Le but de ce travail de thèse a été de développer à MIRC en une capacité à observer deux aspects clefs du métabolisme cérébral chez le rongeur par spectroscopie RMN des noyaux X : le métabolisme mitochondrial du glucose à partir de l’observation du 13C et la synthèse d’ATP par observation du 31P. Ces développements s’inscrivent à la fois dans une recherche fondamentale pour améliorer notre compréhension du signal RMN et raffiner son analyse ainsi que du métabolisme cérébral chez les sujets sains. Ils s’inscrivent aussi dans une recherche translationnelle avec la possibilité d’évaluer certains aspects du métabolisme comme potentiels biomarqueurs de maladies neurodégénératives. Ces travaux ont pu être réalisés à très haut champ (11.7T) permettant d’obtenir un meilleur rapport signal à bruit. Dans un premier temps, nous présenterons le développement d’une séquence de transfert de saturation pour la mesure des flux de synthèse d’adénosine triphosphate (ATP) et de phosphocréatine (PCr). Cette séquence a été optimisée pour sélectionner avec un module de localisation ISIS le signal émis par le cerveau uniquement. Avec l’augmentation de la résolution spectrale à haut champ, cette séquence a pu être utilisée pour caractériser le phosphate inorganique extracellulaire, et prévenir un biais de quantification possible à plus bas champ. De plus, elle a permis l’observation de l’adaptation du métabolisme cérébral chez les rats transgéniques BACHD, modèles de la maladie de Huntington. Ces rats présentent une augmentation d’environ 10% de la concentration de PCr permettant de pallier à leur plus faible taux de synthèse d’ATP qui lui est diminué de moitié. Dans un second temps, la mise en place d’un pipeline automatisé d’analyse des données a permis d’explorer le modèle métabolique bicompartimental de la consommation de glucose observée en spectroscopie 13C, qui prend en compte le cycle de Krebs dans les neurones et les astrocytes. Deux corrections majeures ont été apportées au modèle traditionnel permettant d’expliquer les dynamiques à moyen et long termes. La première est la mise en évidence d’un pool de glutamate vésiculaire agissant comme tampon temporel au marquage du glutamate, la seconde est la présence d’une dilution 6 fois plus importante de pyruvate vers les astrocytes que vers les neurones. Ces résultats viennent renforcer les hypothèses entourant le couplage métabolique entre ces deux types cellulaires. Ces hypothèses ont pu être testées après l’optimisation d’une séquence d’acquisition du signal RMN des noyaux 13C par transfert de polarisation (DEPT), testée in vivo dans le cerveau du rat sain. Finalement, l’utilisation combinée de la spectroscopie 31P et 13C a été appliquée chez le rat sous intoxication chronique au 3-NP, une toxine inhibant le cycle de Krebs et utilisée comme modèle de la maladie de Huntington. / The aim of this thesis was to develop at MIRCen new capabilities to observe two key aspects of energy metabolism in rodent brains using X nuclei NMR spectroscopy: glucose consumption with 13C spectroscopy and adenosine triphosphate (ATP) synthesis with 31P measurements. These developments will be used to both expand general understanding of brain metabolism in healthy subjects but also provide technical tools to search for biomarkers in translational projects of drug development applied to neurodegenerative diseases. This work was done at very high field (11.7T) where signal to noise could be maximized. In the first part, we present the optimization of saturation transfer sequence to measure ATP synthesis rate as well as phosphocreatine (PCr) synthesis rate. With ISIS module, the signal was localized to a voxel containing only the brain, eliminating outside source of signal. With the higher spectral resolution offered by high fields, a second, extracellular pool of Pi was characterized which could prevent possible biases in flux quantification of ATP synthesis. This sequence was also applied to measure metabolic adaptation of BACHD rat models (models of Huntington’s disease, HD) where it was found that the 10% increase in PCr concentration could palliate the ATP synthase activity that is halved in this model. In the second part, we present how deeper analysis of 13C data using automatic differential equation writing script was used to better understand the bicompartmental model of glucose degradation to glutamate and glutamine, which accounts for TCA cycle in neurons and astrocytes. Two major corrections were made to the traditional model, to fit mid- and long-term unexplained dynamics. Looking at glutamate and glutamine isotopomer labeling dynamics, the necessity of adding a vesicular glutamate temporal buffer was made evident. The distinction between astrocytic and neuronal pyruvate dilution also showed that astrocytes use up to 6 times more pyruvate than neurons showing intricate metabolic coupling between the two cell types. These results have then been tested in vivo after optimization of the ISIS-DEPT sequence to observe 13C labeling in the rat brain. Finally, experiments combining 31P and 13C spectroscopy were performed on rats chronically intoxicated with 3-NP, a toxin inhibiting TCA cycle which is used as a model of HD.

