A detailed understanding of the B-cell response to influenza A haemagglutinin is key to the accurate matching of vaccines to seasonal strains, and may inform the development of broader spectrum vaccines. In this study, I develop techniques for predicting the location of the epitopes of protective antibodies by observing the physical locations of amino acid substitutions in human wild-type strains. By linking the understanding gained from this analysis with a large body of assay data, I present a model which can predict antigenic distance from HA1 amino acid sequences and which meets or exceeds the predictive power of previously developed models while retaining generality. An interesting conclusion from the epitope analysis discussed above is that antibodies to the HA head bind in two regions. The antigenic evolution of influenza H3N2 is more punctuated than its genetic evolution. I propose that the dual regions might contribute to the punctuated nature of antigenic evolution, and explore this through the use of a simple simulation. Stalk-binding antibodies to HA have attracted much interest in recent years: a number of broad-binding examples have been isolated, and the slower evolution of the stalk gives hope that these may provide broad protection against future strains. Stalk-binding neutralising antibodies to H3 are known to bind in two regions, and I use data from crystal studies to identify the constituent residues of these regions, which I term antigenic sites F and G, in a manner that is consistent with previous analyses of the constituent residues of HA1 antigenic sites A-E. I analyse the degree of conservation of residues in sites F and G, and conclude that there have been episodes of change in the H3 stalk which are consistent with antigenic evolution.

This study investigates the development of immunochromatographic assays also known as lateral flow assays with paramagnetic particles (PMP) used as a solid phase and label. The PMPs were quantified by virtue of their magnetic properties using a device known as the Resonant Coil Magnetometer (RCM) which is comprised of a capacitor and coil in series. A direct correlation between number ofPMPs present on nitrocellulose membrane and response in the RCM was observed using four coil configurations, the sensitivity and the limit of detection of the RCM varied depending on the coil design. Membrane and paramagnetic particle combinations were chosen following the evaluation of a range of nitrocellulose membranes and paramagnetic particles and used in the development of lateral flow assays for the quantification of hTransferrin (model) and total and free Prostate specific antigen (PSA). Limits of detection of lng/ml and 5ng/ml measured by scanning densitometer or RCM respectively, were at clinically significant concentrations for free PSA and total PSA. A high coefficient of variation was observed with both measuring systems due to the manual preparation of the assay strips, but was greatest with the RCM where the manual positioning of the capture lines in relation to the sensing coil increased variability. Anonymous serum samples evaluated in the total PSA lateral flow assay in comparison to Immulite and FastPack assays showed a negative proportional bias when measured using the scanning densitometer. The RCM was found to underestimate PSA concentrations where PSA concentrations were >18ng/ml, but a positive proportional bias was observed at lower PSA concentrations. Higher CV values were observed in the data obtained from the RCM. This may explain the different profiles obtained using the two measuring systems in comparison to commercially available immunoassays. Differences may also be due to the ability of the RCM to quantify PMPs present throughout the membrane and not just at the membrane surface as seen with the scanning densitometer. This study has shown that PMPs can be used as a solid phase and label in lateral flow assays and the quantification of analyte in standard and serum samples demonstrates the potential of the RCM as a device for the quantification ofmultiple analytes at the point-of-care.

BACKGROUND AND AIMS. The success of stem cell therapies partly depends on the ability to deliver the cells to the site of injury. Circulating endothelial progenitor cells (EPCs) are involved in physiological processes such as vascular re-endothelialisation and post-ischaemic neovascularisation and have been utilised in several clinical trials. Superparamagnetic iron oxide particles have previously been used to label and track cells using magnetic resonance imaging (MRI), as well as to magnetically attract drugs and cells to desired sites. The aim of this PhD was to develop a methodology to magnetically attract EPCs, labelled with a clinically approved iron oxide agent, to a site of arterial injury using magnetic fields originating outside the body. METHODS AND RESULTS. Human EPCs were cultured in the presence of iron oxide superparamagnetic nanoparticles. A labelling method was developed that retained cell survival and differentiation, as indicated by metabolic activity and flow cytometry assays, as well as MRI visibility. Finite element modelling (FEM) computer simulations were performed to investigate the interaction of magnetic forces with hydrodynamic drag forces. FEM indicated successful external magnetic cell targeting from a vessel with flow rate similar to a rat common carotid artery; correspondingly there was a 6- fold increase in cell capture in an in vitro flow system. Angioplasty was performed on rat common carotid arteries to denude the endothelium and EPCs were administered with and without the presence of the external magnetic device during a 10 minute period of flow cessation. Targeting enhanced cell retention at the site of injury by 5-fold. CONCLUSIONS. Using an externally applied magnetic device, it is possible to enhance EPC localisation in a flowing sytem in vitro and to a flow-isolated site of common carotid artery injury in vivo, without affecting cell viability or differentiation in culture. This technology could be more widely adapted to localise and monitor cells in other organs and may provide a useful tool for systemic injection of cell therapies.

