In this thesis, aspects of metabolism of lipids in dairy cows were studied, particularly 18:1 trans fatty acid (tFA) concentrations in plasma and lipoprotein fractions, and transportation of FA in epithelial mammary gland cell cultures. Two in vivo studies were conducted to elucidate which lipoprotein fractions were involved in bovine plasma transport of tFA by infusing oils that induced different plasma tFA profiles. Fatty acid profiles of plasma and lipoprotein fractions [high (HDL), low (LDL), and very low density (VLDL) lipoprotein fractions] were analysed. Results showed that VLDL fraction was the main transport mechanism of tFA in plasma and was more responsive to supply of tFA. MAC-T cell cultures were used to study specific aspects of lipid uptake without interference from the cow’s digestive system and general metabolism. Treatments consisted of adding long-chain FA solutions to lactogenic medium at varying concentrations. After incubation, cystosolic triacylglycerol (TAG), protein and DNA contents were determined. Results demonstrated that cytosolic TAG accumulation is stimulated by addition of long-chain FA, which affects FA profile of cytosolic free FA. Overall, results from this thesis enhance knowledge on plasma transport and mammary uptake of 18:1 tFA in dairy cows. The information strengthens understanding of some aspects of milk fat synthesis and metabolism of FA in dairy cows fed with different sources of tFA. Findings from this thesis are important because saturated FA and tFA in milk fat have caused concern among human health researchers and more recently milk fat has gained appreciation as a functional food due to the health-promoting potential of some FA (vaccenic and conjugated linoleic acids) found specifically in ruminant-derived products.

Colicins are a family of bacterial toxins, which kill Escherichia coli cells and other closely related species. Their mode of action requires binding to an outer membrane receptor, translocation across the cell envelope leading to cytotoxicity at specific targets. The mechanism of colicin cytotoxicity includes a non-specific endonuclease activity or depolarizing of the cytoplasmic membrane by pore-forming activity. The cytotoxic activity can be inhibited by the high affinity binding of an immunity protein. For Group A colicins, translocation requires interaction between the N-terminal domain of the colicin and a series of membrane bound and periplasmic proteins called the Tol system (TolB, TolR, TolA, TolQ and Pal). In order to allow cytotoxicity, the immunity protein of enzymatic colicins must be lost after binding of the colicin to a target cell and the cytotoxic domain has to be translocated through both the outer and inner membranes. This work has studied colicin translocation by using a periplasmic protection assay combined with an in vivo lux-reporter assay and a potassium release assay. Expressing the translocation domain of colicin A in the periplasm and challenging the cells with external colicins showed that the translocation of group A colicins is inhibited as it requires an interaction with the Tol system. Surprisingly, the TolA protein was found to play the major role during the translocation of both ColA and the endonuclease colicin E9 even though the latter colicin has no direct interaction with the TolA protein. This study also suggests that the interaction with TolB is important for both colicins A and E9. Moreover, a series of ColA constructs with a truncated T domain were made by site directed mutagenesis to define the important residues of the TolA and TolB boxes for their interaction with Tol proteins. The results showed that tyrosine 58 residue of the TolA box of colicin A is essential for TolA binding, whilst the glutamate 18 residue of the TolB box of ColA did not show an effect on TolB binding. Colicin-producing cells are protected by the co-expressed immunity protein which prevents killing of the producing cells. The immunity protein must be released from the cytotoxic domain of endonuclease E colicins prior to reaching the E. coli cytoplasm to degrade DNA. Little is known about the mechanism of release of immunity protein from E colicin cytotoxic domains and the role of Tol proteins in the release of immunity protein. Finally in this work an attempt was made to develop a sensitive, real-time assay to study the mechanism by which immunity protein is released from colicin E9 and to study the physiological role of the Tol system in the release process. Further developments are required for improving this assay which would be of great value.

