Temperature dependence of molecular dynamics and supramolecular aggregation in MEH-PPV films: A solid-state NMR, X-ray and fluorescence spectroscopy study

This article presents an investigation of the temperature induced modification in the microstructure and dynamics of poly[2-methoxy-5-(2`-ethylhexyloxy)-1,4-phenylenevinylene] (MEH-PPV) cast films using Wide-Angle X-ray Scattering (WAXS), solid-state Nuclear Magnetic Resonance (NMR), and Fluorescence Spectroscopy (PL). MEH-PPV chain motions were characterized as a function of temperature by NMR. The results indicated that the solvent used to cast the films influences the activation energy of the side-chain motions. This was concluded from the comparison of the activation energy of the toluene cast film, E(a) = (54 +/- 8) kJ/mol, and chloroform cast film, E(a) = (69 +/- 5) kJ/mol, and could be attributed to the higher side-chain packing provided by chloroform, that preferentially solvates the side chain in contrast to toluene that solvates mainly the backbone. Concerning the backbone mobility, it was observed that the torsional motions in the MEH-PPV have average amplitude of similar to 10 degrees at 300 K, which was found to be independent of the solvent used to cast the films. In order to correlate the molecular dynamics processes with the changes in the microstructure of the polymer, in situ WAXS experiments as a function of temperature were performed and revealed that the interchain spacing in the MEH-PPV molecular aggregates increases as a function of temperature, particularly at temperatures where molecular relaxations occur. It was also observed that the WAXS peak associated with the bilayer spacing becomes narrower and its intensity increases whereas the peak associated with the inter-backbone planes reduces its intensity for higher temperatures. This last result Could be interpreted as a decrease in the number of aggregates and the reduction of the interchain species during the MEH-PPV relaxation processes. These WAXS results were correlated with PL spectra modifications observed upon temperature treatments. (C) 2008 Elsevier Ltd. All rights reserved.

Control of aggregation-induced emission by DNA hybridization

Aggregation-induced emission (AIE) was studied by hybridization of dialkynyl-tetraphenylethylene (DATPE) modified DNA strands. Molecular aggregation and fluorescence of DATPEs are controlled by duplex formation.

Fluorescent AlPO(4) gels and glasses doped with Rhodamine 6G: Preparation, structural and optical characterization

Fluorescent AlPO(4) xerogels doped with different amounts of Rhodamine 6G (Rh6G) laser dye were prepared by a one-step sal-gel process. In addition, mesoporous AlPO(4) glasses obtained from undoped gels were loaded with different amounts of Rh6G by wet impregnation. Optical excitation and emission spectra of both series of samples show significant dependences on Rh6G concentration, revealing the influence of dye molecular aggregation. At comparable dye concentrations the aggregation effects are found to be significantly stronger in the gels than in the mesoporous glasses. This effect might be attributed to stronger interactions between the dye molecules and the glass matrix, resulting in more efficient dye dispersion in the latter. The interaction of Rh6G with the glassy AlPO(4) network has been probed by (27)Al and (31)P solid-state NMR techniques. New five- and six-coordinated aluminum environments have been observed and characterized by advanced solid-state NMR techniques probing (27)Al-(1)H and (27)Al-(31)P internuclear dipole couplings. The fractional area of these new Al sites is correlated with the combined fractional area of two new Q(3Al)((0)) and Q(2Al)((0)) phosphate species observed in the (31)P MAS NMR spectra. Based on this correlation as well as detailed composition dependent studies, we suggest that the new signals arise from the breakage of Al-O-P linkages associated with the insertion process. (C) 2010 Elsevier B.V. All rights reserved.

