Solar generated hydrogen as an energy source is green, sustainable, with a high energy density. One day the majority of current fossil fuel based technology could be replaced with hydrogen technology reducing CO2 emission drastically. The goal in this research is to explore hybrid metal oxide photocatalysts in the pursuit of achieving highly efficient photoanodes for use in photoelectrochemical cells (PEC). Achieving high efficiencies of hydrogen production in photoelectrochemical cells is the key challenge for the commercialisation of PEC technology as a viable, sustainable, hydrogen source; limited only by the lifetime of the sun and the resources of the metal oxide materials. In this research TiO2, Fe-Ti-O, ZnO, and Zn2TiO4 are the photocatalysts explored. Alloys of Ti-Fe-O showed improvement over TiO2, whilst a hybrid heterostructure of ZnO/Zn2TiO4/TiO2 enhanced photocurrent densities significantly. A barrier layer in the photoanode achieved localised exciton separation and reduction of recombination rates by inhibiting back flow of electrons after injection into the TiO2 layer. Nanotubes are created by the simple electrochemical process of anodisation. The nanotube composition depends on the anode material. To control the composition ofthe anode, iron and titanium are co-deposited onto a substrate using electron beam evaporation. The introduction of iron into titania nanotubes engineered the band gap, lowering the band gap energy to that of iron oxide whilst the positions of the conduction and valence bands with respect to the oxidation and reduction potentials of water remained favourable. Fe-Ti-O nanotubes showed remarkable photocurrent density improvement compared to TiO2 nanotubes. ZnO nanostructures deposited by vapour transport mechanisms showed variability in the morphology of the structures, as governed by the growth dynamics. Herein, it is shown that an electronically favourable situation arises by the formation of a ZnO-Zn2TiO4-TiO2 heterostructure and a high photocatalytic activity is reported. The structure is composed of a large surface area ZnO nanorod photoabsorber formed on a Ti foil which forms a Zn2TiO4 barrier layer between ZnO and TiO2. The Zn2TiO4 layer inhibits electron transport toward the surface of the photoanode whilst encouraging charge transport to the hydrogenation electrode. The heterostructure interfacial surface area is extended through the utilisation of TiO2 nanotubes, which demonstrated a 20.22 % photoelectrochemical efficiency under UV illumination. Surface modification of ZnO nanorods with aerosol assisted chemical vapour deposited TiO2 nanoparticles enhanced photocurrent densities of the ZnO rods, improving charge separation of excitons created within the TiO2 nanoparticles. ZnO nanotubes formed via a novel route using chemical bath deposition of ZnO is investigated, an annulus ZnO seed layer facilitated the site specific growth of ZnO nanotubes whilst a uniform seed layer formed ZnO nanorods.

Thesis (Ph. D.)--University of Toronto, 2008. / Includes bibliographical references.

This thesis primarily details synthetic and biophysical studies on the tridachiahydropyrone family of natural products. The general aim of this work was to explore an hypothesis regarding the location and function of these metabolites, isolated from sacoglossan molluscs. Specifically, it was hypothesised that tridachiahydropyrone is synthesised photochemically from linear polyene precursors via a selective double bond isomerisation-6pi electrocyclisation sequence which occurs within the cell membrane of the producing organism. Furthermore, it was postulated that this reaction sequence, and subsequent photochemical transformations of tridachiahydropyrone into the related products phototridachiahydropyrone and oxytridachiahydropyrone, serve to protect the producing mollusc from the damaging effects of UV radiation. Firstly, the proposed polyene precursors were synthesised using a convergent strategy dependent upon a late-stage Suzuki coupling. Their photochemical, biomimetic conversion into tridachiahydropyrone, phototridachiahydropyrone and oxytridachiahydropyrone, was then accomplished. The interactions of tridachiahydropyrone and its biomimetic precursors with model membrane systems were next explored, using a fluorescence spectroscopic technique. This work demonstrated that the molecules bind to phospholipid vesicles (PLVs) of varying compositions. The synthesis of tridachiahydropyrone within the PLVs was also achieved The propensity of the compounds to act as sunscreens was lastly investigated, by measuring the degree of protection against photochemically-induced lipid peroxidation they conferred on irradiated PLVs, using the thiobarbituric acid reactive substances assay. At high compound concentrations the compounds were found to act as sunscreens, whilst at lower concentrations pro-oxidant activity was observed. In addition to this main work, methodology for the palladium-catalysed cyanation of vinyl halides with acetone cyanohydrin was developed. Conditions were optimised using beta-bromostyrene, and shown to be applicable to a range of diverse substrates. The protocol proved chemoselective for vinyl bromides in the presence of aryl bromides, which were left unaffected and available for further chemical transformations, adding to the synthetic utility of the reaction.

