Reactive oxygen species (ROS) are increasingly recognised as important signalling molecules that act through the oxidation of protein cysteine residues. Comprehensive identification of redox-regulated proteins and pathways is crucial to understand ROS-mediated events. Identifying cysteine oxidation on a wholeproteome scale remains a technical challenge due to the low abundance of oxidised thiols. Redox proteomics techniques therefore use multistep enrichment protocols, but these have inherent limitations and inform only on the enriched proteome. We developed stable isotope cysteine labelling with iodoacetamide (SICyLIA), a simple, unbiased, and robust mass spectrometry-based workflow for thiol oxidation analysis. We applied SICyLIA to diverse cellular models and primary tissues and generated the most in-depth thiol oxidation profiles to date. Our results demonstrate that acute and chronic oxidative stress causes oxidation of distinct metabolic proteins, indicating that cysteine oxidation plays a key role in the metabolic adaptation to redox stress. Analysis of mouse kidneys showed oxidation of proteins circulating in biofluids, through which cellular redox stress can affect whole-body physiology. Obtaining accurate peptide oxidation profiles from complex organs using SICyLIA holds promise for future analysis of patient-derived samples to study human pathologies. As metabolic proteins were found to be highly sensitive to oxidation, we investigated whether metabolic adaptation to redox stress is regulated through thiol oxidation by combining SICyLIA with metabolomics. We delineated how glycolysis and the pentose phosphate pathway (PPP) are regulated in acute oxidative stress conditions. We generated new biological insight into the mechanism by which GAPDH oxidative inhibition maximises NADPH production in the PPP to help cells adapt to acute oxidative stress, a function previously attributed to PKM2 and TPI. Our results negate a role for PKM2 in this phenomenon: PKM2 is not oxidised and not required in acute oxidative stress. In contrast, we find that PKM2 is oxidised in chronic oxidative stress conditions. Oxidation causes an oligomerisation switch from dimeric to tetrameric PKM2, which depends on a cysteine residue that is conserved in allosterically regulated PK isoforms. Together, this may suggest another adaptation of this PK isoform to confer benefits onto PKM2-overexpressing cancer cells that face high levels of oxidative stress.

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The plasma membrane of Halobactertium, strain 1, an extremely halophilic bacterium, has been isolated and characterised. The cell envelope fraction (CEFB) was isolated from a cell homogenate by differential centrifugation. After dialysing the cell envelope fraction against distilled water and treating with nucleaaes, a fairly pure preparation of plasma membranes (NM) was obtained by centrifuging. The membrane -containing fraction was purified by gel filtration on Agarose, which yielded a purified membrane fraction (R) and a protein-nucleotide fraction (RP). A small molecular weight protein fraction P was separated from the purified membrane fraction either by gel filtration on Sephadex in a buffer containing phosphate and fluoride tons or by ultrafiltration. Protein fractions RUP1 and RUP2 were separated from the purified membrane by gel filtration on Agarose in the presence of 6M-urea. The remaining membrane- containing fraction, which was eluted in the void volume of the urea-Agarose gel, was coded RU. The organism was studied in batch culture; maximal growth was reached after 48 hours, after which time the cells were in the 'stationary phase.' The endogenous respiratory activity of the cells rose to a maximum at 40 hours and then declined steadily, but the viable count remained fairly steady. Analyses of the membrane fraction NM were made at various phases of growth up to a maximum of 160 hours. All the cell lipid was found to be concentrated in the membrane fraction. The amount of lipid expressed as a percentage of the salt-free dry weight of the cell remained constant, but both the total cell protein and the membrane protem fell durtng the period between 16 and 112 hours of growth. Also, the proportion of membrane to whole cell fell during this period. The menaquinone and the carotenoid pigment were localised exclusively in the membrane fraction. Both compounds exhibited maximal concentrations at 64 hours of cell growth and retained a constant molar ratio to each other regardless of the growth phase. The crude (NM) and purified (R) membrane fractions were both affected by magnesium ion. In the absence of the ion, the membrane disaggregates to small lipoprotein particles. Magnesium ions also assist in the binding of the amino sugar layer of the cell envelope and of the P and RP fractions to the membrane. The physico-chemica! properties of the fractions NM, R. RP, RUP and P have been investigated by a combination of amino acid analysts, gel electrophoresis, sucrose density gradient centrifugation and gel filtration on Sephadex or Agarose. In addition, the binding of magnesium ion to the membrane and the isotonic point of the membrane rractton R have been determined. The fraction R was found to be free from amino sugar and nucleotide. Fractions NM, R and RU contain all the cell lipid, and eytechreme, Fractions NM and R, and probably also fraction RU, contain the cell menaquinone and carotenoid. Evidence is presented that suggests that the fraction R contains the NADH oxidase and adenosine triphosphatase of the electron transport system. The action of the detergent sodium dodecyl sulphate (SOS) on the membrane fraction R was to break up the membrane into smaller particles. The disaggregation noccurred in two distinct steps. The disaggregated particles could be reaggregated to a fraction which resembled the original membrane by removing the SDS by dialysis or gel filtration. The disaggregated particles and also the reaggregated membrane fraction were subjected to gel filtration, gel electrophoresis and sucrose density gradient centrifugation, in order to determine whether or not the lipid and protein components of the membrane had been separated. A fraction which may be analogous to mitochondrial structural protein was isolated by ammonium sulphate fractionation of fraction R dissolved in a mixture of sodium deoxycholate, sodium chelate and SDS.

