Nous avons analysé par cryomicroscopie électronique la morphologie et la structure de longues chaines d’ADN condensées par un polycation tétravalent, la spermine (polyamine). Les expériences ont été réalisées i) avec des solutions de chaînes diluées et ii) avec des chaines isolées confinées dans la capside d’un virus.Les expériences ont été réalisées avec de l’ADN Lambda (48kbp) en solution diluée (0.03 mM Ph) et à faible concentration ionique (10 mM Tris HCl, 1 mM EDTA, pH 7.6). Nous avons exploré une large gamme de concentrations en spermine, allant du seuil de précipitation (0.05 mM sp) jusqu’à la limite de re-solubilization et au-delà (400 mM sp). Seize minutes après mélange de l’ADN et de la spermine, les échantillons sont piégés en film mince et vitrifiés à basse température pour garder intactes les conditions ioniques, puis imagés à basse température sous faibles doses d’électrons (cryoMET). La plupart des chaînes d’ADN forment des agrégats de tores de structure hexagonale avec des interdistances entre hélices de 2.93, 2.88, et 2.95 nm pour des concentrations en spermine respectivement égales à 0.05, 1 et 100 mM spermine, ce qui est en bon accord avec les données collectées précédemment par diffraction des rayons X. A concentration plus élevée en spermine (200mM), les tores hexagonaux sont remplacés par des faisceaux cholestériques de structure plus lâche (3.32 nm entre hélices). Nous en déduisons que la forme comme la structure des condensats cristallins liquides ADN-sp sont liées aux interdistances entre hélices et déterminés par les conditions ioniques i.e. par l’énergie cohésive entre chaînes d’ADN. En dehors du domaine de précipitation (400mM sp), les molécules d’ADN forment un réseau soluble de fines fibres (4-6nm de diamètre) qui nous amènent à reconsidérer l’état de ces chaiînes en présence de spermine. Nous avons également conçu des expériences pour visualiser les agrégats formés 6 à 60 sec après addition de la spermine dans les mêmes conditions de tampon. Parmi les nombreuses formes originales que nous avons observées (absentes après 16 min), la présence de fibres étirées ou en hélice, visibles seulement après 9sec, nous conduit à proposer que les chaines d’ADN soient immédiatement étirées après addition de spermine puis relaxent sous forme de fibres hélicoïdales qui donnent naissance à de petits toroids (comprenant quelquefois moins d’une chaine) qui grandissent et fusionnent. Nous avons également analysé les dimensions de l’ensemble des tores observés et montré l’existence de contraintes géométriques qui restent à élucider. Puisqu’il était généralement impossible de prévenir l’agrégation des chaines d’ADN, nous avons choisi une autre approche pour analyser le collapse de chaines d’ADN individuelles. Nous avons utilisé une population de virus T5 contenant une fraction de leur génome initial (12-54 kbp). La molécule d’ADN, initialement confinée dans le petit volume de la capside (de de 80nm diamètre) est collapsée par addition de spermine. Par comparaison avec le premier jeu de données, nous avons travaillé à concentration plus élevée en ADN (0.45 mM Phosphates dans l’ensemble de l’échantillon) et la concentration en spermine a été ajustée entre 0.05 et 0.5 mM (ce qui correspond à des rapports de charges +/- bien inférieurs). Ces expériences ont donc été réalisées au voisinage de la ligne de précipitation, dans la « région de coexistence », entre le domaine où les chaines sont en condition de pelote et le domaine ou les chaines sont toutes collapsées sous forme de tores. Nous avons montré l’existence de formes intermédiaires entre ces deux états que nous appelons « tores chevelus » dans lesquels une partie de la molécule est condensées dans le tore alors que l’autre partie reste non condensée. Les distances entre hélices ont également été mesurées. Elles sont plus grandes dans ces structures intermédiaires que dans les tores formés à plus forte concentration en spermine. Ces deux séries d’expériences montrent l’intérêt des méthodes de cryo-microscopie pour étudier la structure locale des phases condensées de l’ADN. Nous avons