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Abstract: Using a combination of two surface-sensitive spectroscopy techniques, the chemical state of the Ag(111) surface and the nature of the adsorbed species in the presence of ethylene and oxygen gases are identified. In the 10 mbar pressure range and 25–200 °C studied here, Ag(111) remains largely metallic even in O2-rich conditions. The only adsorbed molecular species with a low but discernible coverage is surface carbonate, which forms due to further oxidation of produced CO2, in a similar manner to its formation in ambient air on Ag surfaces. Its formation is also pressure-dependent, for instance, it is not observed when the total pressure is in the 1 mbar pressure range. Production of carbonate, along with carbon dioxide and water vapor as the main gas-phase products, suggests that an unpromoted Ag(111) surface catalyzes mainly the undesired full oxidation reaction.

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Abstract: X-ray photoelectron and absorption spectroscopies can provide valuable interface-sensitive chemical information; however, they are traditionally performed under ultra-high vacuum conditions to maximize the inelastic mean free path of photoelectrons and attenuation lengths of X-rays. Developments in the field, including electron analyzers coupled with sophisticated differentially pumped lenses, have made it possible to extend these techniques to liquid and gas environments (up to tens of mbar). Enclosed environmental cells are an alternative approach that promises access to atmospheric pressures and above, and the potential for low-cost and widespread deployment by incorporation into standard X-ray photoelectron spectroscopy systems. In this chapter we discuss the progress made in the development of such enclosed environmental cells for X-ray spectroscopies. We focus on the main requirements to perform these measurements, including discussion of the implementation of X-ray and photoelectron transparent membranes, common reaction cell designs and materials, and the methods of detection typically employed. We also present some landmark examples which mark progress in this area up to now, before providing an outlook for the topic as a whole.

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Abstract: Pseudomonas aeruginosa (PA) depends on the Entner-Doudoroff pathway (EDP) for glycolysis. The main enzymatic regulator in the lower half of the EDP is pyruvate kinase. PA contains genes that encode two isoforms of pyruvate kinase, denoted PykAPA and PykFPA. In other well-characterized organisms containing two pyruvate kinase isoforms (such as Escherichia coli) each isozyme is differentially regulated. The structure, function and regulation of PykAPA has been previously characterized in detail, so in this work, we set out to assess the biochemical and structural properties of the PykFPA isozyme. We show that pykFPA expression is induced in the presence of the diureide, allantoin. In spite of their relatively low amino acid sequence identity, PykAPA and PykFPA display broadly comparable kinetic parameters, and are allosterically regulated by a very similar set of metabolites. However, the x-ray crystal structure of PykFPA revealed significant differences compared with PykAPA. Notably, although the main allosteric regulator binding-site of PykFPA was empty, the “ring loop” covering the site adopted a partially closed conformation. Site-directed mutation of the proline residues flanking the ring loop yielded apparent “locked on” and “locked off” allosteric activation phenotypes, depending on the residue mutated. Analysis of PykFPA inter-protomer interactions supports a model in which the conformational transition(s) accompanying allosteric activation involve re-orientation of the A and B domains of the enzyme and subsequent closure of the active site.

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Abstract: Bacterial cell division is a complex process that requires tight co-ordination and regulation of chromosome replication and segregation, and the synthesis and remodelling of the bacterial cell wall. The peptidoglycan sacculus is synthesised by peptidoglycan synthases and it is a vital component of the bacterial cell structure, to withstand the turgor of the cell to prevent its lysis FtsZ is one of the first proteins to localise at midcell during cell division where it forms a ring-like structure, the Z-ring, at the inner face of the cytoplasmic membrane. After Z-ring formation, the divisome is recruited to the division site to form the septum and the elongasome is positioned at the side wall for elongation. Both the elongasome and the divisome are dynamic macromolecular complexes composed of numerous proteins at unknown stoichiometries and it is thought there are proteins in both complexes that are yet to be discovered. Understanding bacterial cell division at the molecular and cellular level is an important area of research to enable the development of novel antibiotics as well as comprehending one of biology’s fundamental questions. GpsB is a conserved Gram-positive cytosolic protein that plays an important role in the elongation-division cycle by acting as a scaffold to control the relative spatial arrangements of peptidoglycan synthases (PBPs). In this thesis the molecular interactions of GpsB with cytoplasmic mini-domains of PBPs from two human pathogens (Listeria monocytogenes and Streptococcus pneumoniae) are described using structural and biochemical techniques. The importance of the critical interacting residues for the GpsB:PBP interaction for cell wall growth and viability of L. monocytogenes and S. pneumoniae were analysed in collaboration with the Halbedel, and Massidda and Winkler groups, respectively. A novel function of PBP binding was introduced into DivIVA, a cell division regulator and GpsB homolog, by protein engineering in an attempt to understand the functional divergence between GpsB and DivIVA. Potential building blocks for the development of GpsB:PBP inhibitors were identified in the form of small fragments by an X-ray crystallography-based fragment screening experiment at Diamond Light Source. Eukaryote-like serine/threonine protein kinases (eSTPKs) and partner phosphatases (eSTPs) are conserved in Gram-positive bacteria. They consist of an intracellular N-terminal kinase domain and an extracellular sensing region linked by a short transmembrane helix. The external regulatory region is comprised of three or four PASTA domains that bind to ii peptidoglycans and compounds containing beta-lactams. eSTPKs are involved in the regulation of many cellular processes including development of regulation, control of cell growth, stress response, virulence and sporulation. The eSTPK/eSTP pair from L. monocytogenes are PrkA and PrpC. This thesis concerns the importance of autophosphorylation for the function of the kinase domain of PrkA (PrkA-KD) and that phosphorylation of serine 173 is crucial for activation, agreeing with the mechanism of activation of Stk1 in Staphylococcus aureus. The previously uncharacterised phosphoprotein Lmo1503 (renamed ReoM) is a homologue of IreB from Enterococcus faecalis, a negative regulator of cephalosporin resistance. It is confirmed that ReoM is a substrate of the PrkA/PrpC pair and the crystal structure of the full length ReoM protein is presented. Isothermal titration calorimetry determined the interaction of ReoM and PrkA-KD is within the nanomolar range and there is a ten-fold reduction in affinity with a PrkAKDS173A mutation. The Halbedel group have linked ReoM phosphorylation to the activation of ClpCP-dependent degradation of the primary UDP-GlcNAc 1-carboxyvinyltransferase in L. monocytogenes, MurA.

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Abstract: The complex flow within the molten metallic pool, during advanced manufacturing and joining processes such as welding and additive manufacturing, directly affects the performance of the final components; through the generation of various microstructures and strain gradients. Understanding of the flow within such melt pools can enhance the predictability of the final microstructures and therefore component performance. In situ synchrotron X-ray imaging is employed to observe and map the evolving flow patterns within gas tungsten arc weld pools using tracking particles. The experimentally observed flow patterns are compared with numerical simulations to gain additional insights on accurate predictability in relevance to the physical driving forces within the flow. The spatio-temporal distribution of the flow using the mapped data is analyzed by considering the evolution of driving forces acting throughout the weld pool. The results demonstrate quantitative benchmark flow mapping with confirmation of the overall mechanisms of weld pool flow.