Influenza A virus (IAV) exhibits a high mutation frequency. Mutations in primary viral targets such as hemagglutinin, neuraminidase, and the proton channel M2 have afforded IAV with substantial drug resistance to previously effective drugs such as oseltamivir and amantadine (AMT) along with their analogs. One of the main drug-resistant alterations in the M2 protein that has become ubiquitous is the mutation S31N. Divalent copper has previously been suggested to have anti-IAV properties using in vitro assays involving Xenopus oocytes and SSNMR. In this work, the EC50 of AMT, two AMT analogs, CuCl2 · 2H2O, and four previously published Cu2+ complexes were tested for antiviral activity against the California/07/2009 (H1N1) IAV strain containing the S31N M2 protein in viral mini-plaque assays. AMT, CuCl2 · 2H2O, and two previously published divalent copper complexes were tested for M2 proton-transport blocking efficacy in liposomes using truncated M2 (22-62, S31N). Two novel divalent copper species, NAG101 and NAG107, both derived from AMT analogs were developed and tested for both antiviral activity and M2 blocking efficacy. Cell integrity was maintained at concentrations up to 1 mM (48 hours of exposure) with all compounds except Cu(II) 3. In the viral mini-plaque assay the novel divalent copper complexes NAG101 (EC50 25.7 +/-7.7 µM) and NAG107 (EC50 2.91 +/- 0.29 µM) were 2.5-fold and 21-fold better than AMT (EC50 64.3 +/- 9.3 µM) respectively. In the liposome assay NAG101 (EC50 18.9 +/- 1.5 µM) and NAG107 (EC50 4.5 +/- 0.6 µM) were 2.5-fold and 11-fold better than AMT (EC50 49.3 +/- 2.9 µM respectively. In the viral mini-plaque assay, CuCl2 · 2H2O (EC50 57.2 +/- 10.1 µM) was comparably effective to AMT, but was more than ten times more effective in the liposome assay targeting S31N M2 with an EC50 6.1 +/- 0.8 µM. Divalent copper species possess anti-IAV activity against the ubiquitous S31N M2-containing viral strain California/07/2009 (H1N1) and may prove to be effective against other IAV strains by blocking M2.

Myelin basic protein (MBP) has fascinated researchers and clinicians alike due to its major structural role in myelin and the central nervous system, and its potent auto-immunogenic properties that cause demyelination in animal models. The charge variants of MBP have been of particular interest. The C1 component, the least modified and most cationic of the variants, is the most abundant form of MBP in healthy adult myelin. The C8 component, the most modified and the least cationic variant, has been found in higher proportions in myelin of MS patients and children. Here, an investigation of the structural differences between C1 and C8 components of MBP was conducted. The spectral and hydrodynamic properties of these variants were monitored via a number of biophysical/biochemical techniques. The effect of zinc (Zn2+) on the conformational behaviour of MBP was examined. Zn2+ is an abundant metal in the brain, and had been previously shown to induce hydrodynamic compaction in MBP. Both variants have a loose tertiary arrangement with subtle differences. This arrangement is deficient in secondary structure and undergoes non-cooperative temperature-induced melting. Zn2+ stabilizes a molten globular-like state with enhanced ANS fluorescence, and promotes oligomerization.

Divalent thulium has been doped into CsCaI₃, CsCaBr₃, CsCaCl₃ and RbCaI₃- a series of AMX₃ salts. Using previously published optical spectra, a series of parameterised energy level calculations have been performed. The calculated energy levels, optimised crystal field parameters and simulated optical absorption spectra are presented. Theoretical predictions yield excellent approximation to the experimental data. Temperature dependent fluorescent lifetimes from the (³F₄,t₂g) and (³H₆,t₂g) excited (emitting) states have been measured using a pulsed dye laser. For CsCaBr₃ and RbCaI₃ doped with Tm²⁺, visible emission for the (³F₄,t₂g) state yields 10 K and 28 K lifetimes of 1.7 μs and 0.40 μs respectively. In both cases no emission is observed at room temperature. Considering direct multiphonon relaxation to the lower lying (³H₆,t₂g) levels, a simple energy gap law well accounts for the measured data with effective phonon energies in the range 100-200 cm⁻¹ - consistent with the phonon density of states in these low phonon energy hosts. Monitoring infrared emission from the (³H₆,t₂g) states, 14 K and 10 K lifetimes of 301 μs and 250 μs are found for CsCaBr₃ and CsCaCl₃ respectively. For CsCaBr₃ this value reduces to 270 μs at 200 K and is not quenched until 300 K, whilst for CsCaCl₃ emission is quenched by 170 K. This temperature dependent behavior is interpreted in terms of internal conversion via configurational crossing between the excited and ground state potential energy surfaces. Fitting the fluorescence lifetime data to a modified Mott equation, it is inferred that the potential barrier for non-radiative relaxation is five times larger in CsCaBr₃ compared to CsCaCl₃. This explains the fact that emission is still observable in the bromide host at room temperature.

