Crystal structure of Leishmania tarentolae hypoxanthine-guanine phosphoribosyltransferase

Abstract Background Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) (EC 2.4.2.8) is a central enzyme in the purine recycling pathway. Parasitic protozoa of the order Kinetoplastida cannot synthesize purines de novo and use the salvage pathway to synthesize purine bases, making this an attractive target for antiparasitic drug design. Results The glycosomal HGPRT from Leishmania tarentolae in a catalytically active form purified and co-crystallized with a guanosine monophosphate (GMP) in the active site. The dimeric structure of HGPRT has been solved by molecular replacement and refined against data extending to 2.1 Å resolution. The structure reveals the contacts of the active site residues with GMP. Conclusion Comparative analysis of the active sites of Leishmania and human HGPRT revealed subtle differences in the position of the ligand and its interaction with the active site residues, which could be responsible for the different reactivities of the enzymes to allopurinol reported in the literature. The solution and analysis of the structure of Leishmania HGPRT may contribute to further investigations leading to a full understanding of this important enzyme family in protozoan parasites.

Crystal structures of Leptospira interrogans FAD-containing ferredoxin-NADP+ reductase and its complex with NADP+

Abstract Background Ferredoxin-NADP(H) reductases (FNRs) are flavoenzymes that catalyze the electron transfer between NADP(H) and the proteins ferredoxin or flavodoxin. A number of structural features distinguish plant and bacterial FNRs, one of which is the mode of the cofactor FAD binding. Leptospira interrogans is a spirochaete parasitic bacterium capable of infecting humans and mammals in general. Leptospira interrogans FNR (LepFNR) displays low sequence identity with plant (34% with Zea mays) and bacterial (31% with Escherichia coli) FNRs. However, LepFNR contains all consensus sequences that define the plastidic class FNRs. Results The crystal structures of the FAD-containing LepFNR and the complex of the enzyme with NADP+, were solved and compared to known FNRs. The comparison reveals significant structural similarities of the enzyme with the plastidic type FNRs and differences with the bacterial enzymes. Our small angle X-ray scattering experiments show that LepFNR is a monomeric enzyme. Moreover, our biochemical data demonstrate that the LepFNR has an enzymatic activity similar to those reported for the plastidic enzymes and that is significantly different from bacterial flavoenzymes, which display lower turnover rates. Conclusion LepFNR is the first plastidic type FNR found in bacteria and, despite of its low sequence similarity with plastidic FNRs still displays high catalytic turnover rates. The typical structural and biochemical characteristics of plant FNRs unveiled for LepFNR support a notion of a putative lateral gene transfer which presumably offers Leptospira interrogans evolutionary advantages. The wealth of structural information about LepFNR provides a molecular basis for advanced drugs developments against leptospirosis.

NF-κB activation mediates crystal translocation and interstitial inflammation in adenine overload nephropathy

Adenine overload promotes intratubular crystal precipitation and interstitial nephritis. We showed recently that these abnormalities are strongly attenuated in mice knockout for Toll-like receptors-2, -4, MyD88, ASC, or caspase-1. We now investigated whether NF-κB activation also plays a pathogenic role in this model. Adult male Munich-Wistar rats were distributed among three groups: C (n = 17), receiving standard chow; ADE (n = 17), given adenine in the chow at 0.7% for 1 wk and 0.5% for 2 wk; and ADE + pyrrolidine dithiocarbamate (PDTC; n = 14), receiving adenine as above and the NF-κB inhibitor PDTC (120 mg•kg-1•day-1 in the drinking water). After 3 wk, widespread crystal deposition was seen in tubular lumina and in the renal interstitium, along with granuloma formation, collagen accumulation, intense tubulointerstitial proliferation, and increased interstitial expression of inflammatory mediators. Part of the crystals were segregated from tubular lumina by a newly formed cell layer and, at more advanced stages, appeared to be extruded to the interstitium. p65 nuclear translocation and IKK-α increased abundance indicated activation of the NF-κB system. PDTC treatment prevented p65 migration and normalized IKK-α, limited crystal shift to the interstitium, and strongly attenuated interstitial fibrosis/inflammation. These findings indicate that the complex inflammatory phenomena associated with this model depend, at least in part, on NF-κB activation, and suggest that the NF-κB system may become a therapeutic target in the treatment of chronic kidney disease.