montré comment le confinement modifie le comportement de l’ADN en solution et l’intérêt d’étudier ces effets compte tenu de son importance dans le contexte biologique. / By using cryo-electron microscopy, we analyzed the morphology and structure of long double-stranded DNA chains condensed upon addition of varying amounts of the tetravalent polycation spermine (polyamine). Experiments have been performed i) with chains diluted in the bulk and ii) with individual chains confined in a virus capsid.Bulk experiments have been done with lambda DNA (48.5 kbp) at low concentration (0.03 mM Ph) and in low salt conditions (10 mM Tris HCl, 1 mM EDTA, pH 7.6). We explored a wide range of spermine concentration, from the onset of precipitation (0.05 mM sp) up to above the resolubilization limit (400 mM sp). Sixteen min after mixing spermine and DNA, samples have been trapped in thin films and vitrified in liquid ethane to keep ionic conditions unchanged, and imaged at low temperature with low doses of electrons (cryoTEM). DNA chains mostly form large aggregates of toroids in which DNA chains are hexagonally packed with interhelical spacings of 2.93, 2.88, and 2.95 nm at 0.05, 1 and 100 mM spermine, respectively, in agreement with previous X-ray data. At higher spermine concentration (200 mM), hexagonal toroids are replaced by cholesteric bundles with a larger interhelical spacing (3.32 nm). We conclude that the shape and the structure of the liquid crystalline sp-DNA condensates are linked to the DNA interhelix spacing and determined by the ionic conditions i.e. by the cohesive energy between DNA strands. Outside of the precipitation domain (400 mM spermine), DNA chains form a soluble network of thin fibers (4-6 nm in diameter) that let us reconsider the state of these DNA chains in excess of spermine. We also designed experiments to visualize condensates formed 6-60 sec after mixing Lambda DNA with 0.05 mM spermine, under identical buffer conditions. Among multiple original shapes (not found after 16 min), the presence of stretched and helical elongated fibers seen only 9sec after addition of spermine let us propose that DNA chains are immediately stretched upon addition of spermine, relax into helical structures and finally form small toroids (containing in some cases less than one Lambda chain) that further grow and aggregate. We also analyzed the dimensions and structural details of the complete collection of toroids, and reveal the existence of geometric constraints that remain to be clarified. Since it was only exceptionally possible to prevent the aggregation of DNA in dilute solution, we used another approach to observe the collapse of single DNA chains. We handled a population of T5 viruses containing a fraction of their initial genome (12-54 kbp long). The Na-DNA chain, initially confined in the small volume of the capsid (80nm in diameter) is collapsed by the addition of spermine. Compared to the first set of experiments, we explored a higher DNA concentration range (0.45 mM Phosphates in the whole sample) and the spermine concentration was varied from 0.05 to 0.5 mM (which corresponds to much lower +/- charge ratios). Experiments are thus performed close to the precipitation line, in the coexistence region, between the region where all chains are in a coil conformation, and the region where all chains are collapsed into toroids. We describe the existence of intermediate states between the coil and the toroidal globule that were not reported yet. In these “hairy toroids”, part of the DNA chain is condensed in the toroid and the other part stays uncondensed outside of it. The interhelical spacing was also measured; it is larger in these partly-condensed toroids than in the fully organized toroids formed at higher spermine concentration.These two series of experiments show the interest of cryoEM to analyze the structural polymorphism and local structure of spermine-DNA aggregates. We also demonstrated how the confinement interferes with DNA condensation and the interest to investigate such effects that are important in the biological context.