Targeting Cdc7, a kinase essential for DNA replication initiation, results in potent cancer cell killing. Cancer cells in which CDC7 is silenced by RNAi enter an abortive S phase followed by apoptosis due to loss of a functioning DNA replication origin activation checkpoint. This checkpoint prevents normal cells from entering S phase (reversible G1 arrest) if the DNA replication initiation machinery is perturbed. The pre-clinical anti-cancer effects of CDC7 silencing have highlighted this kinase as an important target for new drug development. Expanding on published reports, I performed further target validation using molecular tools generated in the work of this thesis, including an affinity-purified antibody to the Cdc7 regulator ASK and functional recombinant Cdc7/ASK kinase complex. Making use of fibroblast and HL60 tissue culture model systems, I show that Cdc7 and ASK are amongst a group of essential replication initiation factors that are tightly downregulated to suppress proliferative capacity during exit from cycle into quiescent and differentiated states. This finding is further supported by low expression levels in normal liver and oral squamous epithelium and the lack of Mcm2 phosphorylation at serine 53, a well known Cdc7 target. In liver carcinoma and oral squamous cell carcinoma, on the contrary, the majority of cancer cells are expressing Cdc7 and ASK and show Mcm2 phosphorylation at Ser-53. Thus it can be postulated that Cdc7 inhibitors should selectively kill cancer cells, while normal proliferating cells are reversibly arresting in G1 and quiescent and differentiated cell populations are not affected due to downregulation of the target protein. To screen for compounds that selectively inhibit Cdc7, I developed a sensitive in vitro kinase assay and contributed to the successful transfer of this assay to a high-throughput screening platform and the generation of a structural model of the Cdc7 kinase domain allowing in silico predictions of the most potent inhibitors. On completion of the work for this thesis, the HTS assay and structural model fromed the core of an ongoing drug discovery programme run by Cancer Research Technology. Two series of novel, selective small molecule inhibitors which exhibit low nM activity against Cdc7 and cellular efficacy (apoptosis) have been developed and are currently being tested in mouse xenograft models. The work presented in this thesis provides a strong rationale for targeting the DNA replication initiation pathway, and in particular Cdc7. Future intend to treat clinical trials will establish the potential of pharmacological Cdc7 inhibitors for selective cancer cell killing in patients.

Characterisation of striatal primary cultures using immunocytochemistry (ICC) and calcium imaging has demonstrated that the cultures consist primarily of neurones (70%), and that the remaining cells display morphology typical of astrocytes. Both cell types were found to express the Type I IL-1 receptor. Treatment of striatal cells <I>in vitro</I> with IL-1 stimulated two mitogen activated protein (MAP) kinase families. Extracellular-regulated kinase (ERK) and p38 MAP kinase were both phosphorylated in a time- and concentration-dependent manner. Phosphorylation was found to correlate with induction of kinase activity, and therefore MAP kinase activation. Further studies utilising ICC revealed that p38 MAP kinase phosphorylation occurred specifically within the astrocyte nucleus. Downstream of the MAP kinases, IL-1 activated the transcription factor c-Jun by phosphorylation. The MAP kinase substrates ATF-2 and CREB were not activated upon IL-1 stimulation. Furthermore, IL-1 stimulated phosphorylation and nuclear translocation of the transcription factor NF-kB, an important mediator of immune responses. NF-kB translocation was observed specifically in astrocytes. Gene expression was studied directly using the TaqMan quantitative real time PCR technique. These studies revealed that IL-1 stimulated transcription of the cytokines tumour necrosis factor alpha (TNF-α), IL-6 and IL-1 itself. Gene expression of other cytokines (including IL-1 receptors, the IL-1 receptor antagonist and IL-18) were not stimulated by IL-1 in striatal cells. ELISA analysis of protein levels of selected cytokines (TNF-α and IL-6) revealed that gene expression did not correlate with protein release. These studies reveal for the first time important aspects of IL-1 signalling in a functionally significant region of the rat brain.

