A aplicação de uma sequência de pulsos com tempo de repetição muito menor que os tempos de relaxação Tp << T2; T1, faz com que a magnetização atinja um estado estacionário descrito por H.Y. Carr como Estado Estacionário em Precessão Livre, Steady State Free Precession (SSFP). Nessa condição, o sinal é composto pela complexa sobreposição das componentes FID e eco. Sequências tipo SSFP são utilizadas na aquisição rápida de sinais, resultando em uma boa razão sinal ruído (s/r) em curto intervalo de tempo, porém introduzem fortes anomalias de fase e amplitude devido a complexa interação das componentes que formam o estado estacionário. Neste trabalho, desenvolvemos sequências de pulsos tipo SSFP para RMN em alta e baixa resolução com alternância e incremento de fase. Em alta resolução desenvolvemos as sequências SSFPdx e SSFPdxdt com incremento de fase linear e quadrático respectivamente. Os resultados mostram que espectros de núcleos com baixa sensibilidade podem ser obtidos com mesma razão s/r em menor tempo experimental e as sequências desenvolvidas removem as anomalias espectrais. Em baixa resolução, os resultados mostram que a introdução de alternâncias de fase na Continuous Wave Free Precession (CWFP) possibilita a remoção da dependência da sequência com o offset de frequência e com o tempo entre pulsos. Além disso, mostramos que a sequência CP-CWFPx-x com ângulo de refocalização pequeno (5&deg; a 10&deg;) possibilita a estimativa rápida do tempos de relaxação longitudinal. Apresentamos também resultados dos estudos e desenvolvidos no estágio de pesquisa no exterior, onde as sequências de pulsos no estado estacionário &ndash; DECPMG e Split 180&deg; &ndash; foram estudas numericamente e implementadas nos sistemas magnéticos compactos: mini-Halbach e MOUSE-NMR. Por fim, são apresentados resultados com os métodos de processamento de dados Krylov Basis Diagonalization Method (KBDM) e a Transformada Inversa de Laplace aplicados na análise de sinais SSFP. Resultados mostram que KBDM é uma ferramenta útil no processamento de dados em alta e baixa resolução, tanto na obtenção de espectros como na determinação da distribuição dos tempos de relaxação. / The application of a pulse sequence with repetition time much smaller than the relaxation times, Tp << T2; T1, causes the magnetization to reach a steady state, described by H. Y. Carr as a Steady State Free Precession (SSFP). In this condition, the signal is composed of the complex overlapping of the FID and eco components. SSFP type sequences are used in fast acquisition of NMR signals, resulting in a good signal to noise ratio (s/r) in a short time interval, however, they introduce phase and amplitude anomalies due to the complex interaction between the components of the steady state. In this work, we develop SSFP type pulse sequences for NMR in high and low resolution, with alternation and increment of phase. In high resolution, we develop SSFPdx and SSFPdxdt sequences, with linear and quadratic phase increment respectively. Results show that the low sensitivity nuclei spectra can be obtained with the same s/r ratio in smaller experimental time, about an order of magnitude, and the developed sequences can remove the spectral anomalies. In low resolution, the results show that the introduction of a phase alternation in the Continuous Wave Free Precession (CWFP) allows the elimination of the dependence of the sequence with the offset frequency and the time between pulses. Besides, we show that the CP-CWFPx-x sequence with a small refocalization angle (5° to 10°) allows the fast estimative of the longitudinal relaxation time in a single experiment. The results of the studies conducted during an international research internship are also presented. Steady state pulse sequences &ndash; DECPMG and Split 180° &ndash; were studied and implemented in compact magnetic systems: mini-Halbach and MOUSE-NMR. Finally, the results of the application of the Krylov Basis Diagonalization Method (KBDM) and the Inverse Laplace Transform for the analysis of SSFP signals are presented. The results show that KBDM is a useful tool in data processing for low and high resolution, both for obtaining spectra and determining the relaxation times distribution.

