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Histone H3 lysine 4 (H3K4) methylation is an evolutionarily conserved mark commonly associated with transcription activation in eukaryotes. In mammals, this post-translational modification is deposited by the KMT2 family of H3K4 methyltransferases. Biochemical studies have shown that the enzymatic activity of the KMT2 enzymes is regulated by a core complex of four evolutionarily conserved proteins: WDR5, RbBP5, ASH2L and DPY30, collectively known as WRAD, which are all important for global H3K4 methylation. However, how these proteins interact and regulate the activity of the KMT2 enzymes is not well investigated. During my PhD, I have used structural and biochemical approaches to determine the interactions underlying formation of the core complex and regulation of KMT2 enzymatic activity. My research have shown that 1) WDR5 uses two peptide-binding clefts on opposite sides of its β-propeller domain to bridge the KMT2 enzymes to the regulatory subunit RbBP5, 2) the WDR5 peptidyl-arginine-binding cleft exhibits plasticity to accommodate the binding of all KMT2 enzymes and 3) RbBP5 S350 phosphorylation stimulates formation of the RbBP5-ASH2L complex and H3K4 methylation by the mammalian KMT2 enzymes. Collectively, these studies have provided the structural basis for understanding the important interactions governing KMT2 complex assembly and activity.

The molecular and crystal structures of dibenzobarrelene and a number of its diester derivatives, and three dibenzosemibullvalenes have been determined by the use of X-ray crystallography. The objectives of the study were to investigate whether or not the di-π-methane photorearrangement could be carried out in the solid state, and how such solid state results would differ from results obtained in solution. In addition, we were interested in investigating the extent to which intermolecular steric effects would modulate or change the course of the photorearrangement in the solid state as compared to its solution pathway with the view of developing a structure-reactivity correlation for the reaction in the solid state. All the dibenzobarrelenes underwent the di-π-methane photorearrangement in the solid state to give the corresponding dibenzosemibullvalene photoproduct(s). In the symmetrical 11,12-diester derivatives of dibenzobarrelene, only one di-π-methane photoproduct could be identified for each of the reactants. The ethyl/ethyl and isopropyl/iso-propyl diester derivatives displayed polymorphism. An absolute asymmetric synthesis was performed on one of the dimorphs of the iso-propyl/iso-propyl derivative which crystallized in a chiral space group, and a quantitative enantiomeric excess yield was obtained. The molecular structures of all the compounds studied showed different degrees of conjugation of the ester carbonyl groups to the central vinyl bond. The unsymmetrical 11,12-diester derivatives yielded regioisomeric dibenzosemibullvalene photoproducts. Generally, the reactions in the solid state were found to be more regioselective than the same reactions in solution. The observed differences of the relative quantities of regioisomeric photoproducts in the solid state are interpreted on the basis of intermolecular steric effects. In appropriate systems, intermolecular steric effects may be used to predict successfully not only the major regioisomeric product of a di-π-methane photorearrangement in the solid state, but also the major enantiomeric product. One regioisomeric photoproduct is obtained for each 9,11 and 10,11-diester derivative. The photoproducts obtained from these unsymmetrical 9,11 and 10,11-diester derivatives of dibenzobarrelene show that electronic effects may be the dominant factor which governs the photochemical reaction pathway of this class of compounds in the solid state. Overall, intermolecular steric hindrance and electronic factors affect the solid state photochemical pathway of each compound to different extents. There appears to be an interplay of electronic and steric factors in determining the reaction pathway which leads to the major product in the solid state. The dominance of one factor (steric versus electronic) over the other in the determination of the most favorable photochemical pathway is dependent upon the conformations of the ester groups and their relative extents of conjugation to the central vinyl bond, and the relative intermolecular steric environments of the ester groups or substituents involved in the first step (vinyl-benzo bridging) of the photochemical reaction. / Science, Faculty of / Chemistry, Department of / Graduate

