Lung surfactant is a mixed monolayer of lipids and proteins that reduce the surface tension at the air-lung interface to prevent alveolar collapse. Exposure of lung surfactant to ozone pollution has been linked to an increased risk of death due to respiratory diseases. This work aimed to determine the ozone damage caused to lung surfactant at the air-water interface. A range of analytical techniques showed that peptide mimics of surfactant protein B were rapidly oxidised by ozone but no cleavage of the peptides occurred. Neutron reflectivity revealed that the peptides remained at the air-water interface after oxidation but that their interaction with anionic phospholipids, thought to be crucial to their function, was significantly reduced. Neutron and X-ray reflectivity experiments showed that exposure of different lipid monolayers to ozone led to a loss of unsaturated phospholipid material from the interface, whereas oxidised cholesterol remained but the molecules were considerably reorganised. The ozonolysis of whole animal lung surfactant was then investigated and it was shown that material was lost from the interface during reaction and that the monolayer was much less capable of reducing surface tension. Additionally, molecular dynamics simulations were performed, determining that surfactant protein C can significantly influence the ordering of surrounding phospholipids in a monolayer and that palmitoylation of the protein leads to an increase in this ordering effect. Finally, the uses of neutron scattering to explore the oxidation of lipid membranes were investigated. It was shown that neutron reflectivity of supported phospholipid bilayers at the solid-liquid interface is a useful tool for determining their reactivity with different reactive oxygen species. Small angle neutron scattering and off-specular neutron reflectivity were also shown to be valuable methods for exploring the changes in lipid raft formation upon oxidation of phospholipids.

Azinomycins A and B are bioactive compounds produced by Streptomyces species. These naturally occurring antibiotics exhibit potent in vitro cytotoxic activity, promising in vivo antitumor activity and exert their effect by disruption of DNA replication by the formation of interstrand cross-links. The electrophilic C-10 and C-21 carbons contained within the aziridine and epoxide moieties are known to be responsible for the interstrand cross-links through alkylation of N-7 atoms of guanine bases. The naphthoate moiety (3-methoxy-5-methyl-naphthoate) is believed to play a role in the azinomycins’ biological activity through hydrophobic interactions in the DNA major groove. In this study, precursor directed biosynthesis (PDB) has been investigated for the production of unnatural azinomycin analogues. A series of naphthoic acid analogues were fed to the azinomycin producing bacteria (Streptomyces sahachiroi ATCC 33158). LCMS analyses revealed that 1-naphthoic acid, 4-fluoro-1-naphthoic acid, 4-methyl-1-naphthoic acid and 3-methoxy-1-naphthoic acid were successfully incorporated into the azinomycin A biosynthesis pathway and resulted in the development of novel azinomycin A analogues. These novel products were isolated and purified using preparative HPLC and were characterised by diode array detection and electrospray tandem mass spectrometry. NMR characterisation and biological studies could not yet be conducted due to the low production levels (~100 μg in 500 mL fermentation broth) and the persistence of some impurities in these novel azinomycin A analogues. In the course of this work, additional metabolites were found in the fermentations. LCMS analysis revealed that a range of naphthoic acids were biotransformed into primary amides. Column chromatography has been carried out towards purification of all the produced amides. As consequence, 1-naphthoic amide and 4-fluoro-1-naphthoic amide were successfully purified and characterised by NMR and LCMS. Further amides were so far only characterised by LCMS, as impurities hampered NMR analysis. Antibacterial and antifungal tests were carried out to investigate the biological activity of the purified amide. The results revealed that they were inactive against the tested microorganisms.

