Possible mechanism of charge transport and dielectric relaxation in SrO-Bi2O3-B2O3 glasses

Transparent SrBi2B2O7 glasses were prepared via melt-quenching technique and characterized using differential scanning calorimetry and x-ray powder diffraction. The ac conductivities of the glasses were studied as a function of frequency (100 Hz-10 MHz) at different temperatures. The frequency dependence of conductivity has been analyzed using Almond-West expression. The exponent n was nearly unaffected by temperature. Impedance and modulus spectroscopies were employed to further examine the electrical data. Dielectric relaxation exhibited a stretched exponential behavior with a stretching exponent beta independent of temperature. From conductivity analysis we have proposed that the charge transport occurs through the participation of nonbridging oxygen (NBO), which switches positions in a facile manner. The stretched exponential behavior appears to be a direct consequence of the NBO switching mechanism of charge transport.

Ligand dependent intra and inter subunit communication in human tryptophanyl tRNA synthetase as deduced from the dynamics of structure networks

Homodimeric protein tryptophanyl tRNA synthetase (TrpRS) has a Rossmann fold domain and belongs to the 1c subclass of aminoacyl tRNA synthetases. This enzyme performs the function of acylating the cognate tRNA. This process involves a number of molecules (2 protein subunits, 2 tRNAs and 2 activated Trps) and thus it is difficult to follow the complex steps in this process. Structures of human TrpRS complexed with certain ligands are available. Based on structural and biochemical data, mechanism of activation of Trp has been speculated. However, no structure has yet been solved in the presence of both the tRNA(Trp) and the activated Trp (TrpAMP). In this study, we have modeled the structure of human TrpRS bound to the activated ligand and the cognate tRNA. In addition, we have performed molecular dynamics (MD) simulations on these models as well as other complexes to capture the dynamical process of ligand induced conformational changes. We have analyzed both the local and global changes in the protein conformation from the protein structure network (PSN) of MD snapshots, by a method which was recently developed in our laboratory in the context of the functionally monomeric protein, methionyl tRNA synthetase. From these investigations, we obtain important information such as the ligand induced correlation between different residues of this protein, asymmetric binding of the ligands to the two subunits of the protein as seen in the crystal structure analysis, and the path of communication between the anticodon region and the aminoacylation site. Here we are able to elucidate the role of dimer interface at a level of detail, which has not been captured so far.

Molecular mechanisms of bacteriophage phi6 RNA-dependent RNA polymerase and its utilization in biotechnology

Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.

The role of phosphorus as a regulator of bloom-forming diazotrophic cyanobacteria in the Baltic Sea

Eutrophication favours harmful algal blooms worldwide. The blooms cause toxic outbreaks and deteriorated recreational and aesthetic values, causing both economic loss and illness or death of humans and animals. The Baltic Sea is the world s only large brackish water habitat with recurrent blooms of toxic cyanobacteria capable of biological fixation of atmospheric nitrogen gas. Phosphorus is assumed to be the main limiting factor, along with temperature and light, for the growth of these cyanobacteria. This thesis evaluated the role of phosphorus nutrition as a regulating factor for the occurrence of nitrogen-fixing cyanobacteria blooms in the Baltic Sea, utilising experimental laboratory and field studies and surveys on varying spatial scales. Cellular phosphorus sources were found to be able to support substantial growth of the two main bloom forming species Aphanizomenon sp. and Nodularia spumigena. However, N. spumigena growth seemed independent of phosphorus source, whereas, Aphanizomenon sp. grew best in a phosphate enriched environment. Apparent discrepancies with field observations and experiments are explained by the typical seasonal temperature dependent development of Aphanizomenon sp. and N. spumigena biomass allowing the two species to store ambient pre-bloom excess phosphorus in different ways. Field experiments revealed natural cyanobacteria bloom communities to be predominantly phosphorus deficient during blooms. Phosphate additions were found to increase the accumulation of phosphorus relatively most in the planktonic size fraction dominated by the nitrogen-fixing cyanobacteria. Aphanizomenon sp. responded to phosphate additions whereas the phosphorus nutritive status of N. spumigena seemed independent of phosphate addition. The seasonal development of phosphorus deficiency is different for the two species with N. spumigena showing indications of phosphorus deficiency during a longer time period in the open sea. Coastal upwelling introduces phosphorus to the surface layer during nutrient deficient conditions in summer. The species-specific ability of Aphanizomenon sp. and N. spumigena to utilise phosphate enrichment of the surface layer caused by coastal upwelling was clarified. Typical bloom time vertical distributions of biomass maxima were found to render N. spumigena more susceptible to advection by surface currents caused by coastal upwellings. Aphanizomenon sp. populations residing in the seasonal thermocline were observed to be able to utilise the phosphate enrichment and a bloom was produced with a two to three week time lag subsequent to the relaxation of upwelling. Consistent high concentrations of dissolved inorganic phosphorus, caused by persistent internal loading of phosphorus, was found to be the main source of phosphorus for large-scale pelagic blooms. External loads were estimated to contribute with only a fraction of available phosphorus for open sea blooms. Remineralization of organic forms of phosphorus along with vertical mixing to the permanent halocline during winter set the level of available phosphorus for the next growth season. Events such as upwelling are important in replenishing phosphate concentrations during the nutrient deplete growth season. Autecological characteristics of the two main bloom forming species favour Aphanizomenon sp. populations in utilising the abundant excess phosphate concentrations and phosphate pulses mediated through upwelling. Whilst, N. spumigena displays predominant phosphorus limited growth mode and relies on more scarce cellular phosphorus stores and presumably dissolved organic phosphorus compounds for growth. The Baltic Sea is hypothesised to be in an inhibited state of recovery due to the extensive historical external nutrient loading, extensive internal phosphorus loading and the substantial nitrogen load caused by cyanobacteria nitrogen fixation. This state of the sea is characterised as a vicious circle .