X-ray Computer Tomography (CT) is well suited to the inspection of Fibre-Reinforced-Plastic (FRP) composite materials. However, a range of limitations currently restrict its uptake. The aim of the present research was to develop advanced inspection procedures that overcome these limitations and increase the scope of composite structures that can be inspected by industrial cone beam CT. Region of Interest (ROI) CT inspection of FRP laminated panels was investigated and two data completion methods developed to overcome reconstruction errors caused by truncated projection data. These allow accurate, highly magnified regions to be reconstructed on objects that extend beyond the Field-of-View (FOV) of the detector. The first method extended the truncated projection data using a cosine signal tailing off to zero attenuation. This method removed the strong 'glowing' artefacts but an inherent error existed across the reconstructed ROI. This did not affect the defect detectability of the inspection but was viewed as problematic for applications requiring accurate density measurements. The second method used prior knowledge of the test object so that a model could be created to estimate the missing data. This technique removed errors associated with ROI reconstruction thus significantly improving the accuracy. Techniques for extending the FOV were developed and applied to the inspection of FRP wind turbine blades; over 1.5X larger than the conventional scanning FOV. Two data completion methods were developed requiring an asymmetrically positioned detector. The first was based on the cosine tailing technique and the second used fan beam ray redundancy properties to estimate the missing data. Both produced accurate reconstructions for the 'offset' projection data, demonstrating that it was possible to approximately double the FOV. The cosine tailing method was found to be the more reliable. A dual energy image CT technique was developed to extend the optimum dynamic range and improve defect detectability for multi-density objects. This was applied to FRP composite/Titanium lap joints showing improved detectability of both volumetric and planar defects within the low density FRP. The dual energy procedure was validated using statistical performance measures on a specially fabricated multi-density phantom. The results showed a significant improvement in the detail SNR when compared to conventional CT scans.

Gas cells make up a significant proportion of bread’s volume and are responsible for a number of bread’s characteristics, making their size distribution throughout bread an important quality parameter. The number and size of cells affect the texture and volume of bread, the quantity of sauce mopped up, and how bright the bread appears. Gas cells are incorporated into bread dough during mixing and manipulated throughout the breadmaking process to obtain the desired cellular structure. Due to the fragile nature of bread dough, obtaining accurate quantitative data on its cellular structure is challenging. This thesis investigates the cellular structure of bread, as well as assessing the effect of sugar during breadmaking. Magnetic resonance imaging (MRI), microscopy and X-ray computerised tomography (X-ray CT) have been used throughout research in bread dough to visualise dough’s cellular structure. A non-destructive and non-invasive method giving a high resolution is X-ray CT, in particular when using a synchrotron light source. However, time on a synchrotron beamline is highly competitive, and can require applications more than two years in advance. Running costs of experiments from a synchrotron beamline are also high. This thesis details an alternative X-ray set-up to accurately visualise dough’s cellular structure using a conventional and therefore more easily accessible X-ray source. Three X-ray CT experiments were conducted to investigate dough’s cellular structure throughout mixing, during proving and in different sugar content doughs. The resolution of the scans varied from 7-11 µm. Industrial bread dough mixing is often conducted at a high pressure initially to improve oxygen availability, followed by a period of partial vacuum to favourably manipulate the cell size distribution. Using X-ray CT, dough cell size distribution was measured at different points throughout pressure-vacuum and constant pressure mixing. A simplified population balance model was fitted to the measured cell size distributions and the validity of the assumptions within the simplified model explored. It was shown that the dynamic changes in the cell size distribution within bread dough could be accurately measured during pressure step change mixing with a non-synchrotron X-ray source. Pressure-vacuum mixing was shown to give a finer cell distribution than constant pressure mixing and the observed decrease in cell number density was found to be much more short lived than the decrease in cell size. The model was found to provide a reasonably accurate characterisation of pressure-vacuum mixing. X-ray CT was also used to monitor dough’s changing cellular structure during proving by taking scans every 5 minutes over 145 minutes. Dough voidage increased from 3% to 66%, resulting in a volume increase from 544 mm3 to 1293 mm3. Cell growth was quickest between 40 and 140 minutes, where a steady increase in volume and significant changes in the cell structure occurred. A change in voidage distribution was observed, with greater proportions of gas located in larger cells over time. In addition, over the course of proving cell numbers dropped, a 156-fold increase in mean cell volume occurred, and mean cell Feret shape increased from 1.59 to 1.91. This in-situ method of X-ray imaging of bread dough provides higher resolution images than comparable data from conventional X-ray sources. In addition, the method has proved to be effective in obtaining high resolution and high contrast 3D images of the cellular structure of dough. This technique will help those wanting to investigate cellular changes in the dough dynamically, but without the waiting time and applications that are required with synchrotron X-rays. On investigating the effect of sugar during breadmaking, sugar was found to increase the gas free dough density and dough voidage, change the dough’s rheology, increase its proving time and produce denser bread. Application of a population balance model on the experimental results indicate that the decrease in steady state voidage as the sugar content increases is a result of an increase in disentrainment. This was reflected in the X-ray CT of sugared vs. non-sugared doughs through fewer and smaller cells present in sugared doughs. This is likely to be a result of a weaker dough structure, making cell rupture more likely. The Chorleywood Bread Process (CBP) is used industrially worldwide for the production of bread in less time and using inferior ingredients compared to the traditional bulk fermentation process, making it more cost effective. These results show that simply extending the pressure vacuum mixing used for the production of standard bread loaves in the CBP to sugared doughs should be avoided as aeration of sugared doughs differs to non-sugared doughs. The results suggest that to do so would be detrimental to the product quality.