This thesis investigates the structure and function of the kinetochore centromere binding factor 3 (CBF3) complex. The kinetochore is a multi-protein complex, which controls the chromosome attachment to the mitotic or meiotic spindle and nucleates on the centromere. The latter is a specific chromosomal loci divided in two groups highly divergent in length and composition: regional and point centromeres. Regional centromeres are composed of long arrays of repetitive DNA supporting multiple microtubule nucleations. On the contrary, point centromeres are characterised by a short and conserved sequence supporting a single microtubule attachment. Despite this discrepancy, both point and regional centromere establishment start with the replacement of the histone H3 by the Cse4 histone variant. The point centromeres H3 replacement by Cse4 (loading) solely relies on the recognition of a conserved DNA sequence (licensing) by the CBF3 complex, a crucial centromere element composed by four essential proteins: Ndc10, Cep3, Ctf13 and Skp1. Ctf13 and Skp1 regulate the CBF3 assembly. Cep3, the licensing element, recognises the point centromere DNA sequence. Finally, Ndc10 acts as the loading factor of the CBF3 complex by recruiting Cse4. At the start of this work the structural basis underlying the mechanism of Cse4 loading by Ndc10 was speculative and the structure of the complex was unknown, mainly because of the inherent instability of Ctf13. In this study, I solved the X-ray structure of Ndc10 Nterminal domain (Ndc10NTD) at 1.9 Å and highlighted the unsuspected similarities of the Ndc10NTD fold with the tyrosine recombinase/λ-integrase family. Interestingly, Ndc10 lost the catalytic activity characteristic to the family but conserved a strong DNA binding, which I characterised by structural and mutagenesis studies. Furthermore, I described the Ndc10 C-terminal domain, which displays another DNA binding domain and supports the Ndc10 dimerisation. Ultimately, this work will lead to the structural characterisation of the Ndc10 dimer bound to DNA, of which preliminary crystallisation and data collection results are presented. Finally, I will introduce early results on the purification of the Ctf13 subdomains and the CBF3 reconstitution, a challenging step toward the comprehension of the point centromere establishment.

CD44 is a transmembrane cell surface glycoprotein which has been implicated in various cell processes such as cell adhesion, lymphocyte migration and lymphocyte activation. However, although an important function for CD44 has been suggested by antibody-blocking studies, CD44+/+ mice paradoxically show few defects in immune function. Since compensatory mechanisms, operable in the complete absence of CD44 expression, could account for these apparently contradictory findings, we compared the development and function of CD44+/+ and CD44+/+ lymphocytes when these cells were in direct competition. Radiation bone marrow chimeras made with different combinations of CD44+/+ and CD44"7" donor and recipient mice were employed. CD44"/' progenitor cells were able to fully reconstitute both T and B cell pools when injected into irradiated CD44+/+ or CD44'A recipients. However, chimeras reconstituted with a mixture of CD44"/" and CD44+/+ progenitors showed a deficiency in the generation of CD44"A T cells. This deficit appeared to be due to both reduced thymic homing and impaired intrathymic maturation of CD44+/+ precursors. In contrast, a slight increase in the production of B cells derived from the CD44+/+ progenitors was observed in mixed chimeras, which was independent of precursor trapping in the thymus, as shown in experiments in thymectomised mice. The function of mature CD44+/+ lymphocytes was investigated by assessing immune responses after immunisation or infection of mixed chimeras. As measured by tetramer and intracellular cytokine staining, CD44+/+ CD8+ T cells were shown to respond less well than CD44+/+ cells, while CD44+/+ B cells exhibited a subtle defect in antibody production. Overall, the results demonstrate that CD44 plays a role during lymphocyte development and in the function of mature lymphocytes, which is only apparent when CD44 positive and negative cells are in direct competition.

This thesis describes a novel approach to the rational design of artificial esterases and aldolases. The Introduction provides a literature summary of the previous approaches that have been employed towards the design and synthesis of artificial enzyme systems. Chapter 2 describes the preparation and reactivity of a number of polymer based artificial enzymes, which are capable of catalysing ester hydrolysis. The study has involved the incorporation of a histidine catalytic group together with specifically designed peptide binding groups within a polymeric backbone. The binding groups were specifically selected according to their binding affinity towards an appropriate transition state analogue. The synthesis of peptide binding sites and thus incorporation of these, together with the histidine catalytic group into a polymer backbone, using standard peptide chemistry has been outlined. The results to an investigation of the influence of different pH, solvent and substrate concentration on the activity of artificial esterases are presented. Chapter 3 describes preliminary work undertaken towards the design and synthesis of artificial aldol catalysts. The aldolases, which feature a proline residue attached to a polymer backbone are shown to selectively catalyse aldol reactions using aromatic aldehydes as electrophilic partner. Chapter 4 describes the detailed experimental procedures used.