Sistemi liquido cristallini complessi: simulazioni al calcolatore e studi ESR

The aim of this PhD thesis was to study at a microscopic level different liquid crystal (LC) systems, in order to determine their physical properties, resorting to two distinct methodologies, one involving computer simulations, and the other spectroscopic techniques, in particular electron spin resonance (ESR) spectroscopy. By means of the computer simulation approach we tried to demonstrate this tool effectiveness for calculating anisotropic static properties of a LC material, as well as for predicting its behaviour and features. This required the development and adoption of suitable molecular models based on a convenient intermolecular potentials reflecting the essential molecular features of the investigated system. In particular, concerning the simulation approach, we have set up models for discotic liquid crystal dimers and we have studied, by means of Monte Carlo simulations, their phase behaviour and self­-assembling properties, with respect to the simple monomer case. Each discotic dimer is described by two oblate Gay­Berne ellipsoids connected by a flexible spacer, modelled by a harmonic "spring" of three different lengths. In particular we investigated the effects of dimerization on the transition temperatures, as well as on the characteristics of molecular aggregation displayed and the relative orientational order. Moving to the experimental results, among the many experimental techniques that are typically employed to evaluate LC system distinctive features, ESR has proved to be a powerful tool in microscopic scale investigation of the properties, structure, order and dynamics of these materials. We have taken advantage of the high sensitivity of the ESR spin probe technique to investigate increasingly complex LC systems ranging from devices constituted by a polymer matrix in which LC molecules are confined in shape of nano- droplets, as well as biaxial liquid crystalline elastomers, and dimers whose monomeric units or lateral groups are constituted by rod-like mesogens (11BCB). Reflection-mode holographic-polymer dispersed liquid crystals (H-PDLCs) are devices in which LCs are confined into nanosized (50­-300 nm) droplets, arranged in layers which alternate with polymer layers, forming a diffraction grating. We have determined the configuration of the LC local director and we have derived a model of the nanodroplet organization inside the layers. Resorting also to additional information on the nanodroplet size and shape distribution provided by SEM images of the H-PDLC cross-section, the observed director configuration has been modeled as a bidimensional distribution of elongated nanodroplets whose long axis is, on the average, parallel to the layers and whose internal director configuration is a uniaxial quasi- monodomain aligned along the nanodroplet long axis. The results suggest that the molecular organization is dictated mainly by the confinement, explaining, at least in part, the need for switching voltages significantly higher and the observed faster turn-off times in H-PDLCs compared to standard PDLC devices. Liquid crystal elastomers consist in cross-linked polymers, in which mesogens represent the monomers constituting the main chain or the laterally attached side groups. They bring together three important aspects: orientational order in amorphous soft materials, responsive molecular shape and quenched topological constraints. In biaxial nematic liquid crystalline elastomers (BLCEs), two orthogonal directions, rather than the one of normal uniaxial nematic, can be controlled, greatly enhancing their potential value for applications as novel actuators. Two versions of a side-chain BLCEs were characterized: side­-on and end­-on. Many tests have been carried out on both types of LCE, the main features detected being the lack of a significant dynamical behaviour, together with a strong permanent alignment along the principal director, and the confirmation of the transition temperatures already determined by DSC measurements. The end­-on sample demonstrates a less hindered rotation of the side group mesogenic units and a greater freedom of alignment to the magnetic field, as already shown by previous NMR studies. Biaxial nematic ESR static spectra were also obtained on the basis of Molecular Dynamics generated biaxial configurations, to be compared to the experimentally determined ones, as a mean to establish a possible relation between biaxiality and the spectral features. This provides a concrete example of the advantages of combining the computer simulation and spectroscopic approaches. Finally, the dimer α,ω-bis(4'-cyanobiphenyl-4-yl)undecane (11BCB), synthesized in the "quest" for the biaxial nematic phase has been analysed. Its importance lies in the dimer significance as building blocks in the development of new materials to be employed in innovative technological applications, such as faster switching displays, resorting to the easier aligning ability of the secondary director in biaxial phases. A preliminary series of tests were performed revealing the population of mesogenic molecules as divided into two groups: one of elongated straightened conformers sharing a common director, and one of bent molecules, which display no order, being equally distributed in the three dimensions. Employing this model, the calculated values show a consistent trend, confirming at the same time the transition temperatures indicated by the DSC measurements, together with rotational diffusion tensor values that follow closely those of the constituting monomer 5CB.

Effect of indomethacin on bile acid-phospholipid interactions: implication for small intestinal injury induced by nonsteroidal anti-inflammatory drugs.