Trillions of microorganisms inhabit mucosal surfaces of the human body. Despite increasing evidence of their impact on human health, many of the molecular mechanisms underlying hostmicrobial interactions (HMI) are poorly understood. To contribute to our understanding of HMI at mucosal surfaces, we investigated the novel family of M60-like/PF13402 domain-containing proteins and their putative functional partners. M60-like domains are shared by proteins from several mucosal microbes including two important human mucosal microbes; the bacterial mutualist Bacteroides thetaiotaomicron and the protist pathogen Trichomonas vaginalis, suggesting these proteins are important for interaction with the mucosal layer. We initially tested our hypothesis that these are glycoprotein-targeted metal dependent proteases in both these organisms. The three M60-like domains of B. thetaiotaomicron proteins (BT4244, BT3015 and BT4272) exhibited mucin protease activity. This proteolytic activity was shown to be inhibited in a mutant version of the protein (BT4244-FL-E575D) as well as in the presence of Ethylenediaminetetraacetic acid (EDTA), implying BT4244 and its relatives are metal dependent proteases. All M60-like proteins from B. thetaiotaomicron contained a carbohydrate binding module (CBM) from family 32 and these were shown to be capable of binding galacto-configured sugars that are common to mucin glycans, while in contrast the putative carbohydrate binding PA14 domain of the T. vaginalis TVAG339720 M60-like protein interacted with heparin and its sulphated derivatives. Mucins are glycoproteins and prominent components of the mucus secreted at mucosal surfaces while heparin is a close relative of heparan sulphate which typically exists as part of proteoglycans in the glycocalyx of mucosal epithelia. Although the actual target of the M60-like domain of TVAG339720 and its relatives in T. vaginalis are not currently known, the interaction of the TVAG339720 PA14 domain with heparin suggests that these may be proteases targeting proteoglycans and play a role in adhesion of the pathogen to the epithelial layer, a key initial step in pathogenesis. M60-like domain-containing proteins of B. thetaiotaomicron are also components of Sus-like systems. Sus-like systems are Bacteroidetes specific machinery that comprise a suite of cellenvelope located carbohydrate-active enzymes and sugar binding proteins that target complex glycans, with each Sus-like system tuned to the degradation of a specific glycan. The Sus-like system containing the BT4244 enzyme (BT4240-50), encoded by the polysaccharide locus (PUL) PULBT_4240-50 was characterised in this study. The results demonstrated that BT4244 is a surface protein and that its proteolytic activity is part of a concerted action of BT4240-50 components to utilise complex mucin glycoproteins containing the T (Galβ1-3GalNAc) and F (GalNAcα1-3GalNAc) antigens. Gene deletion studies revealed that PULBT_4240-50 provides a competitive advantage to the organism when grown on mucins, probably through its possession of the N-acetylgalactosamine (GalNAc) kinase BT4240, which was shown to be crucial for GalNAc utilisation. Finally, although variably conserved in closely related Bacteroides, the high frequency of PULBT_4240-50 components in this group of organisms suggests it may be an important evolutionary adaptation for survival at mucosal surfaces. Our findings not only set the stage for future functional studies on the novel M60-like/PF13402 family of proteins and their functional partners, but also further our understanding of host-microbial interactions at mucosal surfaces.