The extracellular glucosyltransferases of Streptococcus sanguis polymerise the glucosyl moiety of sucrose to form high molecular weight complex glucans. The adhesive and agglutinative properties of these glucans are important in the formation of dental plaque and, hence, in cariogenesis. The glucosyltransferases of S. sanguis 804 (NCTC) were extensively purified (182-fold) by hollow fibre ultrafiltration (Bio-Fiber 80) followed by ammonium sulphate precipitation (0–70% of saturation). The enzymes were further purified by hydroxylapatite chromatography and appeared by this technique to consist of at least three enzymes with differing specific activities. It is not known whether these enzymes are, in fact, composed of different polypeptides or are modified forms of one protein. The activity of the glucosyltransferases can be measured as the rate of release of fructose from sucrose or as the rate of synthesis of ethanol-sodium acetate-precipitable polysaccharide (glucan). Using the former method, Kapp for sucrose for (NH4) 2SO4-purified glucosyltransferases was about 6 mmol/l and, using the latter method, Kapp was about 20 mmol/l. Glucosyltransferase activity (as rate of glucan synthesis) was stimulated 2 to 4-fold by low concentrations (0.125-0.50 μmol/l) of T2000 Dextran (Pharmacia; mol. wt. 2 × 106). Glucan synthesis was inhibited slightly by nigerose and was inhibited strongly by metrizamide (85% inhibition at 170 μmol/l metrizamide). The rate of release of fructose was not affected by either xylitol or hydrogen peroxide. The rate of synthesis of precipitable glucan was strongly inhibited by high concentrations of substrate (sucrose); the rate of release of fructose was relatively unaffected. The proposed mechanism for this effect is that sucrose acts as an alternative glucosyl acceptor (as well as donor) and thus inhibits glucosyl transfers to growing glucan chains. The oral concentrations of sucrose during and after consumptions of various sweet foods and beverages were studied and were often sufficient to inhibit glucan synthesis. In such cases, the sucrose concentrations for maximum rate of glucan synthesis only occurred as sucrose was cleared from the mouth, after the food or drink was finished. Glucan synthesis by S. sanguis is important in plaque formation. Thus, these results provide an additional explanation for the clinical finding that the incidence of caries is related to the frequency of dietary intake of sucrose and not merely the total amount of sucrose consumed.