In vitro epithelial cell cultures are increasingly used to model drug permeability, as predictive tools for absorption in humans. Medical regulatory agencies recommend in vitro permeability screening for biopharmaceutical classification of novel therapeutic compounds, and recently published guidelines on investigating interactions of novel therapeutic compounds with clinically relevant transporters. The expression and functionality of drug transporters in the lung is poorly characterised, and insufficient to allow detailed understanding of drug-transporter interactions in the airways. Additionally, as human in vitro permeability is used to predict absorption from rat in vivo, a rat bronchial epithelium in vitro cell line would aid the understanding of interspecies differences in transporter-mediated drug trafficking. This thesis investigates the morphological and physiological barrier properties of Calu-3, normal human bronchial epithelial (NHBE) cell layers and rat airway epithelial cell (RL-65) cultures. The morphology and barrier integrity of RL-65 layers were shown to be in agreement with existing human bronchial epithelial cell models after culture for 8 days at air-liquid interface. The expression of >30 ABC, SLC and SLCO transporters in human models was in general agreement with published expression levels in human lungs. MDR1 functionality was investigated, and whilst no asymmetric transport of 3H-digoxin was observed in RL-65 cell layers, net secretory transport was observed for Calu-3 cell layers at both low (25-30) and high (45-45) passage number and for some batches of NHBE cell layers. Chemical, metabolic and biological inhibitors were employed to evaluate MDR1 contribution to 3H-digoxin trafficking, however the exact transporter(s) involved could not be determined. Whilst MDR1 functionality could not be ruled out, results suggest that it is unlikely to be the main transporter involved in 3H-digoxin trafficking in the bronchial epithelium. These studies have highlighted the need for more specific approaches to investigating transporter functionality in in vitro systems.

Monocytic leukaemia zinc-finger protein (MOZ) is a histone acetyltransferase (HAT) implicated in haematopoiesis and acute myeloid leukaemia, as well as embryonic and postnatal development. MOZ contains multiple domains, including a MYST HAT domain and a double PHD finger domain (DPF) suggesting it interacts with histones. This work has established for the first time that the MOZ DPF exhibits dual functionality in establishing and sensing post-translational modifications (PTMs) of histones. Firstly, our data detected the direct interaction of MOZ with the N-terminal tails of histones H3 and H4 and shows that the MOZ DPF domain mediates such binding. Both PHD fingers are required and functionally cooperate to establish the DPF histone binding preference in terms of PTMs. We demonstrate that H3K4me3 prevents MOZ DPF association with H3, although H3K4me2 is tolerated. Similarly, H4Kac acts as a dominant exit signal that excludes MOZ from chromatin. This ability to sense H3K4 PTM status was confirmed in a collaborative effort establishing the crystal structure of MOZ DPF in complex with an unmodified H3 peptide. The H3 peptide adopted an α-helical conformation in the complex, which has not previously been observed. Secondly, we present novel data showing that the MOZ DPF domain exhibits a mild histone H3-specific acetyltransferase activity. This provides the first report of a possible enzymatic role in chromatin modification attributed to a PHD finger. Furthermore, the combined DPF and MYST domains were found to influence the reaction rate and substrate specificity of MOZ-induced histone acetylation. Our studies revealed that the MOZ DPF could associate with heterochromatic PTMs, namely H3K9me3. We report here that both the H3K9-specific methyltransferase SUV39H1 and heterochromatin protein 1 (HP1) form interactions with MOZ, implicating its function in both corepressor and coactivator complexes. Thus, our data suggest that like several other chromatin-associated proteins, MOZ is a multi-functional regulator of chromatin modification and gene expression.

CELF1 is an RNA binding protein with regulatory roles in translation, alternative splicing and mRNA degradation. This protein is of particular interest as its upregulation is believed to be involved in the pathogenesis of type 1 myotonic dystrophy. CELF1 functions by binding to a specific sequence in the 3’ untranslated region of its target mRNAs. This sequence has been termed the “EDEN” motif, but the exact requirements for binding of CELF1 were not well defined. In this study we therefore aimed to determine the sequence requirements for an RNA substrate to form a high affinity interaction with CELF1, and characterise the structure of the resulting complex. The CELF1 protein is composed of three structured RNA recognition motifs separated by flexible linkers. Our strategy was to investigate the RNA binding properties of each domain in isolation, and then the requirements for tandem binding of the domains in order to build up the complete “EDEN” motif capable of forming a high affinity complex with the wild type protein. This has been accomplished using NMR spectroscopy to map the chemical shift perturbations in each domain on binding to a range of RNA substrates. ITC was also used to investigate the binding affinities of each domain, and the enhancement of affinity when domains bind in tandem. By these methods we have refined the sequence requirements for simultaneous binding of all domains of CELF1, and designed RNA substrates which will bind with higher affinity than any previously reported. We have also shown the potential involvement of RNA secondary structure in forming the CELF1 binding site, and identified two possible examples of this in natural mRNA targets. CELF1 binding triggers deadenylation of its target mRNAs and this is suspected to be via a mechanism involving recruitment of poly (A) ribonuclease. These two proteins have been shown to interact, but no structural information was available to show which domains were interacting, or whether CELF1 was capable of forming a ternary complex with both RNA and poly(A) ribonuclease. Since the ribonuclease exists as a 146 kDa dimer, the complex of it with CELF1 was an ambitious target for NMR. In this study we demonstrate that high resolution NMR data can be acquired on this key regulatory complex. Using this we go on to confirm the interaction between these two proteins, and that the domains involved in binding suggest a ternary complex is possible.

