Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1994. / Vita. Abstract. Includes bibliographical references (leaves 61-73). Also available via the Internet.

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 naturally occurring polyamines play an essential role in cell growth and proliferation. The levels of polyamines have been shown to increase in rapidly proliferating cancer cells. Therefore, compounds that inhibit enzymes in polyamine biosynthetic pathway may have therapeutic potential. Compounds capable of providing both in vitro and in vivo inhibition of almost all enzymes in the polyamine biosynthetic pathway are known. An exception is the lack of an agent that inhibits spermidine/spermine N 1 -acetyltransferase (SSAT), the rate-limiting enzyme in the catabolism of polyamines. The design, synthesis and characterization of five new polyamine analogues as potential inhibitors of SSAT are presented. Three compounds, N 1 -[3-(propenamido) propyl]-1,4-diaminobutane dihydrochloride 5 , N 1 -[3-(maleimido)propyl]-1,4-diamino-butane dihydrochloride 7 and N 1 -[3-(2-bromoacetamido)propyl]-1,4-diaminobutane dihydrochloride 9 , were designed as active-site-directed affinity label inhibitors. Two compounds, N-[N-(5-acetamido-2-hydroxypentyl-3-aminopropyl)]-1,4-diaminobutane trihydrochloride 12 and N-[3-(2-hydroxyethylamino)propyl]-1,4-diaminobutane trihydrochloride 14 , were designed as transition state-like analogue inhibitors. These compounds were synthesized using one key intermediate, N-(3-aminopropyl)-N,N ′ -bis-(tert-butoxycarbonyl)-1,4-diaminobutane 3 . Three of these synthesized compounds, 5 , 7 and 12 were evaluated for their ability to inhibit SSAT. The enzyme used was a crude extract of human large cell undifferentiated lung carcinoma cell line NCI H157 cells. These synthetic analogues when tested against the crude enzyme extract at concentrations of 0.05, 0.1, 1 and 5 μM appeared to show no effects on the activity of SSAT.

L’autophagie est un processus d’autodigestion dans lequel la cellule dégrade ses propres composants dans le but de maintenir l’homéostasie dans ses conditions basales. En absence de nutriments, l’autophagie est activée et favorise la survie cellulaire en fournissant des substrats énergétiques résistant aux conditions de stress. Autophagie et métabolisme communiquent à différents niveaux; une baisse en métabolites richement énergétiques, tels qu’en ATP et en NADH, est détectée par des senseurs cellulaires (AMPK et SIRT1 respectivement) et mène à l’activation de l’autophagie. Ici, nous définissons un niveau supplémentaire de régulation de l’autophagie induite par le jeûne. Dans ce travail, nous montrons que cette privation en nutriments est caractérisée par une diminution rapide de l’Acétyl CoA, intégrateur majeur de l’état nutritionnel au carrefour du catabolisme des graisses, des sucres et des protéines. La baisse en AcCoA s’accompagne de la réduction proportionnelle des niveaux généraux d’acétylation des protéines ainsi que par l’induction de l’autophagie. Les manipulations destinées à augmenter ou diminuer les niveaux cytosoliques d’AcCoA, ciblant soit la synthèse mitochondriale soit son transport dans le cytoplasme, résultent en la suppression ou l’induction de l’autophagie aussi bien dans les cultures cellulaires que dans les tissus de souris. La déplétion en AcCoA impacte directement l’activité des KATs utilisant l’AcCoA comme substrat pour l’acétylation protéique. Nous avont montré que cette baisse en AcCoA réduit spécifiquement l’activité de EP300; cette KAT est en effet nécessaire à la suppression de l’autophagie à des niveaux élevés d’AcCoA, se comportant ainsi comme le senseur des niveaux cytosoliques d’AcCoA. A son tour, EP300 contrôle l’autophagie en inhibant les protéines autophagiques clés. Dans l’ensemble, nos résultats illustrent les fonctions de l’AcCoA cytosolique comme régulateur métabolique central de l’autophagie, délimitant ainsi des stratégies pharmacologiques centrées sur l’AcCoA qui permettent la manipulation thérapeutique de l’autophagie. En effet, la privation en nutriments et la restriction calorique sont connues pour jouer un rôle positif sur la santé et la longévité en promouvant leurs effets. Néanmoins, les stratégies basées sur la restriction calorique sont difficilement applicables en clinique. Ici, nous proposons une