Genomic DNA is under constant assault by environmental factors that introduce a variety of DNA lesions. Cells evolved several DNA repair and recombination mechanisms to remove these damages and ensure the integrity of the DNA material. A variety of specific proteins, called nucleases, processes toxic DNA structures that deviate from the heritable duplex DNA as common pathway intermediates. DNA-induced protein ordering is a common feature in all DNA repair nucleases. Still, the conformational requirement of the DNA and the protein and how they control the catalytic selectivity of the nuclease remain largely unknown. This study focus on the bases of catalytic activity of a protein belongs to the 5’ nuclease super-family called the human Flap endonuclease 1 (FEN1); it removes excess 5’ flaps that are generated during DNA replication. hFEN1 mutations and over-expression had been linked to a variety of cancers. This thesis aims to study the structural and dynamic properties of free hFEN1 and the catalytic activity of DNA-bound hFEN1 in solution utilizing the modern high-resolution multidimensional Nuclear Magnetic Resonance (NMR) spectroscopy. It was possible to depict the secondary structure and backbone conformation in solution of wild type (WT) hFEN1 by the usage of the improved list of assigned resonances, derived from the NMR 2D and 3D ¹⁵N-detected experiments and compared to the assignment with the previously published resonance assignment (BMRB id: 27160). I was successfully assigned the new spectrum and enhanced it by assigning seven more residues. Moreover, we tested the interaction of 1:10 ratio of hFEN1-Ca2+ with DNA by the ¹³C-detected 2D CACO experiment. The results indicate hFEN1:DNA interaction. Furthermore, parts of hFEN1 get more ordered/structured once DNA appears, thus we recorded the protein flexibly by 2D ¹H-¹⁵N TROSY-HSQC using the relaxation rate parameters: longitudinal R1, transverse R2 complemented with ¹⁵N-{¹H} NOEs (heteronuclear Overhauser enhancement). It was found that the overall molecular architecture is rigid, and the highest flexibility lies in the α2-α3 loop and arch (α4-α5) regions. Further analysis is needed to understand more profoundly the activity of hFEN1 in an atomic level by inducing mutations and testing the protein in various environmental conditions.

Bacterial cell division relies on the cell division septum to form at the mid-cell position. In gram negative bacteria, this is mediated by three proteins, MinC, MinD and MinE. Together these proteins interact with each other and the membrane in a dynamic, oscillating process which prevents cell division septum formation at the cell poles. The early phase of this process involves MinD binding to the membrane, which is triggered upon binding of ATP. Subsequent interactions with MinE result in stimulation of the ATPase activity of MinD. After hydrolysis, MinD is released from the membrane and diffuses to a new binding site. Many in silico models have been constructed of the Min system in an attempt to describe its self-organizing behaviour. A limitation of these models is that, in order to prevent rapid re-binding of MinD to the membrane after hydrolysis of ATP, the exchange of bound ADP for ATP is assumed to be a slow process, on the order of 1/s . In order to provide experimental evidence of the rate of nucleotide binding, we performed a series of triple-resonance NMR experiments to complete a partial assignment of backbone atom resonances, which required the application of deuterium labelling and amino acid-specific selective unlabelling. After the introduction of ATP, it was discovered that no dimerization had been induced, in contrast with existing literature. It was proposed that MinD from N. gonorrhoeae only forms a dimer in the presence of a membrane, while literature with MinD from E. coli shows it does not have this requirement. Interestingly while dimerization had not been induced, there was a persistent population of dimeric species even in the absence of nucleotide. This was discovered to be the result of disulfide formation, likely an artifact of established purification protocols. Binding of both ADP and ATP to MinD were studied by titration using NMR, with the relative affinity of both nucleotides to MinD being indistinguishable. By analyzing peak coalescence in the half-bound condition, a maximum rate was determined for nucleotide binding, with the lifetime being on the order of 170ms. Results from this experiment support models requiring a slow nucleotide binding step, and help enhance understanding of how Min proteins sustain oscillations required for normal cell division.

The subject of my thesis is a design of the methods optimizing, the acquisition of MR signals when small objects measure. The thesis is divided into several parts in order to give a deeper knowledge of the problem. The first part focuses on the theory associated with NMR (Nuclear Magnetic Resonance) and SNR (signal-to-noise ratio). The practical verification of the theory follows. The conclusion of this work is focused on MR images filtering by use of wavelet transform to suppress a noise in the image. The method optimization of MR acquisition parameters for the measurement of small objects is then distributed to the entire work.

