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.

Sus1 is a conserved protein involved in chromatin remodeling and mRNA biogenesis. The SUS1 gene of Saccharomyces cerevisiae is unusual, as it contains two introns and is alternatively spliced, retaining one or both introns in response to changes in environmental conditions. SUS1 splicing may allow the cell to control Sus1 expression, but the mechanisms that regulate this process remain unknown. In this thesis project, we have investigated whether the structure adopted by SUS1 RNA sequences contributes to regulate the splicing of this gene. Using in silico analyses together with NMR spectroscopy, gel electrophoresis and UV thermal denaturation experiments, we first show that the downstream intron (I2) of SUS1 forms a weakly-stable, 37-nucleotide stem-loop structure containing the branch site near its apical loop and the 3' splice site after the stem terminus. A cellular assay revealed that two of four mutants containing altered I2 structures had significantly impaired SUS1 expression. Semi-quantitative RT-PCR experiments indicated that all mutants accumulated unspliced SUS1 pre-mRNA and/or induced distorted levels of fully spliced mRNA relative to wild-type. Concomitantly, Sus1 cellular functions in histone H2B deubiquitination and mRNA export were affected in I2 hairpin mutants that inhibited splicing. The second part of the thesis project focuses on the exon located between the two introns of the SUS1 gene. This middle exon (E2) can be skipped during splicing, is generated in circular form, and has been found to influence the splicing of the flanking introns, an unusual situation in budding yeast where splicing mainly relies on intron recognition. Using NMR spectroscopy, gel electrophoresis, UV thermal denaturation and ribose 2'-OH modification experiments combined with computational predictions, we show that E2 of SUS1 comprises a conserved double-helical stem topped by a three-way junction. One of the hairpins emerging from the junction exhibited significant thermal stability and was closed by an unusually structured purine-rich loop. This loop contained two consecutive sheared G:A base pairs and was structurally related to the substrate loop of the VS ribozyme. Cellular assays revealed that three mutants containing altered E2 structures had impaired SUS1 expression and that a compensatory mutation restoring the conserved stem recovered expression to wild-type levels. Semi-quantitative RT-PCR experiments indicated that all mutants were capable of altering the quantities of unspliced and/or fully-spliced SUS1 RNA transcripts relative to wildtype. Overall, the results gathered in this thesis project indicate that RNA structures formed by the middle exon and the second intron of the S. cerevisiae SUS1 gene are relevant for splicing and also influence other processes of SUS1 mRNA biogenesis. / Sus1 es una proteína conservada implicada en remodelación de cromatina y biogénesis de moléculas de ARNm. El gen SUS1 de Saccharomyces cerevisiae es peculiar, ya que contiene dos intrones y sufre un proceso de ayuste (corte y empalme) alternativo, reteniendo uno o ambos intrones en respuesta a cambios en las condiciones ambientales. El ayuste del ARNpre-m de SUS1 puede permitir a la célula controlar la expresión de la proteína Sus1, pero los mecanismos que regulan este proceso son poco conocidos. En este proyecto de tesis hemos investigado si la estructura adoptada por secuencias de ARN de SUS1 contribuye a regular el proceso de ayuste de este gen. Utilizando análisis in silico junto con espectroscopia de RMN, electroforesis en gel y experimentos de desnaturalización térmica monitorizados por UV, primero demostramos que el ARN del segundo intrón (I2) del gen SUS1 forma una horquilla débilmente estable de 37 nucleótidos. Esta horquilla contiene nucleótidos del sitio de ramificación (branch site) en su bucle apical y nucleótidos del sitio 3' de empalme adyacentes al extremo inferior del tallo. A través de ensayos funcionales descubrimos que dos de cuatro mutantes que alteran la estructura de la horquilla I2 exhibían peor expresión de SUS1. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes acumularon ARNpre-m SUS1 no ayustado y/o indujeron cambios en los niveles de ARNm maduro con respecto a la secuencia silvestre. Además, las funciones celulares de Sus1 relativas a desubicuitinación de histona H2B y transporte de ARNm se vieron afectadas en los mutantes de la horquilla I2 que inhibían el proceso de ayuste. La segunda parte de la memoria de tesis se centra en el análisis del exón central (E2) situado entre los dos intrones del gen SUS1. Este exón puede eliminarse durante el proceso de ayuste, se genera en forma circular, e influye en el procesamiento de los intrones adyacentes, una situación inusual para las regiones exónicas de S. cerevisiae, donde el ayuste