Este trabalho consiste em realizar um estudo detalhado das vantagens e desvantagens da utilização do FDM (Filter Diagonalization Method) para a análise de dados obtidos pela sequência de Precessão Livre no Estado Estacionário (Steady State Free Precession - SSFP) para aquisição rápida de espectros de Ressonância Magnética Nuclear (RMN). No caso de RMN de baixa resolução, o procedimento de aquisição rápida, SSFP, é uma poderosa ferramenta para melhorar a relação sinal/ruído, apresentando muitas aplicações práticas. Apesar desse sucesso em baixa resolução, a SSFP não é rotineiramente utilizada para aplicações em RMN de alta resolução, provavelmente devido ao (1) artefatos provenientes do truncamento do sinal e (2) as anomalias causadas pela mistura do FID com o eco dos sinais. Existem na literatura inúmeras possíveis técnicas para suprimir este tipo de problemas, porém, nenhuma delas é capaz de realmente eliminar as anomalias geradas devido ao procedimento de aquisição rápida da SSFP. O FDM é um método paramétrico não-linear para fitar sinais no domínio do tempo. Seu objetivo fundamental é resolver o Problema da Inversão Harmônica, HIP, tornando-se robusto e adequado para a análise espectral de sinais no domínio do tempo nos casos onde a Transformada de Fourier falha. Neste trabalho, demonstramos que o FDM pode ser implementado para análises de sinais SSFP, com mais eficiência que os obtidos pelos procedimentos padrões de TF. A temperatura ambiente, espectros de RMN 13C de amostras de brucina, obtidos com tempo entre pulsos de 100ms, podem ser reproduzidos com boa relação sinal/ruído e alta resolução por meio do FDM. A limitação da análise por FDM é mais relevante nos casos de espectros com alta densidade de picos em uma determinada região espectral. Nestes casos, o curto período de observação do sinal na janela do tempo impõe uma série de limitações na resolução obtida pelo FDM. / This work consists in a detailed study of the advantages and disadvantages of the use of the Filter Diagonalization Method, FDM, for data analysis in Steady State Free Precession, SSFP, technique, usually employed to implement fast acquisition of Nuclear Magnetic Resonance, NMR, spectra. In the case of low resolution NMR using fast acquisition procedures, SSFP is a powerful tool to improve signal-to-noise ratio, presenting several important practical applications. Despite its success in the low resolution regime, SSFP is not a routine technique for high resolution applications, so far, mainly because of (1) truncation artifacts and (2) the intrinsic anomalies caused by admixture of free-induction-decay and echo signals. The literature reports many possible techniques to solve such kind of problems, but, none of them is capable to really eliminate the generated spectra anomalies caused by the fast acquisition procedure used in SSFP. FDM is a parametric method for non-liner fitting performed in the time domain. Its main goal is to solve the Harmonic Inversion Problem, HIP, making it robust and suitable for spectral analysis of time signals in the cases where the Fourier Transform, FT, technique fail. In this work we demonstrate that FDM can be used to implement the analysis of the SSFP data, with more efficiency than that achieve by appropriated FT procedures. Room temperature 13C NMR spectra of brucine samples, obtained from pulse sequences with 100 ms repetition time, can be reproduced with good signal-to-noise ratio and high resolution by means of the FDM. The limitation of the FDM analysis is more relevant in the case of spectra with a high density of peaks in a limited spectral frequency region. In these cases, the reduced short observation time window imposes serious limitation to the resolution achieved by the FDM.

O objetivo deste trabalho é o desenvolvimento e implementação de metodologias de imagens por Ressonância Magnética Nuclear, para diminuição do tempo de aquisição, já que nos exames clínicos convencionais esse tempo é muito superior ao utilizado nessas seqüências, que é da ordem de T_ 2 , essas seqüências são baseadas na varredura única do espaço-k, convencionalmente denominada Echo Planar Imaging. Os propósitos de utilização dessa metodologia compreendem desde exames clínicos convencionais, em que se pretende analisar, em projetos futuros, eventos não periódicos de curta duração e a dinâmica dos sistemas biológicos estudados, até imagens de cavidades utilizando gases hiperpolarizados. As técnicas implementadas em comparação com as inicialmente propostas por Masfield apresentam uma diferença que é a inexistência do pulso de RF de inversão e, com isso, o tempo de duração das seqüências implementadas é ainda menor. Apenas não se deve esperar muito da qualidade das imagens sem o pós-processamento, uma vez que esse trabalho já está em andamento. / The objective of this work is the development and implementation of methodologies of images for Nuclear Magnetic Resonance, for reduction of the time of acquisition, since in the conventional clinical examinations this time is very superior to the used one in these sequences, that are of the order of T_ 2 , these sequences is based on the only sweepings of the space-k, conventionally called Echo Planar Imaging. The intentions of use of this methodology understand since conventional clinical examinations, where if it intends to analyze, in future projects, not periodic events of short duration and the dynamics of the biological systems studied, until socket images using hiperpolarizados gases. The techniques implemented in comparison with initially the proposals for Masfield present a difference that is the inexistence of the pulse of RF of inversion and, with this, the time of duration of the implemented sequences are still lesser. But if it does not have to wait very of the quality of the images without the after-processing, a time that this work already is in progress.