Electroanalysis has proven to be one of the most widely used technologies for point-of-care devices. Owing to the direct recording of the intrinsic properties of biochemical functions, the field has been involved in the study of biology since electrochemistry’s conception in the 1800’s. With the advent of microelectronics, humanity has welcomed self-monitoring portable devices such as the glucose sensor in its everyday routine. The sensitivity of amperometry/ voltammetry has been enhanced by the use of microelectrodes. Their arrangement into microelectrode arrays (MEAs) took a step forward into sensing biomarkers, DNA and pathogens on a multitude of sites. Integrating these devices and their operating circuits on CMOS monolithically miniaturised these systems even more, improved the noise response and achieved parallel data collection. Including microfluidics on this type of devices has led to the birth of the Lab-on-a-Chip technology. Despite the technology’s inclusion in many bioanalytical instruments there is still room for enhancing its capabilities and application possibilities. Even though research has been conducted on the selective preparation of microelectrodes with different materials in a CMOS MEA to sense several biomarkers, limited effort has been demonstrated on improving the parallel electroanalytical capabilities of these devices. Living and chemical materials have a tendency to alter their composition over time. Therefore analysing a biochemical sample using as many electroanalytical methods as possible simultaneously could offer a more complete diagnostic snapshot. This thesis describes the development of a CMOS Lab-on-a-Chip device comprised of many electrochemical cells, capable of performing simultaneous amperometric/voltammetric measurements in the same fluidic chamber. The chip is named an electrochemical cell microarray (ECM) and it contains a MEA controlled by independent integrated potentiostats. The key stages in this work were: to investigate techniques for the electrochemical cell isolation through simulations; to design and implement a CMOS ECM ASIC; to prepare the CMOS chip for use in an electrochemical environment and encapsulate it to work with liquids; to test and characterise the CMOS chip housed in an experimental system; and to make parallel measurements by applying different simultaneous electroanalytical methods. It is envisaged that results from the system could be combined with multivariate analysis to describe a molecular profile rather than only concentration levels. Simulations to determine the microelectrode structure and the potentiostat design, capable of constructing isolated electrochemical cells, were made using the Cadence CAD software package. The electrochemical environment and the microelectrode structure were modelled using a netlist of resistors and capacitors. The netlist was introduced in Cadence and it was simulated with potentiostat designs to produce 3-D potential distribution and electric field intensity maps of the chemical volume. The combination of a coaxial microelectrode structure and a fully differential potentiostat was found to result in independent electrochemical cells isolated from each other. A 4 x 4 integrated ECM controlled by on-chip fully differential potentiostats and made up by a 16 × 16 working electrode MEA (laid out with the coaxial structure) was designed in an unmodified 0.35 μm CMOS process. The working electrodes were connected to a circuit capable of multiplexing them along a voltammetric measurement, maintaining their diffusion layers during stand-by time. Two readout methods were integrated, a simple resistor for an analogue readout and a discrete time digital current-to-frequency charge-sensitive amplifier. Working electrodes were designed with a 20 μm side length while the counter and reference electrodes had an 11 μm width. The microelectrodes were designed using the aluminium top metal layer of the CMOS process. The chips were received from the foundry unmodified and passivated, thus they were post-process fabricated with photolithographic processes. The passivation layer had to be thinned over the MEA and completely removed on top of the microelectrodes. The openings were made 25 % smaller than the top metal layer electrode size to ensure a full coverage of the easily corroded Al metal. Two batches of chips were prepared, one with biocompatible Au on all the microelectrodes and one altered with Pd on the counter and Ag on the reference electrode. The chips were packaged on ceramic pin grid array packages and encapsulated using chemically resistant materials. Electroplating was verified to deposit Au with increased roughness on the microelectrodes and a cleaning step was performed prior to electrochemical experiments. An experimental setup containing a PCB, a PXIe system by National Instruments, and software programs coded for use with the ECM was prepared. The programs were prepared to conduct various voltammetric and amperometric methods as well as to analyse the results. The first batch of post-processed encapsulated chips was used for characterisation and experimental measurements. The on-chip potentiostat was verified to perform alike a commercial potentiostat, tested with microelectrode samples prepared to mimic the coaxial structure of the ECM. The on-chip potentiostat’s fully differential design achieved a high 5.2 V potential window range for a CMOS device. An experiment was also devised and a 12.3 % cell-to-cell electrochemical cross-talk was found. The system was characterised with a 150 kHz bandwidth enabling fast-scan cyclic voltammetry(CV) experiments to be performed. A relatively high 1.39 nA limit-of-detection was recorded compared to other CMOS MEAs, which is however adequate for possible applications of the ECM. Due to lack of a current polarity output the digital current readout was only eligible for amperometric measurements, thus the analogue readout was used for the rest of the measurements. The capability of the ECM system to perform independent parallel electroanalytical measurements was demonstrated with 3 different experimental techniques. The first one was a new voltammetric technique made possible by the ECM’s unique characteristics. The technique was named multiplexed cyclic voltammetry and it increased the acquisition speed of a voltammogram by a parallel potential scan on all the electrochemical cells. The second technique measured a chemical solution with 5 mM of ferrocene with constant potential amperometry, staircase cyclic voltammetry, normal pulse voltammetry, and differential pulse voltammetry simultaneously on different electrochemical cells. Lastly, a chemical solution with 2 analytes (ferrocene and decamethylferrocene) was prepared and they were sensed separately with constant potential amperometry and staircase cyclic voltammetry on different cells. The potential settings of each electrochemical cell were adjusted to detect its respective analyte.