The halophilic archaeon Haloferax volcanii is a carotenogenic organism, native to the dead sea. Its carotenogeneic pathway was characterised by BLAST searching techniques. The pathway shows that isoprenoid production in H. volcanii utilises the mevalonate pathway. The first two carotenogenic enzymes, phytoene synthase (CrtB2) and phytoene desaturase (CrtI) were modelled, and investigated, results indicating a possibility that they are membrane bound. A method for extraction, separation and quantification of the carotenoids of H. volcanii was developed. This approach used phase extraction of an aqueous and an organic layer, followed by HPLC separation of the extractant components and analysis by comparison of peak absorption at 450nm of the carotenoid being studied to a beta-carotene internal standard. This method was used to assess lycopene and phytoene levels throughout growth of the organism and under stress conditions. It was found that lycopene and phytoene are both synthesised at elevated levels in stationary phase growth. Additionally lycopene and phytoene levels show a response to oxidative stress, though not to other stressors, being degraded then accumulating to levels higher than found under none stress conditions. In order to gain insight into the function of lycopene in the organism the gene crtI coding for phytoene desaturase was deleted. This mutant was found to have all carotenoid production eliminated from the cell. This suggests that the carotenogenic enzymes may form a complex associated with the membrane. The mutant was subjected to stress conditions, the results showing it was highly susceptible to oxidative attack. This study suggests that the carotenoids synthesised by H. volcanii are vital to its defence against reactive oxygen species (ROS).

Over 30 different mutations in p62 UBA have been identified in patients with Paget's disease of bone (PDB). The mechanisms which underlie PDB are poorly understood, although impaired ubiquitin binding has been identified as a mechanism in the onset of disease. However, the decrease in affinity is subtle for many PDB mutants. The p62 UBA is unique amongst UBAs since it exists as a highly stable dimer but binds to ubiquitin as a monomer. The dimerization interface partially occludes the ubiquitin binding interface resulting in competing equilibria. The factors which regulate the affinity of p62 UBA were examined in this thesis. In isolated p62 UBA the monomer-dimer equilibrium and the effects of phosphorylation were investigated. By mutating residues at the dimerization interface, weaker dimers which had a higher affinity for ubiquitin were produced. The weak dimers had an increased population of monomer at equilibrium. A phosphorylation site at Ser403 in p62 UBA was recently identified. Phosphomimetic mutants which showed subtle increases in affinity for ubiquitin were generated. The increase was attributed to the close proximity of Ser403 to the ubiquitin binding interface. Factors outside the UBA also have a role in regulating UBA affinity. Binding by p62 UBA was therefore probed in longer p62 constructs. A fragment of p62 encoding residues 300-440 was used to investigate p62 binding to multiple proteins. A ternary complex was formed, but an allosteric relationship was not observed by ubiquitin and MAP-Le3 in binding to the p62 fragment. A model of full length oligomeric p62 was generated to probe avid binding to polyubiquitin chains. The model showed higher affinity for linear diubiquitin than monoubiquitin suggesting avidity effects are influential in oligomeric p62 binding. Since the effects of PDB mutations and phosphorylation are subtle, they are likely to be amplified by avidity in vivo.