Order induced charge carrier mobility enhancement in columnar liquid crystal diodes

Discotic molecules comprising a rigid aromatic core and flexible side chains have been promisingly applied in OLEDs as self-organizing organic semiconductors. Due to their potentially high charge carrier mobility along the columns, device performance can be readily improved by proper alignment of columns throughout the bulk. In the present work, the charge mobility was increased by 5 orders of magnitude due to homeotropic columnar ordering induced by the boundary interfaces during thermal annealing in the mesophase. State-of-the-art diodes were fabricated using spin-coated films whose homeotropic alignment with formation of hexagonal germs was observed by polarizing optical microscopy. The photophysical properties showed drastic changes at the mesophase-isotropic transition, which is supported by the gain of order observed by X-ray diffraction. The electrical properties were investigated by modeling the current−voltage characteristics by a space-charge-limited current transport with a field dependent mobility.

Thiosaccharine disulfide: synthesis, crystal structure, spectroscopic characterization and theoretical study

The title compound, (thiosaccharine disulfide), bis[1,10dioxide-2,3-dihidro-1,2-benzoisothiazol]disulfide, (tsac)2 has been synthesized and fully characterized by UV–Visible, IR, Raman, 1H and 13C NMR spectroscopy elemental analysis and structural X-ray crystallography. A DFT theoretical study has been performed and good agreement between experimental and theoretical values of structural parameters and vibration frequencies have been achieved.

Polymorphic-induced transformations in CaTa2O6 single-crystal fibers obtained by laser-heated pedestal growth

Calcium tantalite (CaTa2O6) single crystal fibers were obtained by the laser-heated pedestal growth method (LHPG). At room temperature, this material can present three polymorphic modifications. The rapid crystallization inherent to the LHPG method produced samples within the Pm3 space group, with some chemical disorder. In order to check for polymorphic-induced transformations, the CaTa2O6 fibers have been submitted to different thermal treatments and investigated by micro-Raman spectroscopy. For short annealing times (15 min) at 1200 °C, the cubic modification was maintained, though with an improved crystalline quality, as evidenced by the enhanced inelastic scattered intensity (by ca. 250%) and narrowing of Raman bands. The polarized Raman spectra respected very well the predicted symmetries and the selection rules for this cubic modification. On the other hand, long annealing times (24 h) at 1200 °C led to a complete (irreversible) polymorphic transformation. The Raman bands became still more intense (ca. 15 times larger than for the as-grown fibers), narrower, and several new modes appeared. Also, the spectra became unpolarized, demonstrating a polycrystalline nature of the transformed crystals. The observed Raman modes could be fully assigned to an orthorhombic modification of CaTa2O6 belonging to the Pnma space group.

Liquid crystal polymers: macromolecular design for enhanced performances

During this work, done mainly in the laboratories of the department of Industrial Chemistry and Materials of the University of Bologna but also in the laboratories of the Carnegie Mellon University in collaboration with prof. K. Matyjaszewski and at the university of Zaragoza in collaboration with prof. J. Barberá, was focused mainly on the synthesis and characterization of new functional polymeric materials. In the past years our group gained a deep knowledge about the photomodulation of azobenzene containing polymers. The aim of this thesis is to push forward the performances of these materials by the synthesis of well defined materials, in which, by a precise control over the macromolecular structures, better or even new functionality can be delivered to the synthesized material. For this purpose, besides the rich photochemistry of azoaromatic polymers that brings to the application, the control offered from the recent techniques of controlled radical polymerization, ATRP over all, gives an enormous range of opportunity for the developing of a new generation of functional materials whose properties are determinate not only by the chemical nature of the functional center (e.g. azoaromatic chromophore) but are tuned and even amplified by a synergy with the whole macromolecular structure. Old materials in new structures. In this contest the work of this thesis was focused mainly on the synthesis and characterization of well defined azoaromatic polymers in order to establish, for the first time, precise structure-properties correlation. In fact a series of well defined different azopolymers, chiral and achiral, with different molecular weight and highly monodisperse were synthesized and their properties were studied, in terms of photoexpansion and photomodulation of chirality. We were then able to study the influence of the macromolecular structure in terms of molecular weight and ramification on the studied properties. The huge amount of possibility offered by the tailoring of the macromolecular structure were exploited for the synthesis of new cholesteric photochromic polymers that can be used as a smart label for the certification of the thermal history of any thermosensitive product. Finally the ATRP synthesis allowed us to synthesize a total new class of material, named molecular brushes: a flat surface covered with an ultra thin layer of polymeric chain covalently bond onto the surface from one end. This new class of materials is of extreme interest as they offer the possibility to tune and manage the interaction of the surface with the environment. In this contest we synthesized both azoaromatic surfaces, growing directly the polymer from the surface, and mixed brushes: surfaces covered with incompatible macromolecules. Both type of surfaces acts as “smart” surfaces: the first it is able to move the orientation of a LC cell by simply photomodulation and, thanks to the robustness of the covalent bond, can be used as a command surface overcoming all the limitation due to the dewetting of the active layer. The second type of surface, functionalized by a grafting-to method, can self assemble the topmost layer responding to changed environmental conditions, exposing different functionality according to different environment.