Nous présentons l'étude par Résonance Magnétique Nucléaire (RMN) du composé NiCl2-4SC(NH2)2, dit DTN, constitué de chaînes de spins 1 faiblement couplées suivant les directions transverses aux chaînes. A basse température et dans un champ magnétique compris entre les deux valeurs critiques Hc1 et Hc2, ce système s'ordonne dans un état de type Condensat de Bose-Einstein (CBE). Dans cette phase, nous décrivons d'une part la détermination expérimentale du paramètre d'ordre (aimantation transverse), dont l'amplitude est bien décrite par la théorie mais dont la phase (orientation) semble fixée par un terme d'anisotropie. D'autre part nous avons étudié les fluctuations des spins électroniques à basse énergie, par la mesure du taux de relaxation RMN 1/T1, et montré que celui-ci obéit à la loi de puissance 1/T1 propto T^5. Ce comportement peut être associé au processus de 2ème ordre lié à des excitations ayant une dispersion linéaire, tels que les quasiparticules de Bogoliubov, mais sa nature n'est pas encore bien comprise. En dehors de la phase CBE, nous décrivons l'étude des fluctuations de spin dans le régime critique quantique (H ~ Hc2), dans lequel nous établissons une loi d'échelle sur 1/T1, identique à celle que l'on a observé dans un autre composé de description équivalente (échelle de spins BPCB), prouvant ainsi l'universalité de ce régime [S. Mukhopadhyay et al., Phys. Rev. Lett. 109, 177206 (2012)]. Nous avons aussi étudié les effets du désordre induit par la substitution Br-Cl dans le composé Ni(Cl1−xBrx)2-4SC(NH2)2, pour lequel des mesures par des techniques macroscopiques ont suggéré l'existence d'une phase "verre de Bose" [R. Yu et al., Nature 489, 379 (2012)]. Cette phase est caractérisée, pour différentes concentrations du dopage x = 4%, 9%, 13%, par un pic de relaxation RMN 1/T1 au champ Hp = 13.5 T, marquant un regain des fluctuations longitudinales et présentant une forte distribution des valeurs de 1/T1 - probablement due à l'aspect vitreux du système. L'indépendance du Hp en fonction de x démontre que la physique y est dominée par les effets locaux liés aux dopants. / We present a Nuclear Magnetic Resonance (NMR) study of the NiCl2-4SC(NH2)2 compound, called DTN, consisting of spin-1 chains that are weakly coupled along the transverse directions. At low temperatures and for magnetic field values between the two critical fields Hc1 and Hc2, this system enters an ordered phase of the Bose-Einstein Condensate (BEC) type. Within this phase, we first describe the experimental determination of the order parameter (transverse magnetization), the amplitude of which is found to be well described by theory while its phase (orientation) seems to be fixed by an anisotropy term. Second, by NMR relaxation rate 1/T1 we have studied the low-energy fluctuations of the electronic spins and found that they obey the power law 1/T1 ~ T 5. Such a behaviour points to a 2nd order process involving linearly dispersing excitations, such as Bogoliubov quasiparticles, but its nature is not yet well understood. Outside the BEC phase, we report a study of the spin fluctuations in the quantum critical regime (H ~ Hc2), demonstrating a scaling law on 1/T1 similar to the one that has already been observed in another equivalent compound, BPCB spin-ladder, thus proving the universality of this regime [S. Mukhopadhyay et al., Phys. Rev. Lett. 109, 177206 (2012)]. We have also studied the effect of disorder induced by the Br-Cl substitution in the compound Ni(Cl1-xBrx)2-4SC(NH2)2 (doped DTN), for which measurements using macroscopic techniques have suggested the existence of a "Bose glass" phase [R. Yu et al., Nature 489, 379 (2012)]. This phase is characterized, for all studied doping concentrations x = 4%, 9%, 13%, by a peak in the NMR relaxation rate 1/T1 at the field value Hp ~ 13.5 T, evidencing an upsurge of the longitudinal <SzSz> spin fluctuations, and presenting strong inhomogeneity of the 1/T1 values – probably reflecting the glassy character of the system. The observed doping-independence of Hp demonstrates that the corresponding physics is dominated by local effects due to the dopants.

A tese de doutorado tem como foco o desenvolvimento de nanopartículas superparamagnéticas (Fe3O4 - magnetita) hidrofílicas e lipofílicas aplicadas à nanotecnologia do petróleo. Inicialmente, os objetivos foram voltados para a elaboração e transferência de tecnologia envolvendo uma rota de síntese de nanopartículas lipofílicas, em escala semi-industrial. Para isso, foram realizados ensaios piloto num reator com capacidade de uma tonelada, visando a produção de nanopartículas magnéticas recobertas com ácido esteárico. Mais tarde, esse trabalho foi otimizado, permitindo sua execução em laboratório, prosseguindo depois, com um escopo mais amplo, incluindo a síntese de nanopartículas recobertas com polímero hidrofílico. Nesse sentido, foram desenvolvidas duas rotas inéditas para produção desses nanomateriais. Em um segundo estágio, as investigações foram voltadas para a utilização das nanopartículas sintetizadas, em estudos de avaliação das condições dos reservatórios de petróleo. Para isso, a técnica de ressonância magnética nuclear (RMN) foi explorada, monitorando o efeito da concentração dessas nanopartículas superparamagnéticas sobre o tempo de relaxação dos prótons, e o consequente efeito de contraste nas imagens em função da magnetização. A aplicação desse tipo de ferramenta (RMN) já vem sendo feita (sem nanopartículas magnéticas) pelas empresas prestadoras de serviço ao setor de petróleo e gás, na avaliação e perfilagem de reservatórios. Isso motivou o estudo dos nanomateriais magnéticos como sondas para melhorar o mapeamento de fluidos em meio poroso. Eles seriam aplicados como aditivos em fluidos de injeção em reservatórios, tanto para imageamento, como para a obtenção de parâmetros petrofísicos. Por fim, devido à presença de grupos carboxílicos na superfície das nanopartículas hidrofílicas, foram investigadas suas interações com microcristais de carbonato de cálcio, pensando no modelo de reservatório petrolífero do tipo carbonáceo. Explorando técnicas de microscopia eletrônica de varredura (MEV) e de microscopia Raman confocal, a presença das nanopartículas magnéticas sobre a superfície da matriz mineral foi constatada, confirmando sua interação efetiva com o CaCO3. Abordando a síntese, caracterização e aplicações das nanopartículas