Cytoplasmic and transmembrane protein tyrosine phosphatases (PTPs) provide the enzymatic counterbalance to protein tyrosine kinase activity. PTP sigma (PTPa) is an adhesion molecule-like receptor PTP (RPTP) that is expressed on the growth cones of developing axons. PTPa binds ligands located in the basement membrane (heparan sulphate proteoglycans: agrin and collagen 18) and developing muscle (Nucleolin). Disruption of ligand-PTPa interactions affects axon guidance, although neither the role of PTPa in neurons nor the effect of ligand binding on PTPa activity is well understood. Further characterisation of PTPa function remains difficult in the absence of an understanding of PTPa biochemistry that would allow a functional assay to measure the effects of an experimental manipulation on PTPa function. PTPa was shown to be dimeric using a combination of disulphide cross-linking and co-immunoprecipitation techniques. This dimeric form of PTPa was principally cell surface localised according to its accessibility to trypsinisation. However, neither co-immunoprecipitation nor glutathione S-transferase (GST) pull-down techniques allowed the identification of proteins that interact with wild type or recombinant substrate-trapping PTPa. Secondly, H202 treatment of PTPa-expressing cells induced the formation of reduction-sensitive, high molecular weight species. In contrast to the formation of PTPa dimers under the same conditions, this did not require the tyrosine phosphatase domain catalytic site cysteines. It may be possible to utilise the formation of high molecular weight species on non-reducing SDS-PAGE analysis as a proxy measure of PTPa oligomerisation. Moreover, unlike co-immunoprecipitation it can be used on wild type and even endogenously expressed proteins. Finally, the type Ila RPTP family consists of PTPa, PTPS and LAR. To allow the simultaneous disruption of type Ila RPTPs, chicken LAR was cloned from embryonic chick whole body mRNA and characterised.

ii Abstract Vangl2 is a key member of the multi-protein planar cell polarity (PCP) pathway. Previous studies using the loop-tail (Lp) mouse, which carries a mutation in the Vangl2 gene, have shown that PCP is required for normal development of the cardiac outflow tract. The main cell types involved in development of the outflow tract are neural crest cells (NCC) and cells derived from the second heart field (SHF). The PCP pathway plays important roles in polarisation of cells within tissues and in directional cell movements. I hypothesised that PCP signalling is required for efficient movement of progenitor cells into the developing heart and that an abnormality in these processes is sufficient to cause common outflow tract defects. Whilst loss of Vangl2 in NCC has no affect on outflow tract development, deletion of Vangl2 from SHF cells (using Vangl2flox crossed with Isl1-Cre mice) recapitulates the shortened outflow tract and malalignment defects seen in Lp mice. The cellular distribution of Vangl2 changes as SHF cells pass from a progenitor state, still expressing Isl1 protein, to differentiated myocardium. When Vangl2 is lost from the cells derived from the SHF, the cells within the distal walls of the outflow tract show altered localisation of polarised molecules such as β-catenin, fibronectin and laminin, as well as PCP proteins including Dvl2 and Celsr1, suggesting disrupted cellular polarity. The expression of PKCζ and E-cadherin is also altered in the distal outflow tract walls of Vangl2flox/flox;Isl1-Cre embryos, supporting the idea that Vangl2 may regulate the polarity of this tissue. Together, these studies suggest that Vangl2 plays a role in imparting polarity on SHF cells as they contribute to the outflow and that this is important for its lengthening. Confirmation of the importance of the PCP pathway in regulating the polarity of the cells in the distal outflow tract, and its importance for outflow tract development, was obtained by examining upstream components (Wnt5a and Ror2) and downstream targets (Rac1 and ROCK) of the pathway, showing outflow defects and a similar expression pattern of polarised molecules.

γ-Tubulin plays a universal role in microtubule nucleation from microtubule organizing centers (MTOCs), such as the centrosome in higher eukaryotes and the spindle pole body (SPB) in fungi. The γ-Tubulin functions as part of a multiprotein complex called the γ-tubulin Ring complex (γ-TuRC), consisting of γ-tubulin complex protein (GCP) 1–6, where GCP1 is γ-tubulin. It has been shown that GCP1–3 are core components as they are essential and are absolutely required for the γ-TuRC assembly, whereas the other three GCPs are not. Recently, a novel component of the γ-TuRC, MOZART1, has been identified in humans and plants. In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-TuRC. The precise functional role of MOZART1 is still yet unknown. In this thesis, I therefore aimed to reveal its mode of action at a molecular level. For this purpose, I chose to exploit a powerful model system, fission yeast, which allows us to take multidisciplinary approaches. The mzt1 (also known as tam4) gene, which encodes fission yeast MOZART1 homologue, is essential. I localised Mzt1 during mitosis and meiosis and found it at the MTOCs. It also coimmunoprecipitates with γ-tubulin from cell extracts. I also determined the starting methionine of the mzt1 gene which encodes a 64–amino acid peptide. Depletion of Mzt1 leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. It also causes cytokinesis defects, suggesting a role of the γ-tubulin complex (γ-TuC) in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1 interacts with GCP3Alp6, a fission yeast homologue of GCP3. Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1 and the N-terminal region of GCP3Alp6. In addition Mzt1 protein tends to oligomerize into trimer or hexamer. Collectively I propose that Mzt1 oligomer directly interacts with GCP3Alp6 and stabilizes the γ-TuC to facilitate microtubule regulation.