The injurious effect of nonsteroidal anti-inflammatory drugs (NSAIDs) in the small intestine was not appreciated until the widespread use of capsule endoscopy. Animal studies found that NSAID-induced small intestinal injury depends on the ability of these drugs to be secreted into the bile. Because the individual toxicity of amphiphilic bile acids and NSAIDs directly correlates with their interactions with phospholipid membranes, we propose that the presence of both NSAIDs and bile acids alters their individual physicochemical properties and enhances the disruptive effect on cell membranes and overall cytotoxicity. We utilized in vitro gastric AGS and intestinal IEC-6 cells and found that combinations of bile acid, deoxycholic acid (DC), taurodeoxycholic acid, glycodeoxycholic acid, and the NSAID indomethacin (Indo) significantly increased cell plasma membrane permeability and became more cytotoxic than these agents alone. We confirmed this finding by measuring liposome permeability and intramembrane packing in synthetic model membranes exposed to DC, Indo, or combinations of both agents. By measuring physicochemical parameters, such as fluorescence resonance energy transfer and membrane surface charge, we found that Indo associated with phosphatidylcholine and promoted the molecular aggregation of DC and potential formation of larger and isolated bile acid complexes within either biomembranes or bile acid-lipid mixed micelles, which leads to membrane disruption. In this study, we demonstrated increased cytotoxicity of combinations of bile acid and NSAID and provided a molecular mechanism for the observed toxicity. This mechanism potentially contributes to the NSAID-induced injury in the small bowel.

The mammalian central nervous synaptic cleft contains a high density of periodically organized complexes.

Cryo-electron microscopy of vitreous section makes it possible to observe cells and tissues at high resolution in a close-to-native state. The specimen remains hydrated; chemical fixation and staining are fully avoided. There is minimal molecular aggregation and the density observed in the image corresponds to the density in the object. Accordingly, organotypic hippocampal rat slices were vitrified under high pressure and controlled cryoprotection conditions, cryosectioned at a final thickness of approximately 70 nm and observed below -170 degrees C in a transmission electron microscope. The general aspect of the tissue compares with previous electron microscopy observations. The detailed analysis of the synapse reveals that the density of material in the synaptic cleft is high, even higher than in the cytoplasm, and that it is organized in 8.2-nm periodic transcleft complexes. Previously undescribed structures of presynaptic and postsynaptic elements are also described.

Native folding of aggregation-prone recombinant proteins in Escherichia coli by osmolytes, plasmid- or benzyl alcohol-overexpressed molecular chaperones.

When massively expressed in bacteria, recombinant proteins often tend to misfold and accumulate as soluble and insoluble nonfunctional aggregates. A general strategy to improve the native folding of recombinant proteins is to increase the cellular concentration of viscous organic compounds, termed osmolytes, or of molecular chaperones that can prevent aggregation and can actively scavenge and convert aggregates into natively refoldable species. In this study, metal affinity purification (immobilized metal ion affinity chromatography [IMAC]), confirmed by resistance to trypsin digestion, was used to distinguish soluble aggregates from soluble nativelike proteins. Salt-induced accumulation of osmolytes during induced protein synthesis significantly improved IMAC yields of folding-recalcitrant proteins. Yet, the highest yields were obtained with cells coexpressing plasmid-encoded molecular chaperones DnaK-DnaJ-GrpE, ClpB, GroEL-GroES, and IbpA/B. Addition of the membrane fluidizer heat shock-inducer benzyl alcohol (BA) to the bacterial medium resulted in similar high yields as with plasmid-mediated chaperone coexpression. Our results suggest that simple BA-mediated induction of endogenous chaperones can substitute for the more demanding approach of chaperone coexpression. Combined strategies of osmolyte-induced native folding with heat-, BA-, or plasmid-induced chaperone coexpression can be thought to optimize yields of natively folded recombinant proteins in bacteria, for research and biotechnological purposes.

MMPBSA decomposition of the binding energy throughout a molecular dynamics simulation of Amyloid-Beta (Aß10-35) aggregation

Recent experiments with amyloid-beta (Aß) peptides indicate that the formation of toxic oligomers may be an important contribution to the onset of Alzheimer's disease. The toxicity of Aß oligomers depend on their structure, which is governed by assembly dynamics. However, a detailed knowledge of the structure of at the atomic level has not been achieved yet due to limitations of current experimental techniques. In this study, replica exchange molecular dynamics simulations are used to identify the expected diversity of dimer conformations of Aß10-35 monomers. The most representative dimer conformation has been used to track the dimer formation process between both monomers. The process has been characterized by means of the evolution of the decomposition of the binding free energy, which provides an energetic profile of the interaction. Dimers undergo a process of reorganization driven basically by inter-chain hydrophobic and hydrophilic interactions and also solvation/desolvation processes.