The initial aim of the work presented in this thesis was to examine smectic layer compressibility with a view to improving our understanding of the stability of intermediate phases. A natural starting point was to investigate the smectic-A phase, as it is the most basic of the smectic phases. The response of the layered structure to external fields is also a focus of this thesis as electric and magnetic fields enable the layer properties to be probed. Investigations into the reorientation dynamics of smectic-A layers in magnetic fields were performed using geometries and cell thicknesses (>50 μm) that are not feasible using electric fields. Data presented in this thesis show that three distinct reorientation mechanisms can occur, one of which is previously unreported and bridges the gap between the previously known mechanisms. The new mechanism observed in 270 μm and 340 μm thickness cells exhibits multiple stage reorientation on a timescale between tens and hundreds of seconds. Using conventional electro-optic techniques combined with a theoretical approach developed by others, this thesis presents a new technique to provide measurement of relative smectic layer compressibility of eight smectic-A liquid crystalline materials. The method presented here combines data on cell thickness, dielectric anisotropy and the measurement of the voltage threshold of the toroidal to stripe domain transition. As expected, the experimental data indicated that materials with shorter molecular lengths had the largest relative layer compressibility. Finally, direct measurement of smectic layer compressibility was investigated and the design of an apparatus capable of such measurements was undertaken. Preliminary results from such an apparatus are presented along with a discussion on the steps taken to develop the design.

The M-shell x-ray production cross section for thorium and uranium have been determined for protons of energy 0.4 - 4.0 MeV, helium ions of energy 0.4 - 6.0 MeV, carbon ions of energy 4.5 - 11.3 MeV and oxygen ions of energy 4.5 - 13.5 MeV. The total cross sections and the cross sections for individual x-ray peaks in the spectrum, consisting of the following transitions Mz (M4-N2, M5-N3, M4-N3), Ma (M5-N6,7), Mb (M4-N6, M5-O3, M4- O2), and Mg (M4-O3, M5-P3, M3-N4, M3-N5), were compared to the theoretical values determined from the PWBA + OBKN and ECUSAR. The theoretical values for the carbon and oxygen ions were also modified to take into account the effects of multiple ionizations of the target atom by the heavier ions. It is shown that the results of the ECUSAR theory tend to provide better agreement with the experimental data.

Experiments were designed for an undergraduate instrumental analysis laboratory course, two in X-ray diffraction and two in electrochemistry. Those techniques were chosen due their underrepresentation in the Journal of Chemical Education. Paint samples (experiment 1) and pennies (experiment 2) were characterized using x-ray diffraction to teach students how to identify different metals and compounds in a sample. in the third experiment, copper from a penny was used to perform stripping analyses at different deposition times. As the deposition time increases, the current of the stripping peak also increases. the area under the stripping peak gives the number of coulombs passed, which allows students to calculate the mass of copper deposited on the electrode surface. the fourth experiment was on the effects of variable scan rates on a chemical system. This type of experiment gives valuable mechanistic information about the chemical system being studied.