In this work, we examine the structure and function of two important human proteins. The first is human kallikrein 6 (hK6), which is a newly identified enzyme in the serine proteinase family that is expressed in the central nervous system. In chapter 2, the X-ray crystal structure of mature, active recombinant human kallikrein 6 at 1.75 Å is presented. This high resolution model provides the first three-dimensional view of one of the human kallikreins and one of only a few structures of serine proteinases predominantly expressed in the central nervous system. Enzymatic and X-ray data provide support for the characterization of human kallikrein 6 as a degradative proteinase with structural features more similar to trypsin than the regulatory kallikreins. In chapter 3, we have re-solved the structure of hK6 to a resolution of 1.56 Å. In addition, a detailed analysis of the preferred substrate specificity of hK6 at the positions P3, P2, P1′, P2′, and P3′ is undertaken using internally quenched fluorescent substrates based on a peptide background sequence of the identified autolysis region. Furthermore, the identified optimized substrate sequence is modeled into the 1.56 Å structure of human kallikrein 6 using docking in order to identify structural aspects of the protein responsible for this preference. The substrate specificity data show that human kallikrein 6 displays little discrimination for particular amino acids at the tested positions with the exception of P2′, where there is a pronounced preference for proline. The second protein studied in this work is human fibroblast growth factor-1 which is a member of the β-trefoil superfold. In chapter 4, a 1.10 Å atomic-resolution x-ray structure of human fibroblast growth factor 1, a member of the β-trefoil superfold, is reported. The FGF-1 structure exhibits numerous core packing defects detectable using a 1.0Å radius probe. In addition to contributing to the relatively low thermal stability of FGF-1, these defects may also permit domain motions within the structure. The availability of refined ADP's permits a translation/libration/ screw (TLS) analysis of putative rigid body domains. The observed rigid body motion in FGF-1 appears related to the ligand-binding functionalities. / Dissertation / PhD

Human acidic fibroblast growth factor (FGF-1) is a member of the £]-trefoil superfamily and exhibits a characteristic three-fold tertiary structure symmetry. However, evidence of this symmetry is not readily apparent at the level of the primary structure. This suggests that while selective pressures may exist to retain (or converge upon) a symmetric tertiary structure, other selective pressures have resulted in divergence of the primary structure during evolution. Using intra-chain and homologue sequence comparisons for 19 members of this family of proteins, we have designed mutants of FGF-1 that constrain a subset of core-packing residues to three-fold symmetry at the level of the primary structure. The consequences of these mutations upon structure, stability, folding and unfolding kinetics have been evaluated using a combination of x-ray crystallography, differential scanning calorimetry, isothermal equilibrium denaturation and stopped flow protein refolding/unfolding kinetics. An alternative core packing group has been introduced into FGF-1. The alternative core is very similar from the wild type (WT) core with regard to structure, stability, folding and unfolding kinetics. The remaining asymmetry within the protein core is related to asymmetry in the tertiary structure. The removal of tertiary structure asymmetry greatly increases protein stability and results in a conversion from three-state to a two-state folding pathway. The tertiary structure asymmetry is intimately linked to functional regions of the protein. Surprisingly, upon deletion of the functional insertions, the mutant protein is approximately 80 times more potent than the wild type form as determined by functional bioassays. The results show that the ƒÒ-trefoil superfold is compatible with a three-fold symmetric constraint upon the core region, as might be the case if the superfold arose as a result of gene duplication/fusion events. Furthermore, this new protein arrangement can form the basis of a structural "building block" that can greatly simplify the de novo design of ƒÒ-trefoil proteins by utilizing symmetric structural complementarity. This study implies that a symmetric architecture of the £]-trefoil fold is kinetically and thermodynamically ¡§fit¡¨. / Dissertation / PhD