L'agrégation toxique des protéines: une forme de "delinquance moléculaire" activement combattue dans la cellule par les chaperones moléculaires et les protéases [The toxic aggregation of proteins: a kind of "molecular delinquency" actively fought in the cell by molecular chaperones and proteases]

Under various stresses, mutation-sensitised proteins may spontaneously convert into inactive, aggregation-prone structures, which may be cytotoxic and infectious. In the cell, this new kind of "molecular criminality" is actively fought against by a network of molecular chaperones that can specifically identify, isolate and unfold damaged (delinquent) proteins and favour their subsequent native refolding. Irreversibly damaged molecules unable to natively refold are preferentially "executed" and recycled by proteases. Failing that, they are "imprisoned" within compact amyloids, or "evicted" from the cell. Thus, striking parallels, although of questionable ethical value, exist between protein and human criminality, and between the cellular and social responses to these different types of criminality. Fundamental differences also exist. Whereas programmed death (apoptosis) is the preferred solution chosen by aged and aggregation-stressed cells, collective suicide is seldom an option chosen by lawless human societies. More significantly, there is no clear cellular equivalent for the role of the family and the education system, which are so essential to the proper shaping of functional individuals in the society, and give rise to humanism, that favours crime prevention, reeducation and reinsertion programs over capital punishment. To the cardiologist and transplantation surgeon, the interest of molecular chaperones, in particular of Hsp70, Hsp90 and Hsp27, lays in their ability to inhibit the signalling pathway of programmed cell death. Their induction before and during ischemia, by various treatments and drugs could significantly reduce damages from the post ischemic reperfusion of organs.

Role of peptide peptide interactions in aggregation: protonectins observed in equilibrium and replica exchange molecular dynamics simulations

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Modelling the influence of diradical character and aggregation on conjugated molecular materials

Molecular materials are made by the assembly of specifically designed molecules to obtain bulk structures with desired solid-state properties, enabling the development of materials with tunable chemical and physical properties. These properties result from the interplay of intra-molecular constituents and weak intermolecular interactions. Thus, small changes in individual molecular and electronic structure can substantially change the properties of the material in bulk. The purpose of this dissertation is, thus, to discuss and to contribute to the structure-property relationships governing the electronic, optical and charge transport properties of organic molecular materials through theoretical and computational studies. In particular, the main focus is on the interplay of intra-molecular properties and inter-molecular interactions in organic molecular materials. In my three-years of research activity, I have focused on three major areas: 1) the investigation of isolated-molecule properties for the class of conjugated chromophores displaying diradical character which are building blocks for promising functional materials; 2) the determination of intra- and intermolecular parameters governing charge transport in molecular materials and, 3) the development and application of diabatization procedures for the analysis of exciton states in molecular aggregates. The properties of diradicaloids are extensively studied both regarding their ground state (diradical character, aromatic vs quinoidal structures, spin dynamics, etc.) and the low-lying singlet excited states including the elusive double-exciton state. The efficiency of charge transport, for specific classes of organic semiconductors (including diradicaloids), is investigated by combining the effects of intra-molecular reorganization energy, inter-molecular electronic coupling and crystal packing. Finally, protocols aimed at unravelling the nature of exciton states are introduced and applied to different molecular aggregates. The role of intermolecular interactions and charge transfer contributions in determining the exciton state character and in modulating the H- to J- aggregation is also highlighted.

Recovery and upgrading bovine rumen protein by extrusion: effect of lipid content on protein disulphide cross-linking, solubility and molecular weight

Bovine rumen protein with two levels of residual lipids (1.9 per cent or 3.8 per cent) was subjected to thermoplastic extrusion under different temperatures and moisture contents. Protein solubility in different buffers, disulphide cross-linking and molecular weight distribution were determined on the extrudates. After extrusion, samples with 1.9 per cent residual lipids content had a higher concentration of protein insoluble by undetermined forces, irrespective of feed moisture and processing temperature used. Lipid content of 3.8 per cent in the feed material resulted in more protein participating in the extrudate network through non-covalent interactions (hydrophobic and electrostatic) and disulphide bonds. A small dependency of the extrusion process on moisture and temperature and a marked dependency on lipid content, especially phospholipid, was observed, Electrophoresis under non-reducing conditions showed that protein extrusion with low feed moisture promoted high molecular breakdown inside the barrel, probably due to intense shear force, and further protein aggregation at the die end

Molecular cloning and biochemical characterization of a myotoxin inhibitor from Bothrops alternatus snake plasma