superparamagnéticas, esta tese proporciona uma base para estudos de aplicação de nanomateriais, assunto cada vez mais relevante, diante dos inúmeros problemas e desafios enfrentados pelo setor de petróleo e gás. / The Ph.D thesis is focused on the preparation of hydrophilic and lipophilic superparamagnetic nanoparticles (Fe3O4 - magnetite) for application in oil nanotechnology. The initial efforts have been directed to the upscaling of a laboratory route of synthesis of lipophilic nanoparticles, aiming technology transfer to the industry. Accordingly, a pilot process, involving a one ton reactor, has been tested for the production of magnetic nanoparticles coated with stearic acid. After this, the research has evolved, allowing the production in the laboratory scale, and continued, pursuing the development of nanoparticles coated with a hydrophilic polymer. Two new routes for the production of these nanomaterials have been developed. In a second step, the investigations were directed to the application of these nanoparticles to the evaluation of oil reservoirs, by monitoring the proton relaxation times, using nuclear magnetic resonance (NMR), and the consequent contrasting effects observed on the images, as a function of the magnetization and the concentration of these particles. Currently, NMR tools are being employed in the oil and gas sector for the evaluation and profiling of reservoirs. This fact has stimulated the use of such nanomaterials for improving the mapping of the fluids in porous media. Introduced as additives for fluid injection into reservoirs, they can enhance the imaging and also perform the rating of petrophysical parameters. Finally, the presence of carboxylic groups on the surface of the hydrophilic nanoparticles has been explored in studies of interaction with calcium carbonate, simulating a carbonaceous type reservoir. Based on electron microscopy (SEM) and confocal Raman microscopy, the presence of magnetic nanoparticles on the surface of the mineral matrix has confirmed the interaction of these particles with the CaCO3 surface. By developing the synthesis, characterization and application of superparamagnetic nanoparticles, this work provides a useful starting point for further research on the use nanoparticles, for solving problems and challenges in the oil and gas sector.

A radioterapia de intensidade modulada (IMRT) é uma das mais modernas técnicas radioterapêuticas que permite a entrega de elevadas e complexas distribuição de doses ao volume tumoral, que necessita de novos métodos para o controle de qualidade dos procedimentos efetuados. Nos serviços de radioterapia costuma-se usar para o controle de qualidade do sistema de planejamento, a câmara de ionização para verificação pontual da dose e um dispositivo com diodos semicondutores (MapCHECK2) para a verificação bidimensional em um plano da fluência planejada, entretanto, para a verificação tridimensional dessas distribuições de doses ainda não há um dosímetro consolidado na rotina clínica. Nesse contexto, para a dosimetria tridimensional se destacam os géis poliméricos. Neste trabalho foram feitas a dosimetria convencional, pontual e bidimensional como se faz na rotina clínica e a dosimetria tridimensional utilizando o gel polimérico Magic-f, que apresenta a distribuição de dose volumétrica. Para este trabalho foi escolhido o tratamento de câncer de próstata, pois na atualidade é um dos tipos de cânceres mais comuns entre os homens. No contexto da dosimetria gel, para se obter a informação volumétrica é necessária uma técnica de imagem, no presente caso foram utilizadas imagens por ressonância magnética (magnetic resonance imaging, MRI). A partir dessas imagens é possível determinar as distribuições de doses processando-as em um software desenvolvido pelo grupo que determina as taxas de relaxação R2 associada à dose absorvida e posteriormente comparar as imagens obtidas com as imagens do sistema de planejamento. Para isso, se obteve dez cortes ao longo de cada simulador físico ou fantom em que sua comparação foi feita com a respectiva fatia do sistema de planejamento, na posição correspondente. Para uma avaliação quantitativa foi utilizado o conceito de índice gama, no critério padrão da radioterapia, 3% da dose e 3mm de distância de concordância. Os resultados obtidos com a dosimetria gel se mostram de acordo com os controles de qualidade convencionais e oferecem uma visão global da distribuição de dose no volume alvo. / The intensity modulated radiotherapy (IMRT) is one of the most modern radiotherapeutic technique that enables the delivery of high and complexes conformational doses to the tumor volume, that requires new methods for the quality assurance of the procedures performed. Radiotherapy services usually perform quality assurance of the planning system with the ionization chamber for spot-checking and an array of semiconductor diodes (MapCHECK2) to check on a two-dimensional plane, however for tridimensional dose verification does not exist an established dosimeter in the clinical routine. In this context, for three-dimensional dosimetry the polymeric gels were used. In This work the conventional one and two-dimensional dosimetry as employed in the clinical routine, and the three-dimensional dosimetry using polymer gel MAGIC- f, which provide the volumetric dose distribution. Prostate cancer clinical cases were chosen for this work because this kind of tumor is one of the most common cases in male individuals. In the context of dosimetry gel to obtain volumetric information an imaging technique is necessary, in this case the magnetic resonance imaging (MRI), was used to measure the dose. From these images it is possible to determine the distributions of doses processing them in a software developed by our research group that determines R2 relaxation rates associated with the absorbed dose and subsequently compare the images obtained with the images of the planning system. For this, ten slices were obtained along each phantom, and comparisons were made with the respective slice of the treatment planning system, in the corresponding position. For a quantitative evaluation of the gamma index , in the standard criterion in radiotherapy, 3 % dose and 3 mm distance to agreement was used. The results obtained shown that gel dosimetry agrees with the conventional quality controls and provide an overview of dose distribution in the target volume.