abstract: ABSTRACT In terms of prevalence, human suffering and costs dengue infections are the most important arthropod-borne viral disease worldwide. Dengue virus (DENV) is a mosquito-borne flavivirus and the etiological agent of dengue fever and dengue hemorrhagic fever. Thus, development of a safe and efficient vaccine constitutes an urgent necessity. Besides the traditional strategies aim at generating immunization options, the usage of viral vectors to deliver antigenic stimulus in order to elicit protection are particularly attractive for the endeavor of a dengue vaccine. The viral vector (MVvac2) is genetically equivalent to the currently used measles vaccine strain Moraten, which adds practicality to my approach. The goal of the present study was to generate a recombinant measles virus expressing structural antigens from two strains of DENV (DENV2 and DENV4) The recombinant vectors replication profile was comparable to that of the parental strain and expresses either membrane bound or soluble forms of DENV2 and DENV4 E glycoproteins. I discuss future experiments in order to demonstrate its immunogenicity in our measles-susceptible mouse model. / Dissertation/Thesis / M.S. Biology 2013

Cette thèse, menée en collaboration avec Michelin dans le cadre de la fabrication de matériaux composites, porte sur la stabilité colloïdale du latex de caoutchouc naturel (NR). Ce latex est une dispersion colloïdale polydisperse d'un polymère naturel dans un sérum aqueux. Les particules sont stabilisées par une couche complexe de phospholipides et de protéines. Cette dispersion peut être coagulée de façon irréversible par voie physique (sous cisaillement) ou par voie physico-chimique (ajout de cations divalents ou de particules hydrophobes). Dans ce travail, nous avons étudié la coagulation du latex par les cations divalents. Pour cela nous avons utilisé des outils physico-chimiques : diagrammes de stabilité en présence de divers cations, suivi cinétique de l'agrégation des particules et mesures rhéologiques. Ensuite, nous avons sondé la surface de particules de NR, par électrophorèse, en présence de tensioactifs et aussi selon la taille des particules. Pour expliquer la coagulation, nous suggérons que l'augmentation de la force ionique écrante les interactions répulsives et que les cations forment des ponts ioniques entre les particules. Ces ponts les maintiennent au contact et l'irréversibilité du phénomène est assurée par l'adhésion entre les chaines de polymères proches de la surface. L'adsorption des tensioactifs modifie la surface des particules et la coagulation du latex. Une légère différence de charge de surface a été observée entre les particules de NR selon leur taille. Enfin, un phénomène de coagulation similaire a été obtenu avec un latex synthétique, ce qui ouvre la voie à l'exploitation industrielle de ce phénomène de coagulation. / This work, in collaboration with Michelin for the fabrication of composite materials, deals with the colloidal stability of the latex of natural rubber (NR). The NR latex is a polydisperse colloidal dispersion of a bio-polymer in an aqueous serum. The particles are stabilized by a complex layer of phospholipids and proteins. This dispersion can be coagulated by a physical way (under shearing), or by a physical-chemical way (addition of divalent cations or hydrophobic particles). In this thesis, we studied the coagulation of the NR latex by divalent cations with physical-chemical tools (stability diagrams with different cations, aggregation kinetic of particles, rheological measuremments). Then, the surface of NR particles has been characterized by electrophoresis, firstly in the presence surfactants and secondly depending on the particles size. From the results of the first part we suggest that the coagulation with divalent cations is due to a screening effect because of the increase of ionic strength and that divalent cations can bridge the particles together. This allows keeping them in contact. Adhesion between polymer chains near the surface ensures the irrversible cohesion. In a second time, we saw that the adsorption of surfactants changes the particles surface and the coagulation of NR latex with cations. A small difference of surface charge is observed between the biggest and the smallest NR particles. Finally, a similar behavior has been obtained with a synthetic latex in presence of divalent cations. Our results could be used to develop an industrial process based on this coagulation phenomenon.

Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules. Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful. The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

No description available.