Liquid crystal polymer brushes and their application as alignment layers in liquid crystal cells

Liquid Crystal Polymer Brushes and their Application as Alignment Layers in Liquid Crystal Cells Polymer brushes with liquid crystalline (LC) side chains were synthesized on planar glass substrates and their nematic textures were investigated. The LC polymers consist of an acrylate or a methacrylate main chain and a phenyl benzoate group as the mesogenic unit which is connected to the main chain via a flexible alkyl spacer composed of six CH2 units. The preparation of the LC polymer brushes was carried out according to the “grafting from” technique: polymerization is carried out from azo-initiators that have been previously self-assembled on the substrate. LC polymer brushes with a thickness from a few nm to 230 nm were synthesized by varying the monomer concentration and the polymerization time. The LC polymer brushes were thick enough to allow for direct observation of the nematic textures with a polarizing microscope. The LC polymer brushes grown on untreated glass substrates exhibited irregular textures (“polydomains”). The domain size is in the range of some micrometers and depends only weakly on the brush thickness. The investigations on the texture-temperature relationship of the LC brushes revealed that the brushes exhibit a surface memory effect, that is, the identical texture reappears after the LC brush sample has experienced a thermal isotropization or a solvent treatment, at which the nematic LC state has been completely destroyed. The surface memory effect is attributed to a strong anchoring of the orientation of the mesogenic units to heterogeneities at the substrate surface. The exact nature of the surface heterogeneities is unknown. The effect was observed for the LC brushes swollen with low molecular weight nematic molecules, as well. Rubbing the glass substrate with a piece of velvet cloth prior to the surface modification with the initiator and the brush growth gives rise to the formation of homogenous alignment of the mesogenic units in the LC polymer side chains. Monodomain textures were obtained for these LC brushes. The mechanism for the homogeneous alignment is based on the transfer of Nylon fibers during the rubbing process. A surfactant was mixed with the azo-initiator in modifying rubbed substrates for subsequent brush generation. Such brushes exhibited biaxial optical properties. Hybrid LC cells made from a substrate modified with biaxial brushes and a rubbed glass substrate show an orientation with a tilt angle of a = –15.6 . This work shows that LC brushes grown on rubbed surfaces fulfill the important criteria for alignment layers: the formation of macroscopic monodomains. First results indicate that by diluting the brush with molecules which are also covalently bound to the surface but induce a different orientation, a system is obtained in which the two conflicting alignment mechanisms can be used to generate a tilted alignment. In order to allow for an application of the alignment layers into a potential product, subsequent work should focus on the questions how easy and in which range the tilt angle can be controlled.

Aufbau des Crystal-Ball-Detektorsystems und Untersuchung der Helizitätsasymmetrie in gamma p p pi 0 pi 0

Seit Frühjahr 2004 wird der Crystal Ball-Detektor am Photonenstrahl des Mainzer Mikrotrons für Koinzidenzexperimente zur Untersuchung der Struktur der Nukleonen genutzt. Aufbau und Inbetriebnahme des Kalorimeters, insbesondere der neuen Detektorelektronik, bilden einen Schwerpunkt dieser Arbeit. Komponenten wurden neu konstruiert oder auf ihre Verwendbarkeit geprüft und nögenfalls modifiziert. Nach erfolgreichem Abschluss der Aufbauphase wurden Experimente zur Produktion von $pi$- und $eta$-Mesonen am Proton mit mehr als 2500 Stunden Strahlbetrieb durchgeführt. Den zweiten Schwerpunkt der Dissertation bildet die erstmalige Messung der Helizitätsasymmetrie I$^odot$ in der Photoproduktion zweier neutraler Pionen. Zum Verstädnis des Anregungsspektrums der Nukleonen müssen Experimente mit polarisierten Photonen und/oder polarisierten Targets durchgeführt werden. Da Modelle trotz unterschiedlicher Annahmen unpolarisiert gemessene Größen vergleichbar gut reproduzieren, ist die Bestimmung der auf Modellunterschiede empfindlichen Polarisationsobservablen unumgäglich. Im Gegensatz zur Einpionproduktion tritt in der Zweipionproduktion eine Einfachpolarisationsobservable auf, die mit zirkular polarisierten Photonen am unpolarisierten Proton gemessen werden kann. Diese wurde in der Reaktion $gamma$ p $rightarrow$ p $pi^0$ $pi^0$ und in $gamma$ p $rightarrow$ p $pi^+$ $pi^-$ energie- und winkelabhägig bestimmt. Die Ergebnisse weichen stark von den Modellvorhersagen ab.