Conventional capillary zone electrophoresis (CZE) methods, with simple buffer solute, natural or derivatized cyclodextrin and organic additive in BGE, have been developed for a group of ten standard chiral pharmaceutical compounds representing different physiochemical properties and pharmaceutical activities. In this study, factors affecting chiral separation in CZE, including BGE pH value, ionic strength, chiral selector type, selector concentration and organic additives, were optimized. A maximum of eight standard compounds were separated by three different standard methods which were developed. The electrophoretic behaviours of the standard compounds observed were in good agreement with the literature. Partial filling technique (PFT) was studied as a complementary approach to conventional CZE methods for enantioseparation of standard compounds. Partial filling time, selector type and concentration were investigated; a maximum of seven standard compounds were separated by optimized filling time and three different chiral selectors. However, for five of the separated pharmaceuticals, the chiral resolutions achieved were much lower than those obtained from conventional CZE methods. Key observations from the experiment were supported by previous research. For the first time, glycidol was evaluated as a covalently bonded coating material on CE capillary for enantioseparation. Hyperbranched polyglycidol brushes were grown directly from Si/SiO2 surface via anionic ring-opening polymerization, using surface Si-OH groups as initiator. This grafting-form technique eliminated the need for initiator functionalized self-assembled monolayers on the surface, and the thickness and complexity of the hyperbranched polymer brushes could be well controlled in this process. Polyglycidol coating was established on the surface of glass slides and then adapted to CE capillary. Both fused silica capillary and etched capillary were used to examine the electrophoretic properties of polyglycidol coating. Chiral polyglycidol coating was compared with the standard CZE method developed and showed excellent chiral selectivity for standard compounds. Nine out of ten standard compounds were separated with poly-S-glycidol coated capillary, using simple buffer solute containing organic additive. Application of etched capillary further improved the enantioseparation resolution and peak efficiency for those standard compounds. Stability and coating regeneration ability were studied. Polyglycidol coating developed on CE capillary gradually lost its chiral selectivity after 50 30-min runs with acidic BGE. Coating regeneration on the remaining surface was difficult. The result indicated that glycidol isomer can be used as monomer for in situ polymerization in CE capillaries and the coating formed on the inner surface has potential chiral selectivity toward various pharmaceuticals, which is equal or better than traditional chiral CE.

Flavoenzymes are a widely diverse group of enzymes that are able to catalyze a variety of different chemical reactions. A growing interest in flavoenzymes capable of reducing aromatic nitro groups may be attributed to their ability to reduce anticancer prodrugs such as CB 1954. Similarly, quinine oxidoreductases are able to activate anticancer prodrugs such as mitomycin derivatives by reducing the quinone derivative to the hydroxyl derivative. Some oxidoreductases are able to reduce nitro aromatic compounds. These enzymes may be either mammalian such as NQO1 and NQO2 which are over expressed in certain cancerous cells or bacterial which can be introduced into the cancerous growth. The bacterial nitroreductases have been previously well characterized and were found to share several similarities. On the other hand, other nitro reducing enzymes have not been investigated to determine the presence of any shared similarities. Another aim of this thesis is to identify the differences between the structure of the oxidized and reduced forms of NQO2. The similarities shared between the flavoenzymes which are known to reduce nitroaromatic compounds to their hydroxylamine or amine derivative were investigated in this thesis. Both the sequences and 3D structures of the selected proteins were compared and investigated using a variety of bioinformatics tools such as sequence and structure alignment, in addition to homology modelling. It was discovered that there are several aromatic residues conserved in different positions, relative to the flavin cofactor, in the catalytic sites of the proteins. The 3D structure of the reduced NQO2 was determined using X-ray crystallography. The oxidized and reduced protein structures were compared to determine the structural differences. The expression, purification and crystallization of the NQO2 protein, as well as the reduction of the protein crystals are described in this work. The determination of the structure of the reduced protein provided new insights into the mechanism of electron transport to and from the flavin cofactor. Moreover, kinetics studies were undertaken on the purified NQO2 protein using fluorescence spectroscopy. These resulted in the identification of several new substrates for NQO2.

The therapeutic benefits of cannabis have been known for centuries of years. Yet it has only been in the last 40 years that an understanding of the system by which its works in our bodies has begun to be defined. This has in turn led to the discovery and understanding of the endogenous cannabinoid (eCB) system, alongside its main synthesizing and hydrolysing enzymes as well as the endogenous ligands. The use of synthetic cannabinoid receptor (CBR) ligands for therapeutic use has provided problems regarding the natural endogenous regulatory tone of the eCBs, which in turn has resulted in unwanted side effects. Part of the reason of this is due to synthetic agonists producing the well documented psychotropic effects at CB t receptors. Alternative targets for the manipulation of the eCB system for therapeutic benefits have been explored. One remains to be the use of FAAH inhibitors, which in turn potentially increase levels of eCBs in the system, hence potentiating their effects at the CBRs, or at other receptor sites. Therefore we have developed two HTS assays for the identification of potential inhibitors of FAAH and MAGL. They prove to be robust, cheap and facile and provide a clear indication of inhibitable levels of FAAH and MAGL activity. The FAAH assay can be further used to establish concentration-response curves of initial `hit' compounds. Yet, the HTS MAGL assay requires further characterization for use in construction of concentration-response curves, as they are not assays specific for MAGL acitivity and include hydrolysis of the substrate 4-NPA by non-specific esterases. Z-factor scores were calculated for both assays, indicating excellent assays, which can potentially be applied to industrial lab robotics for screening of compound libraries.