nouvelle définition biochimique des Mimétiques de la Restriction Calorique, composés imitant l’effet positif du jeûne. Dans notre contexte, un MRC est un composé capable de réduire l’acétylation protéique par des mécanismes distincts mais convergents: premièrement, par diminution des niveaux d’AcCoA, deuxièmement par inhibition directe des KATs, et enfin, par activation des protéines déacétylases. Ces résultats de l’exécution d’un programme cellulaire conduisent finalement à des effets pro-santé liés à la restriction calorique incluant mais non limités à l’autophagie. / Autophagy is a self-digestion process in which cell degrades its own components in order to maintain homeostasis in basal conditions. In absence of nutrients, autophagy is activated and promotes cell survival by providing energetic substrates to sustain stressful condition. Autophagy and metabolism crosstalk at different levels; a drop in energy-rich metabolites, such as ATP and NADH, is detected by cellular sensors (AMPK and SIRT1 respectively) and leads to autophagy activation. Here, we define a further regulatory level of starvation-induced autophagy. In this work, we show that nutrient deprivation is characterized by a rapid depletion of Acetyl CoA, a major integrator of the nutritional status at the crossroads of fat, sugar, and protein catabolism.Decrease in AcCoA is accompanied by the commensurate reduction in overall protein acetylation levels as well as by autophagy induction. Manipulations designed to increase or reduce cytosolic levels of AcCoA, either targeting mitochondrial synthesis or its transport in the cytoplasm, resulted in the suppression or induction of autophagy both in cultured cells and in mice tissues. Depletion of AcCoA directly impacts on the activity of cellular KATs, which use AcCoA as substrate for acetylating proteins. We showed that a drop in AcCoA specifically reduces the activity of EP300; this KAT was indeed required for the suppression of autophagy by high AcCoA levels, thus behaving as the sensor of cytosolic AcCoA levels. In turn, EP300 controls autophagy by inhibiting key autophagic proteins. Altogether, our results indicate that cytosolic AcCoA functions as a central metabolic regulator of autophagy, thus delineating AcCoA-centered pharmacological strategies that allow for the therapeutic manipulation of autophagy. Indeed, nutrient deprivation and caloric restriction are known to play pro-healthy and longevity promoting effects. Nonetheless, CR-based strategies are hardly suitable in clinical settings. Here, we propose a new biochemical definition of Caloric Restriction Mimetics, compounds that mimic the positive effects of nutrient starvation. In our setting, a CRM is a compound able to reduce protein acetylation through distinct but convergent mechanisms: first, by decreasing AcCoA levels, second by directly inhibiting KATs, third by the activation of protein deacetylases. This results in the execution of a cellular program ultimately leading to CR-related pro-healthy effects, including but not limited to autophagy.

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

As our body's guardian, the immune system maintains systemic health through removal of pathogens, damage and cancer. Ageing of the immune system is associated with compromised immune responses as well as decreased tumour surveillance and is therefore a key risk factor for major diseases in the elderly. Adaptive immune responses are mediated by T and B lymphocytes, and failure in adaptive immunity is a particular hallmark of the ageing organism. Here we show that autophagy is impaired in aged murine B lymphocytes, and loss of autophagy causes severely reduced B cell responses. Our data demonstrate that B cell senescence can be reversed in an autophagy-dependent manner by spermidine, a naturally occurring polyamine metabolite. Mechanistically, our study reveals that the translation factor eIF5A, that requires spermidine for its activation, regulates the expression of the master autophagy/lysosomal transcription factor TFEB. Importantly, we show in humans that spermidine, eIF5A and TFEB levels decrease with age and may serve as ageing biomarkers. Taken together our results indicate that the translational control of autophagy by eIF5A is dysregulated with ageing, and identify a novel pathway with therapeutic implications.