Preparation of new crystal forms via photochemical, mechanochemical and sol-gel methods

This work of thesis involves various aspects of crystal engineering. Chapter 1 focuses on crystals containing crown ether complexes. Aspects such as the possibility of preparing these materials by non-solution methods, i.e. by direct reaction of the solid components, thermal behavior and also isomorphism and interconversion between hydrates are taken into account. In chapter 2 a study is presented aimed to understanding the relationship between hydrogen bonding capability and shape of the building blocks chosen to construct crystals. The focus is on the control exerted by shape on the organization of sandwich cations such as cobalticinium, decamethylcobalticinium and bisbenzenchromium(I) and on the aggregation of monoanions all containing carboxylic and carboxylate groups, into 0-D, 1-D, 2-D and 3-D networks. Reactions conducted in multi-component molecular assemblies or co-crystals have been recognized as a way to control reactivity in the solid state. The [2+2] photodimerization of olefins is a successful demonstration of how templated solid state synthesis can efficiently synthesize unique materials with remarkable stereoselectivity and under environment-friendly conditions. A demonstration of this synthetic strategy is given in chapter 3. The combination of various types of intermolecular linkages, leading to formation of high order aggregation and crystalline materials or to a random aggregation resulting in an amorphous precipitate, may not go to completeness. In such rare cases an aggregation process intermediate between crystalline and amorphous materials is observed, resulting in the formation of a gel, i.e. a viscoelastic solid-like or liquid-like material. In chapter 4 design of new Low Molecular Weight Gelators is presented. Aspects such as the relationships between molecular structure, crystal packing and gelation properties and the application of this kind of gels as a medium for crystal growth of organic molecules, such as APIs, are also discussed.

Polymer-liquid crystal interface: a molecular dynamics study

Liquid crystals (LCs) are an interesting class of soft condensed matter systems characterized by an unusual combination of fluidity and long-range order, mainly known for their applications in displays (LCDs). However, the interest in LC continues to grow pushed by their application in new technologies in medicine, optical imaging, micro and nano technologies etc. In LCDs uniaxial alignment of LCs is mainly achieved by a rubbing process. During this treatment, the surfaces of polymer coated display substrates are rubbed in one direction by a rotating cylinder covered with a rubbing cloth. Basically, LC alignment involves two possible aligning directions: uniaxial planar (homogeneous) and vertical (homeotropic) to the display substrate. An interesting unresolved question concerning LCs regards the origin of their alignment on rubbed surfaces, and in particular on the polymeric ones used in the display industry. Most studies have shown that LCs on the surface of the rubbed polymer film layer are lying parallel to the rubbing direction. In these systems, micrometric grooves are generated on the film surface along the rubbing direction and also the polymer chains are stretched in this direction. Both the parallel aligned microgrooves and the polymer chains at the film surface may play a role in the LC alignment and it is not easy to quantify the effect of each contribution. The work described in this thesis is an attempt to find new microscopic evidences on the origin of LC alignment on polymeric surfaces through molecular dynamics (MD) simulations, which allow the investigation of the phenomenon with atomic detail. The importance of the arrangement of the polymeric chains in LCs alignment was studied by performing MD simulations of a thin film of a typical nematic LC, 4-cyano-4’-pentylbiphenyl (5CB), in contact with two different polymers: poly(methyl methacrylate)(PMMA) and polystyrene (PS). At least four factors are believed to influence the LC alignment: 1. the interactions of LCs with the backbone vinyl chains; 2. the interactions of LCs with the oriented side groups; 3. the anisotropic interactions of LCs with nanometric grooves; 4. the presence of static surface charges. Here we exclude the effect of microgrooves and of static surface charges from our virtual experiment, by using flat and neutral polymer surfaces, with the aim of isolating the chemical driving factors influencing the alignment of LC phases on polymeric surfaces.