Le SmI₂ en tant que réactif de transfert monoélectronique a été largement utilisé en chimie organique depuis les premiers travaux de Kagan. Cependant, la quantité stœchiométrique ou en excès de SmI₂ et d'additifs toxique tels que HMPA sont utilisés normalement pour améliorer la réactivité. De plus, à cause de sa sensibilité à l'oxygène, le stockage de la solution de SmI₂ dans le THF est difficile. Récemment, nous avons développé une nouvelle méthode électrocatalytique basée sur la régénération électrochimique de Sm²⁺. Par rapport à la réactivité du SmI₂ classique, notre approche utilise une quantité catalytique de Sm. Premièrement, pour la réduction de nitroarènes, la réaction a sélectivement fourni les composés aromatiques azoïques et les anilines en fonction du solvant choisi. Notamment, c'est la première fois que la réaction Sm²⁺ se produit dans le méthanol dans le cas des anilines. Deuxièmement, dans le cas de la réduction des sulfoxydes par SmI₂, en général, l'HMPA était nécessaire comme additif. Dans notre procédé électrocatalytique, les sulfoxydes ont été transformés en sulfures avec une chimiosélectivité élevée et des excellents rendements toujours à température ambiante sans besoin ni de HMPA ni d’atmosphère protectrice. Enfin, les dérivés d'isoindolinone sont des séries de produits importants en chimie organique, la réduction des phtalimides est l'approche la plus pratique pour les obtenir. Avec les alcools, l'alcoxylation réductrice de phtalimides a eu lieu pour la première fois avec le Sm²⁺ électrocatalytique dans nos conditions. Et si on ajoute d'autres sources de protons, ce procédé a fourni les ω-hydroxylactames et isoindolinones correspondants avec des rendements élevés. / The SmI₂ as a single electron transfer reagent has been widely used in organic chemistry since the pioneering works by Kagan. However, the stoichiometric or excess amount of SmI₂ and harmful additives such as HMPA are used normally to enhance the reactivity, moreover, due to the oxygen sensitive, the storage of SmI₂ solution is difficult.Recently, we have developed a new electrocatalytic method based on the electrochemical regeneration of Sm²⁺. Compared to the classic SmI₂ reaction, our process occurred with a catalytic amount of Sm. In the reduction of nitroarenes, it selectively afforded the azo aromatic compounds and anilines depending on different solvents system. Notably, it’s the first time that the Sm²⁺ reaction occurred in the methanol. Normally, the HMPA was the additive in the reduction of sulfoxides by SmI₂. Under our electrocatalytic process, the sulfoxides were converted into sulfides in high chemoselectivity and yield at room temperature without HMPA and protecting atmosphere.The isoindolinone derivatives are series of important products in organic chemistry, the reduction of phthalimides is the most convenient approach to provide them. With alcohols, the unprecedented Sm²⁺ electrocatalyzed reductive alkoxylation of phthalimides was established. Moreover, adding other proton sources, this process afforded the corresponding ω-hydroxylactams and isoindolinones.

A rotating diffusion cell (RDC) has been used to study the kinetics of extraction of the transition metals cobalt (II), nickel (II), copper (II) and zinc (II) from sulphate solutions into either of two extractants held in n-heptane; di-(2-ethylhexyl) phosphoric acid (D2EHPA) or di-(2- ethylhexyl) dithiophosphoric acid (D2EHDTPA). The metal concentration was 10 mM and the aqueous pH was held at 4.5. The extractant concentration was varied between 0.015 to 0.4 M. In the case of cobalt extraction by D2EHPA, the metal concentration and the pH were varied Different diluents and modifiers were also studied.The rate of extraction by D2EHDTPA was found to be faster than D2EHPA. A comprehensive mathematical model, based upon established two film theory, was developed and used to describe the above experimental results. The model was also used to predict values of the important parameters. ... These values compared well with those found by other authors but using quite different experimental techniques. OS4 In the case of cobalt extraction by D2EHPA, the more polar diluents lowered the initial rate. The overall model predicts such behaviour where the rate is also dependent on the partition coefficients of the extractant. Finally, the theory of the RDC allows the prediction of the diffusion layer thicknesses, this information together with the reaction zone thickness is used to explore the influences of diffusion and chemical reaction on the overall transfer process. The diffusion processes are calculated to be the most important of the two. This is especially so for the D2EHDTPA systems. / University of Bradford Scholarship Award

The possible effects of changes in the feeding pattern on activated sludge properties related to bioflocculation have been analyzed in lab scale sequencing batch reactors (SBR) in order to determine if these changes in effluent water quality and settling and dewatering properties are significant, so they can be considered in future studies or if they can be recommended as crucial when operating and designing wastewater treatment plants. The activated sludge process is widely used to treat wastewater from both industrial and municipal sources. Biomass from industrial facilities containing high monovalent to divalent ion content usually settles poorly, which leads to low quality effluents that fail to meet environmental requirements. Therefore, the combined effect of feeding pattern plus the addition of sodium to activated sludge reactors was studied in this experiment. A series of SBRs were operated at different sodium concentrations that ranged from 1.5 - 15 meq/L and different feeding times that ranged from 1 minute to 4 hours. Biomass samples were taken from each reactor to study the settling and dewatering properties and effluent samples were used to analyze the amount of organic matter and exocellular polymeric substances present due to deflocculation. As expected, the changes in feeding strategies affected all of the properties measured. When the feeding time was maintained low (pulse feed) the effluent quality and settling properties were the best. As the feeding time was increased the effluent quality, settling, and dewatering properties increased suggesting that the way in which the reactors were fed affected the overall bioflocculation process. The causes of the high deflocculation observed are not well understood, but data suggest that a microbial community change could have affected exocellular biopolymers which are believed to play an important role on bioflocculation. This research demonstrates the importance of the interaction between cation content and feeding pattern when operating a wastewater treatment plants and when reporting lab-scaled results related to settling and bioflocculation. / Master of Science