutyronitrile, &#948;-ethylthiovaleronitrile and ethylselenoacetonitrile, are described. The following conclusions and suggestions are presented: 1) in &#945;-alkylthio-nitriles, the association with proton donors, takes place on the cyano group instead of on the sulphur; 2) the &#945;-alkylthio-nitriles are weaker bases than the corresponding unsubstituted nitriles; 3) in ternary systems, &#946;-alkylthio-nitriles are slightly less basic than the corresponding unsubstituted compounds; 4) in binary systems &#946;-alkylthio-nitriles are stronger bases than the corresponding unsubstituted compound; 4) in ternary systems, &#947;- and &#948;-alkylthio-nitriles do not show any change in basicity in confront with unsubstituted compounds, but an increase in basicity is observed in binary systems. The hypothesis of the steric hindrance to proton donor association and the one of the inductive effect of the sulfur atom, as the only factors responsibles for the decrease of cyano-group basicity in &#945;-alkylthio-nitriles, have been excluded. The divergency of the relative basicities of &#946;-, &#947;- and &#948;-alkylthio-nitriles, obtained by measurements in ternary and binary systems is attributed to an intra- or intermolecular charge transfer involving the sulphur atom and the cyano-group. Lastly, an interaction between the cyano-group and the sulphur atom is suggested as responsible for the decrease of the basicity observed for the &#945;-alkylthio-nitriles. The slight decrease of basicity observed for the &#946;-derivatives in ternary systems, is attributed to the inductive effect of the sulphur atom.

O presente trabalho objetivou desenvolver e implementar, em escala laboratorial, uma rotina experimental com base em Imageamento por Ressonância Magnética Nuclear (IRMN) de modo a verificar seu potencial como metodologia não-destrutiva aplicável à inspeção quali- e quantitativa de água e hidrocarbonetos líquidos aprisionados no interior de células de núcleos-colméia utilizados na confecção de painéis-sanduíche compósitos estruturais de grau aeronáutico. Tentativas foram também realizadas no sentido de se observarem e caracterizarem danos por amassamento de núcleos-colméia, assim como de se detectar a presença de resina polimérica na forma sólida, visando, desta feita, verificar o uso do IRMN na inspeção de componentes previamente reparados e/ou contendo excesso de resina por falha do processo de manufatura. Concluiu-se que IRMN é uma poderosa ferramenta para a detecção e a quantificação de líquidos puros e compostos, ricos em hidrogênio, contidos nas células de núcleos de amostras extraídas de painéis-sanduíche compósitos. O potencial do IRMN na identificação, e, portanto, na discriminação entre os diversos fluidos se mostrou bastante promissor, desde que se empreguem ferramentas de processamento e análise computadorizada de imagens a partir de programas computacionais de reconhecimento de padrões via redes neurais artificiais e/ou sistemas com base em conhecimento. A técnica de IRMN utilizada neste estudo não permitiu a captura de imagens de resina polimérica sólida, nem mesmo quando à esta foram adicionadas cargas de elementos intensificadores de sinais de RMN, tais como ferro e gadolíneo. Danos no núcleo-colméia tão pequenos quanto 1,0 mm de profundidade e 1,8 mm de diâmetro foram clara e inequivocamente imageados e delineados pela técnica IRMN, desde que estivessem permeados por fluido hidrogenado (ex. água). A quantificação de líquidos nos núcleos-colméia por intermédio de ferramentas computacionais simples (processadores e analisadores de imagens) foi muito bem sucedida no caso dos líquidos com relativamente alto ponto de fulgor, pois as massas fluidas se mantiveram constantes por períodos de tempo significativamente longos no interior das células analisadas. / This work intended to develop and implement in laboratorial scale an experimental routine funded in Nuclear Magnetic Resonance Imaging (NMRI) in order to verify its potential as a non-destructive methodology for quali- and quantitative inspection of liquid water and hydrocarbons entrapped in honeycomb core cells utilized to build up aeronautical grade structural composite sandwich panels. Attempts were also carried out to observe and characterize crush damage of honeycomb core, as well as to detect solid polymer resin towards the use of NMRI to assess previously repaired components and/or containing in excess resin amount due to manufacturing process faults. It has been concluded that NMRI is a powerful tool in detecting and quantifying hydrogen-rich pure and compound liquids contained in core cells of composite sandwich samples. The NMRI potential in identifying and, therefore, discriminating several fluids has shown very promising as long as computed image processing and analysis tools are employed from pattern recognition software via artificial neural networks and/or knowledge-based systems. The utilized NMRI technique failed in imaging solid polymer resin, even when the latter was loaded with NMR-signal intensifier elements such as iron and gadolinium. Honeycomb core damages as small as 1.0 mm in depth and 1.8 mm in diameter were clearly and unambiguously imaged and delineated by the NMRI technique since they were permeated with hydrogenated fluid (ex., water). The quantification of liquids in honeycomb cores by means of simple computational tools (image processor and analyzer) was very successful in case of relatively high flash point fluids, insofar as their masses remained constant within the analyzed cells for significantly long periods of time.