Les oligonucleotides sont utilisés pour de nombreuses applications dans le domaine du diagnostic et ils peuvent également être utilisés comme traitement pour de nombreuses maladies. Les oligonucléotides sont des polyanions qui viennent s'hybrider sur leurs séquences complémentaires elles aussi anioniques. Les répulsions électrostatiques impliquent que l'addition de charges positives sur les oligonucléotides serait bénéfique pour diminuer les répulsions et améliorer l'hybridation. Dans le but de diminuer ces répulsions électrostatiques, des conjugués oligonucléotide-oligocation sur lesquels sont greffées des unités spermines ont été développés. Les conjugués oligonucléotideoligocation sont synthétisés sur un synthétiseur automatique d'oligonucléotide en utilisant la chimie des phosphoramidites. Les« Zip Nucleic Acid »ou ZNAs sont des oligonucléotides portant une queue cationique de quelques unités de spermine et sont de charge globale négative. Les modifications apportées permettent d'améliorer l'hybridation en accélérant la reconnaissance de la séquence cible et en augmentant la température de fusion linéairement avec le nombre de spermines greffées sans altérer la spécificité. Les ZNAs se révèlent être efficaces utilisés comme amorces ou sondes en PCR et ils apparaissent comme de nouveaux outils intéressants pour la biologie moléculaire. Les petits ARNintérferents (si RNA) induisant l'extinction d'un gène par la voie d' ARN interférence ont suscités un grand engouement ces dernières années, cependant leur très faible pénétration cellulaire est un frein majeur à leur utilisation. C'est pour cela que les conjugués oligonucléotide- oligospermine ont un réel intérêt pour le domaine de la thérapie in vivo. Les duplexes SIRNAPLUS cationiques sont des siRNAs ciblantspécifiquement un ARN messager. Ils sont constitués d'un brin sens ARN-oligospermine de charge globale positive hybridé à un brin antisens. Les résultats ont montré que lesSIRNAPLUS pouvaient entrer seuls dans les cellules sans agent de transfert pour induire l'extinction d'un gène cible et les premières expériences montrent qu'ils sont actifs in vivo. Mes travaux de thèse ont porté sur le développement des conjugués oligonucléotide oligospermine et démontrent des applications potentielles dans le domaine du diagnostic et de la thérapie. / Oligonucleotides are finding an extremely large number of applications in molecular diagnostics and might become a very selective class of drugs for the treatment of a vast palette of diseases. Oligonucleotides are polyanions that exert their specifie activity following hybridization to a complementary sequence borne by another polyanionic nucleic acid. Simple electrostatic considerations imply that hybridization energy and cell binding couId benefit from addition of cationic groups to the oligonucleotide structure. Towards the aim of improving hybridization by decreasing electrostatic repulsions between the negatively charged strands, oligonucleotide-oligocation conjugates whose global charge is modulated by the number of cationic spermine moieties grafted on the oligonucleotides have been developed. Oligonucleotide-oligospermine conjugates are produced using an automated solid-phase synthesis of conjugates that are entirely based on the phosphoramidite coupling chemistry. Zip Nucleic Acids {ZNAs} are oligonucleotides with a short polycationic tail, composed of relatively few spermine units, leading to molecules overall negative in charge. The modification improves hybridisation by accelerating the target recognition and increases the melting temperature linearly with the number of grafted spermines on the oligonucleotide without altering the specificity. ZNAs have been shown to be potent primers and probes for PCR and are new interesting tools for molecular biology and diagnostics applications. Small interfering RNA (siRNA}-mediated gene silencing has become a drug development paradigm. As drug candidates, they must aIso be able to cross the anionic cell membrane. However, still one major limitation of the use of siRNA remains their inability to penetrate efficiently into cells of a particular tissue or tumour. That gives to oligonucleotide-oligospermine conjugates a real interest in this domain and more generally in vivo therapies. Cationic SIRNAPLUS are duplexes of small RNAs targeting a specifie mRNA. They are produced as an oligospermine-RNA sense strand, with positive global charge, associated to an antisense RNA strand. Results have shown that cationic siRNAs are able to enter cells efficiently without vector and to display silencing activity at nanomolar concentration. To have positive global charge, the number of spermine moieties has been increased. Purification and characterization methods have been developed to have cationic siRNAs compatible with in vivo experiments. My thesis will describe the synthesis of oligonucleotide-oligospermine conjugates as well as their applications.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Acrolein is produced endogenously after traumatic brain injury (TBI) and is considered a primary mechanism for secondary damage occurring after TBI. We are using frontal cortex networks derived from mouse embryos and grown on microelectrode arrays in vitro to monitor the spontaneous activity of networks and the changes that occur after acrolein application. Networks exposed to acrolein exhibit a biphasic response profile. An initial increase in network activity, followed by a decrease to 100% activity loss in applications ≥ 50 µM. In applications below 50 µM, acrolein was not toxic but generated activity instability with coordinated but irregular population busts lasting for up to 6 days. The increase in activity preceding toxicity may be linked to a decrease in free spermine, a free radical scavenger that modulates Na+, K+, Ca+ channels as well as NMDA, Kainate, and AMPA receptors. Action potential wave shape analysis after 20 and 30 µM acrolein application revealed a concentration-dependent 15-33% increase in peak to peak amplitude within minutes after exposure. For the same concentrations of acrolein (50 µM), the time required to reach 100% activity loss (IT100) was longer in serum-free medium than in medium with 5% serum, in which IT100 values were reduced by a factor of 4. The greater toxicity in the presence of serum may be explained by acrolein adducts on serum proteins. These reaction products have been shown by other labs to be toxic in cell culture. This in vitro system could be used to expand biochemical analyses such as acrolein-induced spermine depletion and may provide an effective platform for investigating cell culture correlates of secondary TBI damage.