Raman spectroscopy is an optical technique that can interrogate biological tissues. In doing so it gives us an understanding of the changes in the molecular structure that are associated with disease development. The Kerr gating technique uses a picosecond pulsed laser and fast temporal gating of inelastically (Raman) scattered light. The tissue samples used were taken following fully informed consent and ethics approval. Bladder samples were obtained by taking a biopsy during a TURBT or TURP, prostate samples were taken during TURP and the liver and kidney (pigs) were bought at a supermarket. The bladder and prostate samples were snap frozen in liquid nitrogen and stored in an -80°C freezer until required for experimentation. The liver and kidney tissue were used fresh. The constituent samples were bought from Sigma – Aldrich. Multivariate and least squares analysis were used to ascertain the biochemical basis of the differing pathologies within the bladder and the prostate gland, as well as to test diagnostic algorithms produced by a colleague in our group. Depth profiling through the bladder and prostate gland was shown to be feasible by utilizing the Kerr gating technique as was the suppression of fluorescence from dark tissue (liver and kidney). We have shown for the first time, that we can utilise Raman spectroscopy to determine the biochemical basis of pathologies of the bladder and the prostate gland. With the help of the Kerr gating technique we also obtained spectra from different depths through them. We also suppressed fluorescence and resonantly enhanced Raman spectra from dark tissue. These have major implications in terms of understanding pathogenesis and disease progression and also the potential to accurately assess depth of tumour invasion.

This thesis aimed to investigate vibrational spectroscopies for the identification of biochemical markers of leukaemias and lymphomas. In a preliminary study using the blood proteins albumin, fibrinogen and globulin, Drop Coating Deposition Raman Spectroscopy was explored and extended for use with Fourier Transform infrared spectroscopy for leukaemia blood sample analysis. Due to low sample volumes and minimal preparation required it was identified as a potential alternative to blood centrifugation to obtain the buffy coat for analysis. These studies identified that it was capable of detecting low levels of protein from small, highly concentrated droplets. Thus this method, alongside cytospin centrifugation, was used for the spectroscopic analysis of different blood fractions. Due to the low number of lymphoma samples obtained, only a feasibility study is outlined in this thesis. Samples were collected from leukaemia patients and healthy volunteers. Infrared and Raman spectra were measured of whole blood and buffy coat samples cytospun onto slides and whole blood and plasma pipetted by drop coating deposition. Multivariate statistical analysis was employed to extract key spectral differences between the pathologies and develop classification models for diagnosing chronic lymphoblastic leukaemia from previously treated and untreated patient groups. Principal component analysis followed by linear discriminant analysis was employed to identify the largest variances in the data and leave one sample out cross validation evaluated the performance of the spectral models measured on different blood components in diagnosing leukaemia. The buffy coat infrared model correctly classified 59% of the spectra, and blood droplet Raman 62%. The treated and untreated groups were then combined, which improved classification to 83% for buffy coat infrared and 71% for blood droplet Raman. These findings highlight the potential of drop coating deposition spectroscopy of whole blood for leukaemia diagnosis, although further work is required to achieve a clinically validated method.