Phospholipases A(2) (PLA(2)s) are important components of Bothrops snake venoms, that can induce several effects on envenomations such as myotoxicity, inhibition or induction of platelet aggregation and edema. It is known that venomous and non-venomous snakes present PLA(2) inhibitory proteins (PLIs) in their blood plasma. An inhibitory protein that neutralizes the enzymatic and toxic activities of several PLA2s from Bothrops venoms was isolated from Bothrops alternatus snake plasma by affinity chromatography using the immobilized myotoxin BthTX-I on CNBr-activated Sepharose. Biochemical characterization of this inhibitory protein, denominated alpha BaltMIP, showed it to be a glycoprotein with Mr of similar to 24,000 for the monomeric subunit. CD spectra of the PLA(2)/inhibitor complexes are considerably different from those corresponding to the individual proteins and data deconvolution suggests that the complexes had a relative gain of helical structure elements in comparison to the individual protomers, which may indicate a more compact structure upon complexation. Theoretical and experimental structural studies performed in order to obtain insights into the structural features of aBaltMIP indicated that this molecule may potentially trimerize in solution, thus strengthening the hypothesis previously raised by other authors about snake PLIs oligomerization. (C) 2010 Elsevier Masson SAS. All rights reserved.

Cyclic Guanosine Monophosphate Signaling Cascade Mediates Pigment Aggregation in Freshwater Shrimp Chromatophores

The cell signaling cascades that mediate pigment movements in crustacean chromatophores are not yet well established, although Ca(2+) and cyclic nucleotide second messengers are involved. Here, we examine the participation of cyclic guanosine monophosphate (cGMP) in pigment aggregation triggered by red pigment concentrating hormone (RPCH) in the red ovarian chromatophores of freshwater shrimp. In Ca(2+)-containing (5.5 mmol l(-1)) saline, 10 mu mol l(-1) dibutyryl cGMP alone produced complete pigment aggregation with the same time course (approximate to 20 min) and peak velocity (approximate to 17 mu m/min) as 10(-8) mol l(-1) RPCH; however, in Ca(2+)-free saline (9 X 10(-11) mol l(-1) Ca(2+)), db-cGMP was without effect. The soluble guanylyl cyclase (GC-S) activators sodium nitroprusside (SNP, 0.5 mu mol l(-1)) and 3-morpholinosydnonimine (SIN-1, 100 mu mol l(-1)) induced moderate aggregation by themselves (approximate to 35%-40%) but did not affect RPCH-triggered aggregation. The GC-S inhibitors zinc protoporphyrin IX (ZnPP-XI, 30 mu mol l(-1)) and 6-anilino-5,8-quinolinedione (LY83583, 10 mu mol l(-1)) partially inhibited RPCH-triggered aggregation by approximate to 35%. Escherichia coli heat-stable enterotoxin (STa, 1 mu mol l(-1)), a membrane-receptor guanylyl cyclase stimulator, did not induce or affect RPCH-triggered aggregation. We propose that the binding of RPCH to an unknown membrane-receptor type activates a Ca(2+)-dependent signaling cascade coupled via cytosolic guanylyl cyclase and cGMP to protein kinase G-phosphorylated proteins that regulate aggregation-associated, cytoskeletal molecular motor activity. This is a further example of a cGMP signaling cascade mediating the effect of a crustacean X-organ neurosecretory peptide.

Towards a molecular epidemiology of alcohol dependence analysing the interplay of genetic and environmental risk factors

Background Progress in identifying genetic factors protective against alcohol dependence (AlcD) requires a paradigm shift in psychiatric epidemiology. Aims To integrate analysis of research into the genetics of alcoholism. Method Data from prospective questionnaire and interview surveys of the Australian twin panel, and from a subsample who underwent alcohol challenge, were analysed. Results In men, effects of alcohol dehydrogenase ADH2*1/*2 genotype or high alcohol sensitivity (risk-decreasing), and of history of childhood conduct disorder, or having monozygotic co-twin or twin sister with AlcD (risk-increasing) were significant and comparable in magnitude. Religious affiliation (Anglican versus other) was associated with the ADH2 genotype, but did not explain the associations with AlcD symptoms. No protective effect of the ADH2*1/*2 genotype was observed in women. Conclusions The early onset and strong familial aggregation of AlcD, and opportunity for within-family tests of genetic association to avoid confounding effects, make epidemiological family studies of adolescents and young adults and their families a priority.