Abstract not available

With the rise of nanotechnology in the last decade, nanofluidics has been established as a research field and gained increased interest in science and industry. Natural aqueous nanofluidic systems are very complex, there is often a predominance of liquid interfaces or the fluid contains charged or differently shaped colloids. The effects, promoted by these additives, are far from being completely understood and interesting questions arise with regards to the confinement of such complex fluidic systems. A systematic study of nanofluidic processes requires designing suitable experimental model nano – channels with required characteristics. The present work employed thin liquid films (TLFs) as experimental models. They have proven to be useful experimental tools because of their simple geometry, reproducible preparation, and controllable liquid interfaces. The thickness of the channels can be adjusted easily by the concentration of electrolyte in the film forming solution. This way, channel dimensions from 5 – 100 nm are possible, a high flexibility for an experimental system. TLFs have liquid IFs of different charge and properties and they offer the possibility to confine differently shaped ions and molecules to very small spaces, or to subject them to controlled forces. This makes the foam films a unique “device” available to obtain information about fluidic systems in nanometer dimensions. The main goal of this thesis was to study nanofluidic processes using TLFs as models, or tools, and to subtract information about natural systems plus deepen the understanding on physical chemical conditions. The presented work showed that foam films can be used as experimental models to understand the behavior of liquids in nano – sized confinement. In the first part of the thesis, we studied the process of thinning of thin liquid films stabilized with the non – ionic surfactant n – dodecyl – β – maltoside (β – C₁₂G₂) with primary interest in interfacial diffusion processes during the thinning process dependent on surfactant concentration 64. The surfactant concentration in the film forming solutions was varied at constant electrolyte (NaCl) concentration. The velocity of thinning was analyzed combining previously developed theoretical approaches. Qualitative information about the mobility of the surfactant molecules at the film surfaces was obtained. We found that above a certain limiting surfactant concentration the film surfaces were completely immobile and they behaved as non – deformable, which decelerated the thinning process. This follows the predictions for Reynolds flow of liquid between two non – deformable disks. In the second part of the thesis, we designed a TLF nanofluidic system containing rod – like multivalent ions and compared this system to films containing monovalent ions. We presented first results which recognized for the first time the existence of an additional attractive force in the foam films based on the electrostatic interaction between rod – like ions and oppositely charged surfaces. We may speculate that this is an ion bridging component of the disjoining pressure. The results show that for films prepared in presence of spermidine the transformation of the thicker CF to the thinnest NBF is more probable as films prepared with NaCl at similar conditions of electrostatic interaction. This effect is not a result of specific adsorption of any of the ions at the fluid surfaces and it does not lead to any changes in the equilibrium properties of the CF and NBF. Our hypothesis was proven using the trivalent ion Y3+ which does not show ion bridging. The experimental results are compared to theoretical predictions and a quantitative agreement on the system’s energy gain for the change from CF to NBF could be obtained. In the third part of the work, the behavior of nanoparticles in confinement was investigated with respect to their impact on the fluid flow velocity. The particles altered the flow velocity by an unexpected high amount, so that the resulting changes in the dynamic viscosity could not be explained by a realistic change of the fluid viscosity. Only aggregation, flocculation and plug formation can explain the experimental results. The particle systems in the presented thesis had a great impact on the film interfaces due to the stabilizer molecules present in the bulk solution. Finally, the location of the particles with respect to their lateral and vertical arrangement in the film was studied with advanced reflectivity and scattering methods. Neutron Reflectometry studies were performed to investigate the location of nanoparticles in the TLF perpendicular to the IF. For the first time, we study TLFs using grazing incidence small angle X – ray scattering (GISAXS), which is a technique sensitive to the lateral arrangement of particles in confined volumes. This work provides preliminary data