Estudos envolvendo o oxigênio molecular singlete (1O2) tem uma relevância biológica, uma vez que esta espécie, devido ao caráter eletrofílico, reage com moléculas ricas em elétrons como proteínas, lipídeos e DNA provocando danos que resultam em perdas de função e integridade celular. Em sistemas biológicos, a presença de outras espécies reativas de oxigênio e nitrogênio, dificultam a identificação de lesões específicas causadas por 1O2 .Neste contexto, este trabalho foi desenvolvido objetivando a síntese de endoperóxidos isotopicamente marcados com 17O para serem utilizados como fonte geradora limpa de 17[1O2] em estudos mecanísticos. A capacidade de geração de 17[1O2] pelo endoperóxido N,N\'-di(2,3-dihidroxipropil)-3,3\'-(1,4 naftilideno) dipropanamida 17O (DHPN17O2) foi confirmada utilizando o captador químico sulfato mono-{2-[10-(2-sulfoxi-etil)-antracen-9-il]-etil}éster de sódio e o nucleosídeo 2\'- desoxiguanosina. Os produtos isotopicamente marcados com 17O formados foram analisados por espectrometria de ressonância magnética nuclear e cromatografia líquida de alta eficiência acoplado ao espectrômetro de massa. Os lipídeos, em especial o colesterol ao reagir com o 1O2 geram hidroperóxidos de colesterol como produtos de oxidação primária e na presença de metais resulta em compostos de maior reatividade e toxicidade, como os radicais peroxila, que contribuem para a propagação da peroxidação lipídica. Neste trabalho, demonstramos que os hidroperóxidos de colesterol são capazes de gerar 1O2 na presença de metal através de medidas de luminescência, utilização de supressores e captador químico de 1O2. Os mecanismos de reação envolvidos foram estudados e determinados por espectrometria de massa acoplada a cromatografia líquida de alta eficiência. Por fim, a caracterização detalhada dos produtos formados por espectrometria de ressonância magnética nuclear e massa na reação do colesterol com 1O2 mostrou que além dos hidroperóxidos a reação também produz um aldeído, o 3&#946; -hidroxi-5&#946;-hidroxi-B-norcolestano-6&#946;-carboxialdeído. Até o momento, este composto havia sido identificado como um produto específico da ozonização do colesterol. Neste estudo, baseado nos estudos por reações de quimiluminescência, é proposto o mecanismo de formação deste aldeído em reações de oxidação de colesterol por 1O2 envolvendo intermediário dioxetano. / Studies involving singlet molecular oxygen (1O2) has biological relevance, once this species, due to its eletrophylic character, reacts with rich electron molecules such as proteins, lipids and DNA causing damages that result in loss of function and cellular integrity. In biological system, the presence of other reactive species of oxygen and nitrogen impair the identification of lesions caused by 1O2. In this context, this work was developed with the aim of synthesizing <SUP17O-labeleded endoperoxides to be used as a clean source of (1O2) in mechanistic studies. The ability of 17[1O2]generation by N,N\'-di(2,4-dihydroxypropyl)-1,4-naphthalene-dipropanamide labeled with 17O(DHNP17O2) was observed using the disodium salt of anthracene-9,10-diyldiethyl disulfate as a chemical trap and the nucleoside 2\'-deoxyguanosine. The products isotopically labeled with 17O were analyzed by nuclear magnetic resonance spectroscopy and high performance liquid chromatography coupled to a mass spectrometer. Lipds, in special the cholesterol, when reacting with singlet molecular oxygen generate cholesterol hydroperoxides as primary products and in the presence of metals result in compounds of higher reactivity and toxicity, such as peroxyl radicals which contribute to the propagation of lipid peroxidation. In this work, we demonstrated that cholesterol hydroperoxides are able to generate singlet molecular oxygen in the presence of metal by chemiluminescence measurements by testing the effect of singlet molecular oxygen quencher and by chemical trap. The involved reaction mechanisms were studied and determined by mass spectrometry coupled to the high performance liquid chromatography. Finally, we detailed characterization of the products formed in the reaction of cholesterol with 1O2 by nuclear magnetic resonance and mass spectroscopy showed that besides cholesterol hydroperoxides, the reaction also produces an aldehyde, 3&#946;hydroxy-5&#946;-hydroxy-B-norcholestan-6&#946;-carboxyaldehyde which had been identified as a specific product of cholesterol ozonization. In this study, based on the studies of chemiluminescence reactions, the mechanism of formation of this aldehyde in reaction of oxidation of cholesterol by 1O2 involving a dioxetane intermediate has been proposed.