Mutations as molecular tools : The metabolic-rate dependent molecular clock and DNA barcoding of allied species

Mutation and recombination are the fundamental processes leading to genetic variation in natural populations. This variation forms the raw material for evolution through natural selection and drift. Therefore, studying mutation rates may reveal information about evolutionary histories as well as phylogenetic interrelationships of organisms. In this thesis two molecular tools, DNA barcoding and the molecular clock were examined. In the first part, the efficiency of mutations to delineate closely related species was tested and the implications for conservation practices were assessed. The second part investigated the proposition that a constant mutation rate exists within invertebrates, in form of a metabolic-rate dependent molecular clock, which can be applied to accurately date speciation events. DNA barcoding aspires to be an efficient technique to not only distinguish between species but also reveal population-level variation solely relying on mutations found on a short stretch of a single gene. In this thesis barcoding was applied to discriminate between Hylochares populations from Russian Karelia and new Hylochares findings from the greater Helsinki region in Finland. Although barcoding failed to delineate the two reproductively isolated groups, their distinct morphological features and differing life-history traits led to their classification as two closely related, although separate species. The lack of genetic differentiation appears to be due to a recent divergence event not yet reflected in the beetles molecular make-up. Thus, the Russian Hylochares was described as a new species. The Finnish species, previously considered as locally extinct, was recognized as endangered. Even if, due to their identical genetic make-up, the populations had been regarded as conspecific, conservation strategies based on prior knowledge from Russia would not have guaranteed the survival of the Finnish beetle. Therefore, new conservation actions based on detailed studies of the biology and life-history of the Finnish Hylochares were conducted to protect this endemic rarity in Finland. The idea behind the strict molecular clock is that mutation rates are constant over evolutionary time and may thus be used to infer species divergence dates. However, one of the most recent theories argues that a strict clock does not tick per unit of time but that it has a constant substitution rate per unit of mass-specific metabolic energy. Therefore, according to this hypothesis, molecular clocks have to be recalibrated taking body size and temperature into account. This thesis tested the temperature effect on mutation rates in equally sized invertebrates. For the first dataset (family Eucnemidae, Coleoptera) the phylogenetic interrelationships and evolutionary history of the genus Arrhipis had to be inferred before the influence of temperature on substitution rates could be studied. Further, a second, larger invertebrate dataset (family Syrphidae, Diptera) was employed. Several methodological approaches, a number of genes and multiple molecular clock models revealed that there was no consistent relationship between temperature and mutation rate for the taxa under study. Thus, the body size effect, observed in vertebrates but controversial for invertebrates, rather than temperature may be the underlying driving force behind the metabolic-rate dependent molecular clock. Therefore, the metabolic-rate dependent molecular clock does not hold for the here studied invertebrate groups. This thesis emphasizes that molecular techniques relying on mutation rates have to be applied with caution. Whereas they may work satisfactorily under certain conditions for specific taxa, they may fail for others. The molecular clock as well as DNA barcoding should incorporate all the information and data available to obtain comprehensive estimations of the existing biodiversity and its evolutionary history.