Pseudomonas aeruginosa inhibitor of cysteine peptidases (PA-ICP) is a potent protein inhibitor of papain-like cysteine peptidases (CPs) identified in Pseudomonas aeruginosa, an opportunistic pathogenic bacteria that can cause severe infections in human. It belongs to the newly characterized natural CP inhibitors of the I42 family, designated the ICP family. The members of this family are present in some protozoa and bacterial pathogens. They can inhibit both parasite and mammalian CPs with high affinity and specificity. Whether the main biological function of the proteins in the pathogens is to regulate the hydrolytic activity of the organisms’ endogenous CPs or exogenous CPs so as to facilitate the pathogens’ invasion or survival is still under investigation. Although Pseudomonas aeruginosa contains a CP inhibitor, no CP genes are found in its genome, suggesting that the targets of PA-ICP may be exogenous. This hypothesis is supported by the presence of a putative secretion signal peptide at the N-terminus of PA-ICP which may be involved in exporting the protein to target exogenous CPs. In order to shed light on the biological function and inhibitory specificity of PA-ICP, the structure and backbone dynamics of this protein were characterised using NMR spectroscopy. In this project, the inhibitory activity of PA-ICP to a range of mammalian model CPs was also studied. Like its previously studied homologs, PA-ICP adopts an immunoglobulin fold comprised of seven β-strands. Three highly conserved sequence motifs located in mobile loop regions form the CP binding site. The inhibitor exhibits higher affinity toward the mammalian CP cathepsin L than cathepsins H and B. Homology modelling of the PA-ICP-cathspin L interaction based on the crystal structure of the chgasin-cathpsin L complex shows that PA-ICP may inhibit the peptidases by blocking the enzyme’s active site and that the interactions between chagasin and CPs may be conserved in PA-ICP-peptidase complexes. The specificity of the inhibitors may be determined by the relative flexibility of the loops bearing the binding site motifs and the electrostatic properties of certain residues near the binding sites.

The behaviour of animal cells in vitro is affected by both the chemistry and the shape of the surface (“topography”) to which they adhere. Culturing animal cells on nanopatterns of different shape, dimensions and chemistry considerably modifies cell attachment, spreading, proliferation, migration and gene expression. This work was primarily aimed at elucidating the influence of nanopatterning on some physical properties of the substrate. The contact angle of water on nanopatterned silicon was measured, and the predicted DLVO (Derjaguin-Landau-Verweey-Overbeek) interaction between a nanopatterned silica plate and a microsphere was calculated. After the physical measurements, the silicon nanopatterns were replicated into a biocompatible polymer, and further experimental investigations of the response of biological cells to nano-pillared samples were carried out. Finally, in the last chapter a flow system was designed, in order to determine the influence of a nano-pitted interface on the initial adhesion of cells subjected to hydrodynamic forces. Surface texture has a great influence on both the wetting and the interfacial properties of the substrate. In this thesis, I show that the contact angles on nano-topographies are linked to the geometry and chemistry of the pattern by defined analytical rules. Contact angle measurements also proved that air-trapping can happen at a nanopatterned biomaterial surface. On the other hand, a SEI (Surface Element Integration) study predicts that the adhesion of a microsphere onto a plate should be strongly favoured by nanopatterned regular protrusions, and that the shape of the protrusions is a determining factor in this process. My results on cell behaviour confirm previous observations that some particular nano-patterns can inhibit the proliferation of fibroblasts in vitro. It is also shown how cell-specific this response can be, and possible explanations for this behaviour, including air-trapping at the interface, are discussed.