Our understanding of the folding of membrane proteins lags behind that of soluble proteins due to the challenges posed by the exposure of hydrophobic regions during in vitro chemical denaturation and refolding experiments. While different folding models are accepted for soluble proteins, only the two-stage model and the long-range interactions model have been proposed so far for helical membrane proteins. To address our knowledge gap on how different membrane proteins traverse their folding landscapes, Chapter 2 investigates the structural features of SDS-denatured states and the kinetics for reversible unfolding of sensory rhodopsin II (pSRII), a retinal-binding photophobic receptor from Natronomonas pharaonis. pSRII is difficult to denature, and only SDS can dislodge the retinal chromophore without rapid aggregation. Even in 30% SDS (0.998 $\mathit{\Chi}_{SDS}$), pSRII retains the equivalent of six out of seven transmembrane helices, while the retinal binding pocket is disrupted, with transmembrane residues becoming more solvent-exposed. Folding of pSRII from an SDS-denatured state harbouring a covalently-bound retinal chromophore shows deviations from an apparent two-state behaviour. SDS denaturation to form the sensory opsin apo-protein is reversible. This chapter establishes pSRII as a new model protein which is suitable for membrane protein folding studies and has a unique folding mechanism that differs from those of bacteriorhodopsin and bovine rhodopsin. In Chapter 3, SDS-denatured pSRII, acid-denatured pSRII and sensory opsin obtained by hydroxylamine-mediated bleaching of pSRII were characterised by solution state NMR. 1D $^1$H and $^{19}$F NMR were first used to characterise global changes in backbone amide protons and tryptophan side-chains. Residue-specific changes in backbone amide chemical shifts and peak intensities in 2D [$^1$H,$^{15}$N]-correlation spectra were analysed. While only small changes in the chemical environment of backbone amides were detected, changes in backbone amide dynamics were identified as an important feature of SDS- and acid-denatured pSRII and sensory opsin. $^{15}$N relaxation experiments were performed to study the backbone amide dynamics of SDS-denatured pSRII, reflecting motions on different timescales, including fast fluctuations of NH bond vectors on the ps-ns timescale and the lack of exchange contributions on the µs timescale. These studies shed insight on differences in the unfolding pathways under different denaturing conditions and the crucial role of the retinal chromophore in governing the structural integrity and dynamics of the pSRII helical bundle. Hydrogen bonds play fundamental roles in stabilising protein secondary and tertiary structure, and regulating protein function. Successful detection of hydrogen bonds in denatured states and during protein folding would contribute towards our understanding on the unfolding and folding pathways of the protein. Previous studies have demonstrated residue-specific detection of stable and transient hydrogen bonds in small globular proteins by measuring $^1{\it J}_{NH}$ scalar coupling constants using NMR. In Chapter 4, different methods for measuring $^1{\it J}_{NH}$ scalar coupling were explored using RalA, a small GTPase with a mixed alpha/beta fold, as proof-of-concept. Detection of hydrogen bonds was then attempted with OmpX, a beta-barrel membrane protein, both in its folded state in DPC micelles and in the urea-denatured state. While $^1{\it J}_{NH}$ measurement holds promise for studying hydrogen bond formation, further optimisation of NMR experiments and utilisation of perdeuterated samples are required to improve the precision of such measurements in large detergent-membrane protein complexes. Naturally occurring split inteins can mediate spontaneous trans-splicing both in vivo and in vitro. Previous studies have demonstrated successful assembly of proteorhodopsin from two separate fragments consisting of helices A-B and helices C-G via a splicing site in the BC loop. To complement the in vitro unfolding/folding studies, pSRII assembly in vivo was attempted by introducing a splicing site in the loop region of the beta-hairpin constituting the BC loop of pSRII. The expression conditions for the N- and C-terminal pSRII-intein segments were optimised, and the two segments co-expressed. However, the native chromophore was not observed. Further optimisation is required for successful in vivo trans-splicing of pSRII and application of this approach towards understanding the roles of helices and loops in the folding of pSRII.

Les principales caractéristiques neuropathologiques de la maladie d’Alzheimer (MA) sont les plaques séniles extracellulaires composées de peptide amyloïde β (Aβ) et les enchevêtrements neurofibrillaires intracellulaires composés de Tau hyperphosphorylé. Les mécanismes conduisant à la formation de ces lésions sont encore peu connus et le laboratoire a récemment caractériser le gène “bridging integrator 1” (BIN1), deuxième facteur de risque génétique le plus associé au risque de MA, comme facteur de risque potentiellement associé à la pathologie Tau. Une interaction entre les deux protéines a été décrite in vitro et in vivo suggérant que BIN1 pourrait être impliqué dans le développement de la pathologie associée à Tau dans le cadre de la MA. Cependant, ce rôle de l'interaction BIN1-Tau dans le processus pathophysiologique de la MA n'est pas connu et il reste ainsi à déterminer si cette interaction constitue une cible thérapeutique potentielle. Ce projet a visé alors à mieux comprendre les acteurs de cette interaction en identifiant les modulateurs et les voies moléculaires impliquées dans le contrôle de l'interaction BIN1-Tau, puis de déterminer comment cette interaction est modulée dans le contexte de la MA. Nous avons utilisé pour cela des approches complémentaires de biochimie, de résonance