Amantadine is a cationic aliphatic primary amine eliminated by the kidneys, excreted predominantly unchanged into the urine, and undergoes limited metabolism. Renal tubule secretion has an important role in its elimination. We studied two aspects of amantadine disposition, firstly acetylation, by developing a model to induce the enzyme spermidine/spermine N1-acetyltransferase (SSAT1) with N1, N11-diethylnorspermine (DENSPM) and alcohol (Alc) as representative agents reported to induce its activity, and secondly renal secretion, by studying the effect of intravenous bicarbonate infusion on its renal elimination. We drew two conclusions, firstly, longer exposure to Alc combined with DENSPM administration provided the greatest potentiation of SSAT1 enzyme activity than each agent alone, which indicates a high likelihood of synergy between Alc and DENSPM; and secondly, bicarbonate load administered to healthy male volunteers impairs amantadine renal secretion in the absence of a clinically important change in blood pH, serum creatinine concentration or urinary creatinine clearance.

Amantadine is a cationic aliphatic primary amine eliminated by the kidneys, excreted predominantly unchanged into the urine, and undergoes limited metabolism. Renal tubule secretion has an important role in its elimination. We studied two aspects of amantadine disposition, firstly acetylation, by developing a model to induce the enzyme spermidine/spermine N1-acetyltransferase (SSAT1) with N1, N11-diethylnorspermine (DENSPM) and alcohol (Alc) as representative agents reported to induce its activity, and secondly renal secretion, by studying the effect of intravenous bicarbonate infusion on its renal elimination. We drew two conclusions, firstly, longer exposure to Alc combined with DENSPM administration provided the greatest potentiation of SSAT1 enzyme activity than each agent alone, which indicates a high likelihood of synergy between Alc and DENSPM; and secondly, bicarbonate load administered to healthy male volunteers impairs amantadine renal secretion in the absence of a clinically important change in blood pH, serum creatinine concentration or urinary creatinine clearance.

Thesis (Ph.D. in Pharmaceutical Sciences) -- University of Colorado at Denver and Health Sciences Center, 2006. / Typescript. Includes bibliographical references (leaves 130-149, 177-182). Free to UCDHSC affiliates. Online version available via ProQuest Digital Dissertations;

The purpose of this study was to prepare and characterize complexes produced by the reactions of spermine and spermidine with palladium(II) chloride. A first analysis of the problem seemed to indicate that preparation of the complexes should be straightforward and not difficult. Bonding of the nitrogen atoms to the metals allows sterically for formation of two six-membered rings separated by a tetramethylene chain in a spermine complex, and by a six-membered chelate ring in a spermidine complex.

The effects of spermine, an endogenous polyamine, were examined in area CA1 of the rat hippocampal slice preparation. Spermine, at low millimolar concentrations, rapidly and potently depressed NMDA and K/AMPA-mediated population EPSPs, and GABA-mediated monosynaptic population IPSPs. These effects contrast with its well-known potentiation of NMDA currents at lower concentrations. Our results raise the possibility that the large intracellular stores of spermine that are released after various neural insults could act as an endogenous neuroprotective mechanism by limiting excessive calcium entry.

The polyamines spermine and spermidine play important roles in many cellular processes, and unusual levels of these polyamines have been associated with numerous human diseases. Spermidine/spermine N¹-acetyltransferase (SSAT) is an enzyme involved in polyamine regulation, where acetylation of polyamines by SSAT ultimately leads to their degradation or export from the cell. In this dissertation, x-ray crystallography and nuclear magnetic resonance (NMR) are used to provide insights into the structure and function of this important enzyme. X-ray crystallography provided two distinct views of SSAT: one of the enzyme in complex with coenzyme A (CoA), and another of the enzyme in complex with CoA and the polyamine spermine. Together, the two structures reveal structural plasticity in the active site of the enzyme. The complex with spermine provides a direct view of polyamine binding by SSAT, and shows that the enzyme relies heavily on associated water molecules to bind spermine; these water molecules also appear to form a "proton relay" between the primary amine of spermine and the side-chain of a conserved glutamate residue. Guided by the structural results, NMR methods were used to test hypotheses regarding the enzyme mechanism of SSAT. The activity of the enzyme over a range of solution conditions, and towards different polyamine substrates, was determined; the effects of mutating single amino acids in the enzyme were also evaluated. The enzyme appeared to be most active between pH 8.5 and 9.5, and mutation of the aforementioned glutamate significantly altered this behavior. This suggests the glutamate is directly involved in the acetyltransfer reaction, where it likely functions as a catalytic base though the proton relay in the enzyme active site. These studies advance our general understanding of how polyamines are regulated in mammalian cells, and have the potential to assist in developing new therapeutic options for human diseases involving polyamines. / text