The reader will first be introduced to the principles of positron emission tomography (PET) as a state of the art medical imaging technique. Particular emphasis will be made on the application of the positron emitting isotope fluorine-18 e8F) and how novel binding sites for the insertion of the isotope into biomolecules are currently emerging to overcome the complex formation of C_[18F] bonds. The synthesis of a range of labelled prosthetic groups for the insertion of 18F into peptides was completed. e8F]-fluorobenzaldehyde ([18F]-FBA) was synthesised using three synthetic strategies, including the use of a robotic synthesiser for high activity syntheses. This molecule was subsequently studied in conjugation reactions with a salmon calcitonin derivative formed by solid phase peptide synthesis to include a hydrazine function at the lysine-18 residue. The hydrazine moiety was introduced to the peptide through derivatisation of Fmoc-Iysine-OH with 6-hydrazinonicotinic acid (HYNIC) or 4-hydrazinobenzoic acid (HYBA) in a three-step synthesis. Conjugation of [18F]-FBA to HYNIC-salmon calcitonin and HYBA-salmon calcitonin was studied and produced multiple radiolabelled species as determined by radio-HPLC. The formation of 4-maleimido trimethylammonium triflate as a precursor for 4-fluorophenyl maleimide was completed successfully alongside the formation of a non-radioactive standard. Radiolabelling of this molecule proved unsuccessful and only [18F]-fluoride was returned from reaction mixtures. A thorough study of the formation of e8F]-trifluoroborates was completed to assess the effect of concentration, pH, temperature and the effect of varied fluoride carrier sources on the radiofluorination of arylboronic acids and arylboronate esters. These molecules offer a novel binding site for [18F]-fluoride for the radiolabelling of biomolecules such as peptides and proteins without C_[18F] bond formation. Maximum radiochemical yields in the range of 50%-60% could be achieved in the case of the formation of 4-carboxphenyl-[18F]-trifluoroboate from the corresponding pinacol ester. However these reactions needed to be carried out utilising potassium hydrogen fluoride as a unique source of carrier fluoride and required mM concentrations of boron precursors for the reaction to yield significant radiolabelled product on the range of small molecule boronic acids and boronate esters studied. The formation of cold standard organotrifluoroborates was studied and the use of 19F NMR to attempt to detect mono- and bis- fluorinated intermediates was applied to prove that trifluoroborate formation is a rapid process where no intermediates can be detected between within 2-3 minutes when reacting boronate esters with potassium hydrogen fluoride. Several strategies for the formation of boron containing amino acids were studied to yield precursors suitable for the introduction of boronic acids or boronate esters into peptides through solid phase peptide synthesis. Direct conjugation via amide formation to the side-chain carboxylic acid of Fmoc-Glu-OtBu proved successful when conjugating an aryl boronate ester. The formation of aryl and alkyl boronic acid azide was also shown to be possible for subsequent triazole formation by copper catalyzed reaction with a propargyl derivatised glutamic acid residue {"click" reaction}. A small molecule boronic acid maleimide derivative was synthesised for the sitespecific addition to thiol containing peptides and proteins. This molecule was shown to bind to the free cystiene residue in c{RGDfC) and to a modified construct of the C2A domain of Synaptotagmin {C2Ac}. Radiofluorination of the C2Ac-boron-maleimide derivative was completed to demonstrate the feasibility of kit-based [18F]_ trifluoroborate technology.

The identification of the ASPP family of proteins (ASPP1, ASPP2 and iASPP) has helped facilitate a better understanding of how the functions of the tumour suppressor p53 are specifically regulated. ASPP1 and ASPP2 bind to p53 and specifically enhance its transcriptional activities on proapoptotic genes, while iASPP, by a similar mechanism, inhibits p53. However, iASPP is also an inhibitor of NFκB and may, therefore, inhibit cell proliferation. Many other proteins have also been reported to bind with iASPP, suggesting that p53- or NFκB-independent functions may also exist. However, the mechanisms by which iASPP activity is regulated remain unclear. In this thesis, iASPP has been shown to be phosphorylated by at least two families of non-receptor tyrosine kinases: Src and Abl. Tyrosine phosphorylation of iASPP at Y132 is mainly due to the activation of Src family kinases. Upon phosphorylation at Y132, iASPP is translocated to adherens junctions and focal adhesions, where it forms stable multiprotein complexes with either cadherin/catenins or focal adhesion proteins. Importantly, phosphorylation deficient Y132F-iASPP increases cell motility in MCF-7 cells. A potential negative effect of pY132-iASPP on cell motility has, therefore, been suggested. Oncogenic Bcr-Abl kinase is the underlying cause of chronic myeloid leukaemia (CML). pY-iASPP levels are increased with Bcr-Abl expression, while a specific Abl kinase inhibitor called STI-571, which has been successfully used to treat CML, efficiently inhibits this production of pY-iASPP. These data suggest that iASPP may be a downstream effector by which Bcr-Abl transforms blood cells, and a potential marker to predict the response of CML patients to STI-571 treatment. In addition, iASPP’s functions may be regulated by caspase cleavage in death receptor induced apoptosis of leukaemia cells. This cleavage mechanism produces a 80kDa iASPP fragment (295-828), which loses all identified tyrosine phosphorylation sites at the N-termini, suggesting that cells may prevent the production of pY-iASPP by caspase cleavage to affect the progress of apoptosis. Taken together, the results presented in this thesis provide insights into the posttranslational regulation of iASPP activity, which will lead to a better understanding for both cancer biology and cancer therapy.