Crystal phases and glassy dynamics in monodisperse hard ellipsoids

We have performed Monte Carlo and molecular dynamics simulations of suspensions of monodisperse, hard ellipsoids of revolution. Hard-particle models play a key role in statistical mechanics. They are conceptually and computationally simple, and they offer insight into systems in which particle shape is important, including atomic, molecular, colloidal, and granular systems. In the high density phase diagram of prolate hard ellipsoids we have found a new crystal, which is more stable than the stretched FCC structure proposed previously . The new phase, SM2, has a simple monoclinic unit cell containing a basis of two ellipsoids with unequal orientations. The angle of inclination is very soft for length-to-width (aspect) ratio l/w=3, while the other angles are not. A symmetric state of the unit cell exists, related to the densest-known packings of ellipsoids; it is not always the stable one. Our results remove the stretched FCC structure for aspect ratio l/w=3 from the phase diagram of hard, uni-axial ellipsoids. We provide evidence that this holds between aspect ratios 3 and 6, and possibly beyond. Finally, ellipsoids in SM2 at l/w=1.55 exhibit end-over-end flipping, warranting studies of the cross-over to where this dynamics is not possible. Secondly, we studied the dynamics of nearly spherical ellipsoids. In equilibrium, they show a first-order transition from an isotropic phase to a rotator phase, where positions are crystalline but orientations are free. When over-compressing the isotropic phase into the rotator regime, we observed super-Arrhenius slowing down of diffusion and relaxation, and signatures of the cage effect. These features of glassy dynamics are sufficiently strong that asymptotic scaling laws of the Mode-Coupling Theory of the glass transition (MCT) could be tested, and were found to apply. We found strong coupling of positional and orientational degrees of freedom, leading to a common value for the MCT glass-transition volume fraction. Flipping modes were not slowed down significantly. We demonstrated that the results are independent of simulation method, as predicted by MCT. Further, we determined that even intra-cage motion is cooperative. We confirmed the presence of dynamical heterogeneities associated with the cage effect. The transit between cages was seen to occur on short time scales, compared to the time spent in cages; but the transit was shown not to involve displacements distinguishable in character from intra-cage motion. The presence of glassy dynamics was predicted by molecular MCT (MMCT). However, as MMCT disregards crystallization, a test by simulation was required. Glassy dynamics is unusual in monodisperse systems. Crystallization typically intervenes unless polydispersity, network-forming bonds or other asymmetries are introduced. We argue that particle anisometry acts as a sufficient source of disorder to prevent crystallization. This sheds new light on the question of which ingredients are required for glass formation.

Influence of ice crystal habit and cirrus spatial inhomogeneities on the retrieval of cirrus optical thickness and effective radius

Diese Dissertation untersucht den Einfluss von Eiskristallform und räumlicher Inhomogenität von Zirren auf das Retrieval von optischer Wolkendicke und effektivem Eispartikelradius. Zu diesem Zweck werden flugzeuggetragene spektrale Messungen solarer Strahlung sowie solare und langwellige Strahlungstransfersimulationen durchgeführt. Flugzeuggetragene spektrale aufwärtsgerichtete Radianzen (Strahldichten) sind mit dem SMART-Albedometer (Spectral Modular Airborne Radiation measurement sysTem) während des CIRCLE-2 (CIRrus CLoud Experiment-2) Feldexperiments im Mai 2007 gemessen worden. Basierend auf diesen Radianzdaten werden mittels eines Wolkenretrievalalgorithmus optische Wolkendicken und effektive Eispartikelradien anhand von eindimensionalen Strahlungstransferrechnungen bestimmt. Die Auswirkung der Annahme unterschiedlicher Eiskristallformen auf die retrievten Parameter wird durch Variation der Einfachstreueigenschaften der Eispartikel untersucht. Darüber hinaus wird mittels Strahlungstransferrechnungen auch der Einfluss der Eiskristallform auf den Strahlungsantrieb von Eiswolken ermittelt. Die Frage nach dem relativen Einfluss von räumlicher Wolkeninhomogenität und Eiskristallform wird anhand von dreidimensionalen und independent pixel approximation (IPA) Strahlungssimulationen untersucht. Die Analyse basiert auf einer Modelleiswolke, die aus Daten des NASA (National Aeronautics and Space Administration) TC4 (Tropical Composition, Cloud, and Climate Coupling) Feldexperiments im Sommer 2007 in Costa Rica erzeugt wurde. Lokal gesehen können beide Effekte - Eiskristallform und räumliche Eiswolkeninhomogenität - die gleiche Grössenordnung haben und zu einer Unter- bzw. Überschätzung der retrievten Parameter um 40 – 60% führen. Gemittelt über die ganze Wolke ist jedoch der Einfluss der Eiskristallform viel bedeutender als der von räumlichen Inhomogenitäten.