The aim of the project was to characterise known wnt signalling molecules during the early pronephros patterning using novel and effective bioassays in Xenopus embryos. Anterior somites have been shown previously to have unique biological activity to induce pronephros formation in Xenopus embryos (Seufert et al., 1999). A molecular approach consisted of analysing the expression of canonical wnts (wnt6, wnt7b, wnt8, wnt9a and wnt9b) and the non-canonical wnts (wnt4, wnt5a, wnt11 and wnt11b) genes in isolated pronephric anlagen and pronephros from stage 12.5 to stage 35 and in anterior and posterior somites. This allowed the identification of potential candidate wnt genes which could act as pronephric inducers. Their potential to induce pronephros was tested in vitro using the Holtfreter sandwich culture pronephrogenesis assay, which consists of unspecified intermediate mesoderm cultured inside two animal caps over-expressing the wnt molecule of interest. Results suggest that the canonical wnt molecules (wnt6 and wnt8) are capable of modifying the intermediate mesoderm leading to formation of somite and neural tissue that in turn, can secondarily induce pronephric tubule formation. By contrast, signals of the non-canonical wnt11 and wnt11b are sufficient to directly specify the intermediate mesoderm to become kidney. In vivo, the role of the canonical wnt6, non-canonical wnt11b and the closely related wnt11 gene was investigated by gain and loss-of-function experiments. Results suggest that mis-expression of these genes disturb the normal formation of the pronephric tubules and suggest that both canonical and non-canonical wnt molecules are required for formation of functional pronephros. This thesis also reports the identification of the novel Xlwnt9a and Xlwnt9b genes, their temporal and spatial expression in both X. laevis and X. tropicalis embryos and dissected adult organs and analyses the effects of Xtwnt9b mis-expression on X. laevis pronephros development.

The technique of continuous culture has been used to study the segregatlonal stability of plasmids in Escherichia coli in the absence of selective pressure. Conditions were established that allowed the detection of plasmid-free cells no matter how low their Initial frequency. Using these conditions the segregational stability of two related multicopy plasmids (pDSII09 and pBR322) was examined. pDSII09 was found to be stably Inherited throughout 120 generations of nutrient limited growth despite the observation that the plasmid copy number fell 4 to 5 fold during the culture period. By contrast, pBR322 was lost from chemostat culture after a lag of between 30 and 40 generations, a period during which (by analogy with pDSII09) its copy number had fallen about 2 fold. The functional basis of the differential segregation exhibited by these plasmids was ascribed to the presence (on pDSII09) or absence (on pBR322) of a functional par (partition) signal that ensured the efficient segregation of plasmid molecules into daughter cells at division. Based on this hypothesis, experiments were done to examine the possibility for correction of the defective partitioning of pBR322 by complementation in cis and in trans. In only two cases (both in cis) was complementation achieved. The first using a previously characterised par function from plasmid pSCIOI and the second using a fragment of plasmid pDSII09 that was considered (by argument) to be the region involved in its observed segregational stability. Supportive evidence for the existence of partition elements amongst multicopy plasmids is cited, as is confirmatory work since published by other workers. A possible mechanism of par action is discussed. Chemostat culture has also been used to examine the possibility of plasmid transfer by transformation within the chemostat. This study examined the effects of both growth rate and nutrient limitation on the transformability of Escherichia coli grown in continuous culture. The results obtained are discussed in relation to previously published transformation work using batch grown cells and a possible mechanism of plasmid transformation is suggested.