Flavonoids are a specialized group of compounds produced by plants that give them greater adaptability to their environment and ultimately enhance their ability to survive. In plants, one function of flavonoids is to attract pollinators by their various flavor and scent profiles. They also protect the photosynthetic machinery from photo-oxidation. In humans, flavonoids have been shown to act as antioxidants, exhibit antimicrobial activity, and have shown potential as cancer treatments. In nature, flavonoids are most often found coupled with a sugar group (glucose, rhamnose, and others) which imparts stability and increases bioactivity. The process of adding a sugar (glycosylation) is catalyzed by a class of enzymes called glycosyltransferases (GT). One such enzyme found in grapefruit only glucosylates the flavonol class of flavonoids at the 3-OH position and is of interest due to its unique substrate and regio-specificity. Called Cp3GT (Citrus paradisi flavonol 3-O glucosyltransferase), this enzyme is similar in structure to other plant GT’s yet differs in the flavonoids it can glucosylate and where the glucose can be added. To date, the literature has not reported a structural mechanism for a flavonol specific 3-O glucosyltransferase’s unique catalytic activity. High-resolution structural imagery of enzymes, elucidated using X-ray crystallography, can be used to direct custom enzyme development to produce bioavailable natural products. Furthermore, structural research on enzymes with high specificity strengthens enzyme-ligand docking simulations, which are commonly used to test the binding affinity of potential pharmaceuticals. This research hypothesizes Cp3GT has structural features that confer its unique substrate and regiospecificity that are not revealed by homology modeling. This hypothesis will be tested using x-ray crystallography of purified Cp3GT protein bound to its preferred flavonol substrates. The gene for Cp3GT was transformed into Pichia pastoris and was recombinantly expressed using methanol induction. Cp3GT was purified to 80% purity using cobalt metal affinity chromatography. Cp3GT was subjected to additional purification measures using anion exchange chromatography with the goal of increasing purity to ≥95% for crystallization experiments. Purity analysis was conducted using SDS-PAGE (Coomassie/silver stain, western blot) and UV-Vis spectrophotometry. While initial results are promising, additional purification steps may be needed to achieve the purity necessary for crystallization.

In prokaryotes, protein glycosylation can be a determinant of pathogenicity as it plays a role in host adherence, invasion, and colonization. Impairment of glycosylation in some organisms, for example N-linked glycosylation in Campylobacter jejuni, leads to decreased pathogenicity; thus, opening new avenues for the development of antivirulence agents. A member of the protein glycosylation (pgl) gene locus in C. jejuni, PglC, is predicted single-pass transmembrane (TM) protein, that catalyzes the phosphoglycosyl transferase (PGT) reaction in the first membrane-committed step of the N-linked glycosylation pathway. The small size of PglC (201 aa) compared to homologous PGTs suggests it may represent the minimal catalytic unit for the monotopic PGT superfamily. Herein, the structure of C. concisus PglC including its putative TM domain has been solved to 2.74 Å resolution to reveal a novel protein fold with a unique alpha-helix-associated beta-hairpin (AHABh) motif and largely solvent-exposed structure. There is noted a parsimony of fold in the form of short-range motifs underpinning the structural basis for critical functions of PglC: membrane association and active-site geometry. Biochemical and bioinformatics studies support structural evidence suggesting the crystallographically-observed, kinked TM helix is re-entrant on the cytoplasmic face of the membrane rather than membrane spanning. Thus, PglC represents a first-in-class structure of a novel membrane interaction mode for monotopic membrane proteins. Additionally, the AHABh-motif and active-site helical geometry establishes co-facial positioning of the catalytic-dyad. Molecular docking of PglC substrates, undecaprenyl phosphate (UndP) and UDP-N,N-diacetylbacillosamine (UDP-diNAcBac), within the active-site reveals co-incident binding sites, consistent with the proposed ping-pong enzymatic mechanism. Loading of PglC into membrane-bilayer nanodiscs (ND) allows for the investigation of PglC structure and function within a native-like membrane environment by small-angle x-ray scattering (SAXS). Observation of PglC in ND via SAXS confirms the application of the method for studying small, integral, monotopic membrane proteins in a membrane environment. Moreover, development of a mathematical approach by which resident-protein: ND stoichiometry can be deduced from measured scattering intensity enables independent confirmation of loading stoichiometry. Overall, the membrane-interaction modality observed for PglC is the first structurally characterized example of a new membrane association mode for monotopic proteins with the membrane. These studies provide insight into the structural determinants of the chemical mechanism and substrate-binding for C. concisus PglC and for the extensive homologous monotopic PGT superfamily, thus allow homology modeling and enabling future inhibitor design. / 2019-06-12T00:00:00Z