The circadian cycle plays an important role in homeostasis by regulating the gene expression of rate-limiting enzymes and controlling the release of hormones. This means that disruption to this cycle can lead to metabolic and psychological disorders, such as diabetes or depression. In this thesis, major components of the circadian cycles have been studied using a combination of in vitro and in vivo techniques. CLOCK, NPAS2 and BMAL1 are transcription factors which utilise PAS domains to regulate their dimerisation and the binding of DNA via their helix-loop-helix domains. Constructs of the human forms of these proteins were expressed in E. coli and purified under native conditions. This permitted the study of the heme and carbon monoxide (CO) binding properties of the isolated domains from CLOCK and NPAS2 proteins. Similar heme binding characteristics were observed for both proteins, with a K[subscript D] values for heme binding in the micromolar range, biphasic dissociation curves and comparable UV-visible absorption maxima. All PAS domains tested were shown to bind CO but none reacted with oxygen to form a ferrous-oxy species. The heme chemistry in CLOCK and NPAS2 was examined further by determining the reduction potential of the heme iron. This was done using a novel method developed through the adaption of a previously described equilibrium reaction procedure, which was then altered by the addition of enzymes to remove oxygen, reduce the heme iron and reduce a redox dye. From the known reduction potential of the redox dye, it was possible to derive the reduction potential of the heme group. The results showed that heme bound to CLOCK and NPAS2 has a relatively low reduction potential, a property which likely contributes to the lack of a ferrous-oxy species. DNA-binding and functional assays were also used to show that the binding of heme observed in vitro has a physiological role. The DNA-binding behaviour of CLOCK and NPAS2 constructs were shown to be regulated by the addition of heme in vitro. Furthermore, the use of a heme synthesis inhibitor in a report assay demonstrated for the first time a direct link between intracellular heme concentrations and the transcriptional activity of the CLOCK:BMAL1 complex. It was seen that higher heme concentrations promoted the transcriptional activity of CLOCK and BMAL1. This is consistent with the observation made in vitro that heme disrupts the interaction between the inactive CLOCK:CLOCK homodimer and DNA.

Nitric oxide (NO) signalling has been recently linked with cardiac repolarisation. Studies from isolated heart preparations have demonstrated a protective effect from ventricular fibrillation following vagus nerve stimulation, which is NO- dependent. Genome-wide association studies have linked NO signalling components to arrhythmias. A role for NO signalling has also been described in the diurnal variation of cardiac muscle responses to sympathetic stimulation. The cellular and molecular mechanisms for NO regulation of ventricular repolarisation by cGMP-dependent signalling were investigated using BAY 60-2770, a novel NO/haem-independent soluble guanylyl cyclase (sGC) activator and compared with the NO donor SNAP. Experiments were performed on guinea pig isolated left ventricular myocytes. Cellular cGMP was selectively quantified by radioimmunoassay. Action potentials and the slow (I[subscript Ks]) and rapid (I[subscript Kr]) delayed rectifier K[superscript +] currents were recorded using the perforated patch-clamp technique. BAY 60-2770 applied under basal conditions, modestly increased cellular cGMP levels and shortened action potential duration (APD), but failed to modulate I[subscript Ks] or I[subscript Kr]. In contrast, SNAP failed to modulate APD despite greater increases in cGMP compared with BAY 60-2770. When phosphodiesterases (PDEs) were inhibited, BAY 60-2770 increased cGMP levels much more, prolonged APD and inhibited I[subscript Ks] (but not I[subscript Kr]). The mechanism for inhibition of I[subscript Ks] did not involve protein kinase G. These results demonstrate that PDE activity suppresses elevations of cGMP in response to sGC activation, and also compartmentalises cGMP-dependent signalling. Thus, PDEs uncouple changes in cGMP levels from ion channels located at the sarcolemma. In addition, APD shortening in response to isoprenaline exhibited diurnal variation, with greater shortening in active compared to resting period myocytes. However, this effect was not blocked by nitric oxide synthase inhibition. In conclusion, NO signalling pathways may be able to regulate cardiac repolarisation through a cGMP-dependent mechanism, but PDEs act to limit these responses in the healthy myocardium.