\(Escherichia\) \(coli\) encounters nitrosative stress from various sources. It was shown in a previous study that a possible role for Hcp in the nitrosative stress response. The focus of this study was to determine the function of Hcp. The growth of the \(hcp\) mutant lacking all known NO reductases was inhibited by various sources of nitrosative stres. The growth defect was complemented by native Hcp protein, but not by mutated Hcp protein with a disrupted hybrid cluster. The role of Hcp was shown to protect \(E\). \(coli\) from nitrosative stress, and the hybrid cluster is critical for its function. Direct interaction between Hcp and its oxidoreductase Hcr was demonstrated. The \(hcp\)\(^+\)\(hcr\) strain showed that it was still resistant to nitrosative stress. Possibly alternative oxidoreductase of Hcp exists in \(E\). \(coli\). Gas analysis of the headspace of the anaerobic cultures showed that when treated with NO, the Hcp\(^+\) strain lacking all known NO reductases was still capable of reducing sub-micro molar NO into N\(_2\)O, while the further deletion of Hcp completely abolished NO reduction. This provides the first in vivo evidence that Hcp is a high affinity, low capacity NO reductase in \(E\). \(coli\).

Transhydrogenase is an enzyme located in the cytoplasmic membrane of bacteria or the inner membrane of animal mitochondria. Using the energy of the proton electrochemical gradient (Δp), transhydrogenase translocates protons across the membrane whilst undergoing its redox reaction, in which hydride ion equivalents are transferred from NADH to NADP+ producing NAD+ and NADPH. Transhydrogenase comprises three components; dI binds NA(H), dIII binds NADP(H) and dII spans the membrane. Transhydrogenase is thought to function by way of a binding-change mechanism, which involves “open” and “occluded” conformations of the enzyme. In the open conformation, nucleotides can readily bind and dissociate from the enzyme but the hydride transfer reaction is blocked. In the occluded conformation, hydride transfer is permitted but the binding and release of nucleotides is blocked. Hydride transfer and proton translocation are coupled. The coupling is not well understood due to the lack of structural information about the membrane-spanning dII component. However, it is believed to involve conformational changes of the enzyme, particularly the dII and dIII components, resulting in the switch between the open and occluded conformations. Enzyme assays and tryptophan fluorescence experiments using apo-dIII in complex with dI revealed two features: Firstly, the binding of NADP(H) to dIII is very slow and is probably limited by the conversion from the occluded to the open conformation. Since the switch between the occluded and open conformations is thought to be central in the coupling of hydride transfer and proton translocation, the results presented here give an insight into the binding-change mechanism of transhydrogenase. Secondly, NAD(H) is able to slowly bind into the NADP(H)-binding site of dIII (the “wrong” site). This brought into question the specificity of the dIII component of transhydrogenase for NADP(H). The significance and likelihood of NAD(H) binding to dIII in the intact enzyme in the living cell are discussed.

Existing methodologies for biomolecular detection are limited in several key areas. Heterogeneous assays struggle with various wash steps which can prolong assay time, while other assays require costly reagents, lack mobility and can be highly complex in nature. This project demonstrates how a bio-nano particle in the form of M13 bacteriophage (M13) can be used for the basis of a novel homogeneous immunoassay which incorporates the use of linear dichroism spectroscopy (LD). M13 has a high aspect ratio which allows it to align easily in shear flow, this in turn generates a large LD signal. This property of M13 has been manipulated for use in a new in-vitro diagnostic technique. Existing M13 production yields are much lower than those required for this assay. A new method was developed which increased the yield 10 fold. Chemical modifications were made by covalently attaching chromophores, this enabled the M13 LD signal to be visualised in the visible region and develops the potential for multiplexing. By chemically modifying M13 with chromophores and antibodies it was possible to create an assay capable of detecting 10⁵ cells/mL of Escherichia coli O157. This is 100 times more sensitive than the M13 based assay developed by Pacheco-Gomez et al. (2012). The assay was reassembled to detect small molecules and was found to have a sensitivity of 0.01 mM. The assays presented form a sensitive, specific, fast diagnostic tool capable of detecting pathogens and small molecules. It offers significant improvements over existing methods, and could act as a platform in developing a multimodal detection system.