CArG box is a highly conservative DNA motif found in Serum Response Element (SRE), which regulates expression of c-fos gene. In this thesis, short oligodeoxynucleotides containing CArG box were measured using nuclear magnetic resonance spectroscopy to evaluate their ability to preferentially form hairpin over duplex. 1H spectra were measured at temperature range (274-360) K. We acquired thermodynamic parameters of the transition between hairpin and single strand by fitting the temperature- dependent chemical shifts. The hairpin structure of our oligonucleotide samples was confirmed by non- B-DNA patterns in NOESY spectra, absence of concentration dependence of melting, and other pieces of evidence. Thus, occurrence of unusual DNA conformation of CArG box in native DNA, potentially even cruciform is highly possible. This could explain the high affinity between SRE and its transcription factor. Powered by TCPDF (www.tcpdf.org)

Temperature dependences of nuclear magnetic resonance spectra of isotopes 207 Pb, 137 Ba, and 93 Nb in polycrystalline samples of PbxBa1-x(FeNb)0.5O3, with x = 0 (BFN), 0.5 (PBFN), 1 (PFN) and in Pb(FeNb)0.5O3 single crystal (PFN SC) were acquired. Measured nuclear magnetic resonance spectra are analyzed in this work. The temperature dependence of the spectra show strong broadening with decreasing temperature. Part of the temperature dependences of nuclear magnetic resonance spectra did not comply with expected characteristics. Possible hypotheses for interpretation of such behavior are discussed in the text. Probable arrangement of Fe and Nb cations in PFN and BFN phases is deduced and, based on these differences, the contrast in Néel temperatures of these phases is explained.

Serum response factor (SRF) is a transcription factor which binds to a highly conserved DNA sequence called the CArG box. According to the nucleotide sequence of CArG box it could form a hairpin structure or a cruciform. In this master thesis, the structure of the CArG box in a human gene c-fos was studied by nuclear magnetic resonance. 1 H spectra at temperatures 274{356 K, two-dimensional 1 H{1 H NOESY spectra, and two-dimensional 1 H{13 C HMBC spectra for DNA sequences with lengths of 12, 14 and 16 nucleoti- des were acquired. The thermodynamic parameters of formation of the secondary structure in the samples were determined from the measured temperature depen- dencies. The hairpin formation in the samples was con rmed based on the NOESY spectra and the lack of dependency of the melting temperature on concentration. The observed difference of the secondary structure from B-DNA could serve as a possible explanation of the high a nity of SRF to CArG box. 1

Protein-protein interactions are involved in various biological processes and detailed characterization of their structural basis by the means of structural biology is often instrumental for rigorous understanding of underlying molecular mechanisms. This information is important not only for fundamental biology but also plays an important role in search for sites amenable for therapeutic intervention. Nuclear magnetic resonance spectroscopy is alongside X-ray crystallography and single-particle cryo-electron microscopy one of the key high-resolution techniques in structural biology. Although its applicability to larger systems has a well-known physical limit, it offers unique capabilities in addressing highly dynamic or inherently heterogeneous systems. In this doctoral thesis, the solution-based NMR approach was used for detailed structural characterization of selected biologically important proteins and their complexes that provided important insights into their biological roles. In three distinct projects, I (i) studied the relationship between the structural effects of particular modifications in the insulin-like growth factor II (IGF-II) and their selectivity to the insulin axis receptors; (ii) the specific binding mechanism of the SH3 domain from the Crk-associated substrate (CAS); (iii) and...

Glucans are (1→3)-β-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.

The Candida albicans cell wall provides an architecture that allows for the organism to survive environmental stress as well as interaction with host tissues. Previous work has focused on growing C. albicans on media such as Sabouraud or YPD at 30 °C. Because C. albicans normally colonizes a host, we hypothesized that cultivation on blood or serum at 37 °C would result in structural changes in cell wall mannan. C. albicans SC5314 was inoculated onto YPD, 5% blood, or 5% serum agar media three successive times at 30 °C and 37 °C, then cultivated overnight at 30 °C in YPD. The mannan was extracted and characterized using 1D and 2D 1H NMR techniques. At 30 °C cells grown in blood and serum contain less acid-stable terminal β-(1→2)-linked d-mannose and α-(1→2)-linked d-mannose-containing side chains, while the acid-labile side chains of mannan grown in blood and serum contain fewer β-Man-(1→2)-α-Man- (1→ side chains. The decrement in acid-stable mannan side chains is greater at 37 °C than at 30 °C. Cells grown on blood at 37 °C show fewer →6)-α-Man-(1→ structural motifs in the acid-stable polymer backbone. The data indicate that C. albicans, grown on media containing host-derived components, produces less complex mannan. This is accentuated when the cells are cultured at 37 °C. This study demonstrates that the C. albicans cell wall is a dynamic and adaptive organelle, which alters its structural phenotype in response to growth in host-derived media at physiological temperature.