magnétique nucléaire et de microscopie confocale. Comme modèle cellulaire, des cultures primaires de neurones de rat ont été utilisées, et la méthode “proximity ligation assay” (PLA) a été développée comme approche principale pour observer l'interaction BIN1-Tau dans ces cellules. Nous avons déterminé que l'interaction se produit entre les domaines SH3 de BIN1 et le PRD de Tau et nous avons démontré que l’interaction est modulée par la phosphorylation de Tau et BIN1: la phosphorylation de la Thréonine 231 de Tau diminue son interaction avec BIN1, tandis que la phosphorylation de BIN1 à la Thréonine 348 (T348) augmente son interaction avec Tau. Nous avons mis au point une approche de criblage d’haut contenu semi-automatisée et basé sur une bibliothèque de composés commerciaux. Ce criblage s’est basé sur des cultures primaires de neurones comme modèle cellulaire et le PLA pour détecter l'interaction BIN1-Tau. Nous avons identifié plusieurs composés capables de moduler l'interaction BIN1-Tau, notamment U0126, un inhibiteur de MEK-1/2, qui diminue cette interaction, et la cyclosporine A, un inhibiteur de la calcineurine, qui au contraire augmente celle-ci en augmentant la phosphorylation de T348 de BIN1. Par ailleurs les “Cyclin-dependent kinases” (CDK) ont été montré comme contrôlant aussi ce site de phosphorylation. Nous avons donc mis en évidence le couple Calcineurine/CDK comme contrôlant la phosphorylation T348 de Bin1 et donc l’interaction BIN1-Tau. Nous avons également développé un modèle murin de tauopathie dans lequel nous avons surexprimé BIN1 humain. Nous avons observé que la surexpression de BIN1 résorbait les déficits de mémoire à long terme et réduisait la présence d'inclusions intracellulaires de Tau phosphorylée, provoquées par la surexpression de Tau, ce qui était associé à une augmentation de l'interaction BIN1-Tau. En utilisant des échantillons de cerveau humain post-mortem, nous avons observé que les niveaux de l’isoforme BIN1 neuronal étaient diminués dans les cerveaux d’AD, alors que les niveaux relatifs de BIN1 phosphorylé à T348 étaient augmentés, suggérant un mécanisme compensatoire. Cette étude a démontré la complexité et la dynamique de l’interaction BIN1-Tau dans les neurones, a révélé des modulateurs et des voies moléculaires potentiellement impliquées dans cette interaction, et a montré que les variations de l’expression ou de l’activité de BIN1 ont des effets directs sur l’apprentissage et la mémoire, possiblement liés à la régulation de son interaction avec Tau. / The main neuropathological hallmarks of Alzheimer’s disease (AD) are the extracellular senile plaques composed of amyloid-β peptide (Aβ) and the intracellular neurofibrillary tangles composed of hyperphosphorylated Tau. The mechanisms leading to the formation of these lesions is not well understood and our lab has recently characterized the bridging integrator 1 (BIN1) gene, the second most associated genetic risk factor of AD and the first genetic risk factor to have a potential link to Tau pathology. The interaction between BIN1 and Tau proteins has been described in vitro and in vivo, which suggests that BIN1 might help us to understand Tau pathology in the context of AD. However, the role of BIN1-Tau interaction in the pathophysiological process of AD is not known, and whether this interaction is a potential therapeutic target remains to be determined. The aim of this project is to better understand the actors of BIN1-Tau interaction through the identification of the modulators and the molecular pathways involved therein, as well as to understand how BIN1-Tau interaction is modulated in the context of AD. We employed biochemistry, nuclear magnetic resonance, and confocal microscopy. We used rat primary neuronal cultures (PNC) as the cellular model and developed the proximity ligation assay (PLA) as the main readout of the BIN1-Tau interaction in cultured neurons. We determined that the interaction occurs between BIN1’s SH3 domain and Tau’s PRD domain, and demonstrated that it is modulated by Tau and BIN1 phosphorylation: phosphorylation of Tau at Threonine 231 decreases its interaction with BIN1, while phosphorylation of BIN1 at Threonine 348 (T348) increases its interaction with Tau. We developed a novel, semi-automated high content screening (HCS) assay based on a commercial compound library, also using PNC as the cellular model and PLA as the readout of BIN1-Tau interaction. We identified several compounds that are able to modulate the BIN1-Tau interaction, most notably U0126, an inhibitor of MEK-1/2, which reduced the interaction, and Cyclosporin A, an inhibitor of Calcineurin, which increased the interaction through increasing the BIN1 phosphorylation at T348. Furthermore, Cyclin-dependent kinases (CDK) were also shown as regulator of this phosphorylation site. These results suggest that the couple Calcineurin/CDK regulates BIN1 phosphorylation at T348 and consequently the BIN1-Tau interaction. We also developed a mouse model of tauopathy in which we overexpressed human BIN1. We observed that the overexpression of BIN1 rescued the long-term memory deficits and reduced the presence of intracellular inclusions of phosphorylated Tau, caused by Tau overexpression, and this was associated with an increase of BIN1-Tau interaction. Also, using post-mortem human brain samples, we observed that the levels of the neuronal BIN1 isoform were decreased in AD brains, whereas the relative levels of BIN1 phosphorylated at T348 were increased, suggesting a compensatory mechanism. Altogether, this study demonstrated the complexity and the dynamics of BIN1-Tau interaction in neurons, revealed modulators of and molecular pathways potentially involved in this interaction, and showed that variations in BIN1 expression or activity have direct effects on learning and memory, possibly linked to the regulation of its interaction with Tau.