on a lateral ordering of particles in the film. / Mit dem Heranwachsen der Nanotechnologie in den vergangenen zehn Jahren hat sich die Nanofluidik als Forschungsbereich etabliert und erfährt wachsende Aufmerksamkeit in der Wissenschaft, sowie auch in der Industrie. Im biomedizinischen Bereich, wo intrazelluläre Prozesse häufig komplexer, nanofluidischer Natur sind, wird sich vermehrt für ein detailliertes Verständnis von nanofluidischen Prozessen interessiert, z.B. für den Einfluss von Kolloiden verschiedenster Form oder elektrischer Ladung auf die Kanäle und auf das Fließverhalten oder die Auswirkungen der Einengung von Flüssigkeiten und Kolloiden in Nanometer Geometrien. In der vorliegenden Arbeit werden dünne flüssige Filme, hinsichtlich ihrer Funktion als nanofluidische Modelle untersucht. Im ersten Teil der Arbeit wurde die Fließgeschwindigkeit des Fluids aus dem dünnen Film, abhängig von der Konzentration der filmstabilisierenden Tensidmoleküle n – Dodecyl β – D – Maltoside ( β – C₁₂G₂) bei einer konstanten Elektrolytkonzentration von 0.2 mM NaCl untersucht. Mit einem theoretischen Modell konnte das Dünnungsverhalten nachgezeichnet werden. Es wurde eine kritische Tensidkonzentration gefunden, unter der die Oberflächen lateral mobil sind und über der sie sich wie fest verhalten. Dadurch konnten wir Aufschluss darüber erlangen, wie die Oberfläche des Films unter verschiedenen Bedingungen geschaffen ist, und das in Bezug zur Verteilungsdichte der Moleküle an den Oberflächen setzen. Im weiteren wurden komplexere, nanofluidische Systeme untersucht, wobei zum einen ~ 1 nm lange, stäbchenförmige, multivalent geladene Spermidin - Moleküle die punktförmigen Elektrolyte ersetzten. Es konnte eine deutliche Veränderung der Stabilität zwischen Filmen mit und ohne Stäbchen festgestellt werden. Die Filme, mit NaCl, blieben länger in dem metastabilen „Common Film“ (CF) Zustand als die Filme, die eine vergleichbare Konzentration von Spermidin Stäbchen beinhalteten. Die Ergebnisse deuteten auf eine zusätzliche Anziehungskraft durch Brückenbildung zwischen zwei geladenen Oberflächen durch gegensätzlich geladene Stäbchenförmige Moleküle hin. Es konnte gezeigt werden, dass dieser Effekt weder ein Ergebnis von spezifischer Ionenadsorption an die Filmoberfläche war, noch ein Unterschied in den Gleichgewichtszuständen von den Dicken der CFs und der Newton Black Films (NBFs) hervorrief, was auf die korrekte Annahme der Ionenstärke in der Lösung schließen ließ. Auch in Versuchen mit ebenfalls trivalenten Ionen YCl3 wurde festgestellt, dass kein vergleichbarer Überbrückungseffekt auftritt. Die Ergebnisse wurden mit theoretischen Simulationen verglichen und es wurde eine quantitative Übereinstimmung gefunden bezüglich der Größe des Systeminternen Energiegewinns durch den Überbrückungseffekt. Desweiteren wurde das Fließverhalten von Fluiden mit Kolloiden eingeengt in Nanometer Geometrien untersucht. Für zwei verschiedene Arten von Nanopartikeln (Fe3O4 stabilisiert mit Oleinsäure und polymerstabilisierte Goldpartikel) wurde eine Verlangsamung der Fließgeschwindigkeit festgestellt. Mit einem theoretischen Modell konnte das Fließverhalten nur für enorm erhöhte Viskositätswerte des Fluids erklärt werden. Die Viskositätserhöhung wurde mit Partikelaggregaten, die den Ausfluss behindern, erklärt und diskutiert, unter der Annahme eines nicht - Newtonischen Fließverhaltens der Dispersionen. Gleichermaßen wurde die strukturelle Anordnung der Partikel in den Filmen hinsichtlich ihrer vertikalen und lateralen Verteilung untersucht. In dieser Arbeit werden vorläufige Ergebnisse präsentiert, die noch weiteren Studien bedürfen. Mit Neutronenreflexion sollte die Anordnung der Partikel orthogonal zur Oberfläche im Film analysiert werden. Eine qualitative Analyse lässt schließen, dass bei einer höheren Konzentration von Partikeln in Lösung, sich auch eine erhöhte Konzentration von Partikeln im dünnen Film befindet. Leider konnten die Daten nicht hinsichtlich der Lage der Partikel analysiert werden. Zum ersten Mal wurden dünne flüssige Filme mit Kleinwinkelröntgenstreuung unter streifendem Einfall (GISAXS) analysiert. Mit Hilfe dieser Methode sollte eine laterale Anordnung der Partikel im Film untersucht werden. Es konnten erfolgreiche Messungen durchgeführt werden und mit Hilfe der rechnergestützten Analyse konnte eine Aussage gemacht werden, dass ~ 6 nm große Teilchen in ~ 43 nm Abstand sich im Film befinden. Die Aussage bezüglich der kleinen Teilchen könnte sich auf einzelne, kleinere Partikel beziehen, allerdings könnten auch die 43 nm eine relevante Strukturgröße darstellen, da es in der Dispersion gehäuft Aggregate mit dem Durchmesser in dem Größenbereich vorliegen. Zusammenfassend können sich mit dieser Arbeit die dünnen flüssigen Filme als eine wichtige Kernmethode der Untersuchung von nanofluidischen Prozessen, wie sie häufig in der Natur vorkommen, behaupten.