NUPARM and NUCGEN: software for analysis and generation of sequence dependent nucleic acid structures

Software packages NUPARM and NUCGEN, are described, which can be used to understand sequence directed structural variations in nucleic acids, by analysis and generation of non-uniform structures. A set of local inter basepair parameters (viz. tilt, roll, twist, shift, slide and rise) have been defined, which use geometry and coordinates of two successive basepairs only and can be used to generate polymeric structures with varying geometries for each of the 16 possible dinucleotide steps. Intra basepair parameters, propeller, buckle, opening and the C6...C8 distance can also be varied, if required, while the sugar phosphate backbone atoms are fixed in some standard conformation ill each of the nucleotides. NUPARM can be used to analyse both DNA and RNA structures, with single as well as double stranded helices. The NUCGEN software generates double helical models with the backbone fixed in B-form DNA, but with appropriate modifications in the input data, it can also generate A-form DNA ar rd RNA duplex structures.

Vitrification, relaxation and free volume in glycerol-water binary liquid mixture:Spin probe ESR studies

Glass transition and relaxation of the glycerol-water (G-W) binary mixture system have been studied over the glycerol concentration range of 5-85 mol% by using the highly sensitive technique of electron spin resonance (ESR). For the water rich mixture the glass transition,sensed by the dissolved spin probe, arises from the vitrified mesoscopic portion of the binary system. The concentration dependence of the glass transition temperature manifests a closely related molecular level cooperativity in the system. A drastic change in the mesoscopic structure of the system at the critical concentration of 40 mol is confirmed by an estimation of the spin probe effective volume in a temperature range where the tracer reorientation is strongly coupled to the system dynamics.

Precipitate growth with composition-dependent diffusivity: Comparison between theory and phase field simulations

We consider the growth of an isolated precipitate when the matrix diffusivity depends on the composition. We have simulated precipitate growth using the Cahn-Hilliard model, and find good agreement between our results and those from a sharp interface theory for systems with and without a dilatational misfit. With misfit, we report (and rationalize) an interesting difference between systems with a constant diffusivity and those with a variable diffusivity in the matrix.

Temperature dependent structural properties of nanocrystalline SnS structures

This letter explores the structural behavior of nanocrystalline tin mono sulfide (SnS) structures with respect to temperature (100-600 K). These studies emphasize that the structural properties of SnS nanocrystalline structures depend on the surrounding temperature. The lattice parameters of SnS nanocrystals slightly varied like their microstructures with the increase of temperature. These changes strongly influence the optical properties of SnS nanostructures. On the other hand, the structures exhibited higher strain (similar to 0.44%) than that of microstructured (0.3%) and bulk (0.12%) counterparts. The observed results are discussed under the light of existing concepts and reported.

RecA Protein of Mycobacterium tuberculosis Possesses pH-Dependent Homologous DNA Pairing and Strand Exchange Activities: Implications for Allele Exchange in Mycobacteria

To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria.

Characterization of anomalous relaxation using the time-fractional Bloch equation and multiple echo T2 *-weighted magnetic resonance imaging at 7T

PURPOSE To study the utility of fractional calculus in modeling gradient-recalled echo MRI signal decay in the normal human brain. METHODS We solved analytically the extended time-fractional Bloch equations resulting in five model parameters, namely, the amplitude, relaxation rate, order of the time-fractional derivative, frequency shift, and constant offset. Voxel-level temporal fitting of the MRI signal was performed using the classical monoexponential model, a previously developed anomalous relaxation model, and using our extended time-fractional relaxation model. Nine brain regions segmented from multiple echo gradient-recalled echo 7 Tesla MRI data acquired from five participants were then used to investigate the characteristics of the extended time-fractional model parameters. RESULTS We found that the extended time-fractional model is able to fit the experimental data with smaller mean squared error than the classical monoexponential relaxation model and the anomalous relaxation model, which do not account for frequency shift. CONCLUSIONS We were able to fit multiple echo time MRI data with high accuracy using the developed model. Parameters of the model likely capture information on microstructural and susceptibility-induced changes in the human brain.

H-1 and F-19 NMR relaxation time studies in (NH4)(2)ZrF6 superionic conductor

H-1 and F-19 spin-lattice relaxation times in polycrystalline diammonium hexafluorozirconate have been measured in the temperature range of 10-400 K to elucidate the molecular motion of both cation and anion. Interesting features such as translational diffusion at higher temperatures, molecular reorientational motion of both cation and anion groups at intermediate temperatures and quantum rotational tunneling of the ammonium group at lower temperatures have been observed. Nuclear magnetic resonance (NMR) relaxation time results correlate well with the NMR second moment and conductivity studies reported earlier.