Les acides gras de la série n-3 et notamment l’acide docosahexaénoïque (DHA) jouent un rôle important dans la prévention des maladies cardiovasculaires. Un de ses métabolites, la protectine DX (PDX), qui est un isomère de la protectine D1 (PD1), inhibe l’agrégation des plaquettes sanguines. D’autres composés similaires appelés "poxytrins", qui possèdent aussi un triène conjugué avec une géométrie E,Z,E, ont également été synthétisés à partir d'autres acides gras polyinsaturés (AGPI) via la lipoxygénase de soja. Ces composés présentent des propriétés anti-agrégantes en inhibant la cyclo-oxygénase plaquettaire et le récepteur du thromboxane A2. Dans cette thèse, nous décrivons de nouveaux composés dihydroxylés synthétisés par la 15-lipoxygénase de soja à partir de l’acide alpha-linolénique (18:3n-3), un acide gras polyinsaturé indispensable consommé au niveau du gramme chez l’Homme adulte. Il est converti en acides gras monohydroxylés et dihydroxylés. Ces composés ont été séparés par HPLC en phase inverse et caractérisés par GC-MS après dérivation adéquate. Un acide gras monohydroxylé, majoritaire, l’acide 13(S)-octadécatriénoïque et quatre acides gras dihydroxylés ont été détectés. Ces derniers présentent tous un spectre UV caractéristique avec une absorption maximale à 270 nm et deux épaulements à 260 et 280 nm. Les spectres UV de deux d'entre eux sont superposables à celui de la PDX, ce qui suggère une géométrie E,Z,E des doubles liaisons de leur triène. La caractérisation complète de ces composés a été réalisée par RMN à haut champ et par GC-MS. Ce sont les acides 9(R),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12E,14E-triénoïque, 9(S),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque et 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïque. Ils sont également synthétisés par la 15 lipoxygénase recombinante humaine de type 2. Ces composés dihydroxylés 9,16-diHOTEs ont été testés sur les plaquettes isolées à partir du sang humain. Nous avons observé que seules les molécules ayant la géométrie E,Z,E du triène conjugué inhibent l'agrégation plaquettaire induite par le collagène et inhibent la cyclooxygénase-1 (COX-1) de mouton. Les propriétés anti-inflammatoires de ces produits ont également été étudiés. Tous les isomères 9,16-diHOTEs, possédant un triène conjugué avec une géométrie E,Z,E, inhibent la COX-2 recombinante humaine et seul l’acide 9(R),16(S)-dihydroxy-octadéca-10E,12Z,14E-triénoïques inhibe la 5-lipoxygénase des leucocytes, siège de la synthèse des leucotriènes issus de l’acide arachidonique. En conclusion, les composés dihydroxlés possédant un triène conjugué E,Z,E, issus du 18:3n-3, ainsi que la PDX, inhibent l’activité des COX-1 et 2, et seraient anti-agrégants et anti-inflammatoires. Ces résultats donnent des perspectives pharmacologiques aux recommandations nutritionnelles promouvant la consommation d’acide alpha linolénique. / N-3 fatty acids, especially docosahexaenoic acid (DHA), play an important role in the prevention of cardiovascular diseases. One metabolite of DHA, protectin DX (PDX), an isomer of protectin D1 (PD1) (Chen P et al., 2009),possesses inhibits blood platelet aggregation. Similar compounds called "poxytrins", which have a conjugated triene with a E,Z,E geometry have also been synthesized from other polyunsaturated fatty acids (PUFA) by soybean lipoxygenase. They have anti-aggregating properties by inhibiting platelet cyclooxygenase and thromboxane A2 receptor (Chen P et al., 2011). In this thesis, we describe new dihydroxy compounds synthesized by the soybean 15-lipoxygenase from alpha-linolenic acid (18:3n-3), an essential PUFA that is consumed in the gram range in human adults . It is converted into monohydroxylated and dihydroxylated derivatives. These compounds were separated by reverse phase high performance liquid chromatography (HPLC) and characterized by gas chromatography-mass spectrometry (GC-MS) after appropriate derivatization. A main monohydroxylated fatty acid, 13(S)-octadecatrienoic acid (13(S)-OH-18:3) and four dihydroxylated fatty acids were detected. The last ones have all a characteristic UV spectrum with a maximum absorbance at 270 nm with two shoulder peaks at 260 and 280 nm. The UV spectra from two of them are superimposable to that of PDX, suggesting a E,Z,E geometry for their conjugated triene. The complete characterization of these compounds was performed by high field nuclear magnetic resonance (NMR) and by GC-MS. These are the 9(R),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12E,14E-trienoic, 9(S),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic and 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acids. They can also be synthesized by the (type 2) 15 human recombinant lipoxygenase. These dihydroxylated compounds (9,16-diHOTEs)were tested on isolated human blood platelets. We observed that only molecules containing a conjugated triene with a E,Z,E geometry are able to inhibit platelet aggregation induced by collagen, and inhibit sheep cyclooxygenase-1 (COX-1). The anti-inflammatory properties of these products were also studied. All 9,16-diHOTEs isomers having a conjugated triene with a E,Z,E geometry, inhibit human recombinant cyclooxygenase-2 (COX-2) and only 9(R),16(S)-dihydroxy-octadeca-10E,12Z,14E-trienoic acid inhibits polymorphonuclear leukocytes (PMN) 5-lipoxygenase which is involved in the leukotriene synthesis from arachidonic acid. In conclusion, the E,Z,E dihydroxlated compounds from 18:3n-3, as well as PDX, inhibiting the COX-1 and 2 activities appear to be anti-aggregatory and anti-inflammatory agents. These results provide pharmacological perspectives to nutritional recommendations promoting the intake of alpha-linolenic acid.