Putrescine, spermidine and spermine are the polyamines biosynthesized by human cells via ornithine decarboxylase (ODC) and are also sourced from the diet. Polyamines are required for malignant and normal cell growth and development. Pharmacological suppression of polyamine biosynthesis, by difluoromethylornithine, and inflammation, via sulindac, has demonstrated ~70% efficacy in preventing premalignant colorectal adenomas (CRA) in a clinical trial; however, high polyamine intakes mitigated this preventative action. Further, dietary polyamines increase the dysplasia of CRA in initiated animal models of colorectal cancer (CRC) and are hypothesized to function as tumor promoters. Human research on dietary polyamines was limited until the development of a dietary database in 2007 but, continues to be limited by the lack of a biomarker of exposure. Chapter 1 of this dissertation tests the hypothesis that dietary polyamines increase risk of CRA in polyp-formers (n = 1164) and found evidence to support this hypothesis. However, only women, younger participants and certain genotypes experienced more risk of CRA with high polyamine exposure. Chapter II tests the hypothesis that dietary polyamines increase the risk for CRC in an average risk cohort of post-menopausal women (n = 87,620) and did not find evidence to support this hypothesis in the whole population. Rather, dietary polyamines were non-significantly protective against CRC and significantly protective when paired with aspirin use and against CRC-specific death. There was some evidence to support an increase in risk of CRC in younger participants with high polyamine exposure. Overall, the first two chapters suggest that dietary polyamines protect the colorectum in normal risk individuals but promote carcinogenesis in high risk individuals. Chapter III tests the hypothesis that dietary polyamine intake correlates with urinary polyamine output in a group of overweight/obese, older men (n = 36) and Chapter IV tests the hypothesis that intake of highly ripe sweet cherries will increase urinary polyamine output in a subgroup of 10 men from Chapter III. The findings from these chapters suggest there may be a positive correlation, but that a better measure of dietary polyamine intake is needed to determine if urinary polyamines are biomarkers of exposure to polyamines.

The developmental profiles of mRNA and protein expression for ornithine decarboxylase (ODC), the Na$ sp+$-dependent glucose co-transporter (SGLT1), sucrase isomaltase (SI), and the Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoforms in the postnatal rat small intestine, as well as the effects of exogenous spermidine on their precocious development, were examined. Postnatal age had a significant effect with all enzymes and the nutrient transporter maturing around weaning. Consecutive exposure to exogenous spermidine during suckling precociously induced ODC mRNA, SI protein, and SGLT1 gene expression in the proximal and distal small intestine. Levels of Na$ rm sp+K sp+$ ATPase $ alpha sb1$ and $ beta sb1$ subunit isoform mRNA were precociously induced in the proximal small intestine only. These findings show that exposure to exogenous spermidine can promote precocious alterations in intestinal enzyme and nutrient transporter expression; however, it appears that spermidine must be continuously supplied for these alterations to persist in suckling rats.