The rationalisation of drug potency using three-dimensional structures of protein-ligand complexes is a central paradigm in medicinal research. For over two decades, a major goal has been to find the rules that accurately relate the structure of any protein-ligand complex to its affinity. Addressing this problem is of great concern to the pharmaceutical industry, which uses virtual screens to computationally assay up to many millions of compounds against a protein target. A fast and trustworthy affinity estimator could potentially streamline the drug discovery process, reducing reliance on expensive wet lab experiments, speeding up the discovery of new hits and aiding lead optimization. Water plays a critical role in drug-protein interactions. To address the often ambiguous nature of water in binding sites, a water placement method was developed and found to be in good agreement with X-ray crystallography, neutron diffraction data and molecular dynamics simulations. The method is fast and has facilitated a large scale study of the statistics of water in ligand binding sites, as well as the creation of models pertaining to water binding free energies and displacement propensities, which are of particular interest to medicinal chemistry. Structure-based scoring functions employing the explicit water models were developed. Surprisingly, these attempts were no more accurate than the current state of the art, and the models suffered from the same inadequacies which have plagued all previous scoring functions. This suggests a unifying cause behind scoring function inaccuracy. Accordingly, mathematical analyses on the fundamental uncertainties in structure-based modelling were conducted. Using statistical learning theory and information theory, the existence of inherent errors in empirical scoring functions was proven. Among other results, it was found that even the very best generalised structure-based model is significantly limited in its accuracy, and protein-specific models are always likely to be better. The theoretical framework developed herein hints at modelling strategies that operate at the leading edge of achievable accuracy.

Charles University in Prague, Faculty of Science, Department of Teaching and Didactics of Chemistry, Hlavova 8, Prague 2 Author: Bc. Eva Hrobařová Name: The teaching materials for chemical education - experimental and practical chemistry with different kind of food ABSTRACT: In this thesis I elaborated the theme "The teaching material for active chemical education - experimental and practical chemistry with different kind of food". I created a questionnaire with name "Availability of laboratories and schoolrooms of chemistry in college". The results of this questionnaire were a base for choice of the experiments with different kind of food. Each experiment is completed by photos and a few questions for students. In this work I also analysed several thesis about practical and experimental chemistry. In this thesis I lay stress on importance of experimental and practice chemistry in colleges in Czech Republic. My task was to create easy teaching materials for active education and the utilization of easy experiments to illustrate theory. The experiments are very easy, it's not necessary many tools and it's not even necessary to use chemical laboratory. .

Protein structure models obtained from X-ray crystallography are subject to radiation damage. The resulting specific alterations to protein structures can be mistaken for biological features, or may obscure actual protein mechanisms, leading to misidentification or obscuration of biological insight. The radiation chemistry behind this site-specific damage is not well understood. Radiation damage processes progress in proportion to the dose absorbed by the crystal in the diffraction experiment. Doses can be estimated using existing software, but these assume idealised experimental conditions. To simulate complex diffraction experiments, including treatment of imperfect X-ray beam profiles and inhomogeneous dose distributions, a new program, RADDOSE-3D, was developed. RADDOSE-3D can be integrated into beamline software to provide convenient, more accurate, comparative, and publishable dose figures, also facilitating informed data collection decisions. There is currently no method to automatically detect specific radiation damage in protein structure models in the absence of an 'undamaged' reference model. Radiation damage research therefore generally relies on detailed observation of a few model proteins. A new metric, B_Damage, is designed and used to identify and quantify specific radiation damage in the first large-scale statistical survey of 2,704 published protein models, which are examined for the effects of local environments on site-specific radiation damage susceptibility. A significant positive correlation between susceptibility and solvent accessibility is identified. Current understanding of radiation damage progression is mostly based on a few consecutive structure model 'snapshots' at coarse dose intervals. The low sampling rate considerably limits the ability to identify varying site susceptibility and its causes. Real space electron density data are obtained for crystals of different mutants of a RhoGDI protein with very high sequence identity, to determine sensitising and stabilising factors for radiation induced structural changes. Utilising a newly developed data collection and analysis protocol, these changes could be tracked with unprecedented time resolution.