se basa principalmente en el reconocimiento de intrones. Utilizando experimentos de espectroscopía de RMN, electroforesis en gel, desnaturalización térmica y modificación química combinados con predicciones computacionales, demostramos que el ARN del exón E2 de SUS1 forma un tallo conservado de doble hélice coronado por una intersección de tres hélices. Una de las horquillas que emergen de esta intersección presentó una estabilidad térmica significativa, así como un bucle apical rico en purinas inusualmente estructurado. Este bucle contiene dos pares de bases G:A consecutivos y está estructuralmente relacionado con el bucle de substrato de la ribozima VS. Ensayos celulares revelaron que tres mutantes con estructuras modificadas de E2 exhibían peor expresión de SUS1, y que una mutación compensatoria que restauraba el tallo conservado recuperaba la expresión a los niveles de la secuencia silvestre. Experimentos de RT-PCR semicuantitativos indicaron que todos los mutantes de E2 eran capaces de alterar las cantidades de transcritos ayustados y no ayustados de SUS1 con respecto a la secuencia silvestre. En general, los resultados obtenidos en este proyecto de tesis indican que las estructuras de ARN formadas por el exón central y el segundo intrón del gen SUS1 de S. cerevisiae son relevantes para el ayuste y otros procesos implicados en la biogénesis del ARNm del gen SUS1. / Sus1 és una proteïna conservada implicada a la remodelació de la cromatina i la biogènesi de l'ARNm. El gen SUS1 de Saccharomyces cerevisiae és inusual, ja que conté dos introns i s'empalma de manera alternativa, retenint un o ambdós introns en resposta a canvis en les condicions ambientals. L'empalmament de SUS1 pot permetre a la cèl·lula controlar l'expressió de Sus1, però els mecanismes que regulen aquest procés són segueixen sent desconeguts. En aquest projecte de tesi investiguem si l'estructura adoptada per seqüències d'ARN de SUS1 contribueix a regular l'empalmament d'aquest gen. Emprant anàlisi in silico juntament amb espectrometria de RMN, electroforesi en gel i experiments de desnaturalització tèrmica d'UV, es mostra primer que l'intró aigües a baix (I2) de SUS1 forma una estructura de forqueta de 37 nucleòtids feblement estable que conté el lloc de la branca a prop del seu bucle apical; i el lloc d'empalmamnet 3¿ després de l'extrem de la forqueta. Un assaig cel·lular va revelar que dos de quatre mutants que contenien estructures alterades de l'I2 havien modificat significativament l'expressió de SUS1. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants acumulaven el pre-ARNm madur respecte al tipus salvatge. Concomitantment, les funcions cel·lulars de Sus1 a la desubiqüitinació de la histona H2B i l'exportació d'ARNm es van veure afectats als mutants de la forqueta d'I2 que inhibeixen l'empalmament. La segona part del projecte de tesi se centra a l'exó situat entre els dos introns del gen SUS1. Aquest exó (E2) es pot ometre durant l'empalmament, es genera amb forma circular, i s'ha trobat que influeix a l'empalmamet dels introns que flanquegen, una situació inusual al llevat on l'empalmament està basat principalment al reconeixement d'introns. Emprant espectroscòpia de RMN, electroforesi en gel, desnaturalització tèrmica d'UV i experiments de modificació de ribosa 2¿-OH combinats amb prediccions computacionals, mostrem que E2 de SUS1 comprén un tall conservat de doble hèlix corornat per una unió de tres vies. Una de les forquetes que emergeixen de la unió, va mostrar una estabilitat tèrmica significativa i va ser tapada per un bucle ric en purina inusualment estructurat. Aquest bucle contenia dos pars de bases G:A tallats consecutivament i estava estructuralment relacionat amb el bucle de substrat del ribozim VS. Els assajos cel·lulars van revelar que tres mutants que contenien estructures alterades de E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivells del tipus salvatge. Els assajos cel·lulars van revelat que tres mutants que contenien estructures alterades d'E2 havien alterat l'expressió de SUS1 i que una mutació compensatòria que restaurava el tall conservat recuperava l'expressió a nivell d'un tipus salvatge. Els experiments semi-quantitatius de RT-PCR van indicar que tots els mutants eren capaços d'alterar les quantitats de transcrits d'ARN de SUS1 no empalmats i/o empalmats en relació amb el tipus salvatge. En general, els resultats obtinguts en aquesta investigació indiquen que les estructures d'ARN formades per l'exó mitjà i el segon intró de SUS1 de S. cerevisiae són rellevants per l'empalmament i també influeixen a altres processos de biogènesi de l'ARN de SUS1 / Abuqattam, AN. (2017). IDENTIFICATION OF RNA STRUCTURES MODULATING THE EXPRESSION OF THE mRNA BIOGENESIS FACTOR SUS1 [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90524 / TESIS

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.