The bacterial cell wall surrounds the cytoplasmic membrane and protects the cell against osmolysis in addition to providing shape. The cell wall is comprised of peptidoglycan, repeating units of N-acetly glucosamine and N-acetyl muramic acid form glycan strands and are crosslinked by short peptides that contain both L- and D-amino acids. Owing to the unique nature of peptidoglycan, and its absence in eukaryotic organisms, the cell wall has become an important target for many antibiotics, including the β-lactams and glycopeptides. Newly synthesised peptidoglycan contains pentapeptides, which extend from the lactyl moiety of the MurNAc sugar. These chains consist of L-alanine-D-γ- glutamate/glutamine-L-lysine/meso-diaminopimelic acid-D-alanine-D-alanine. The terminal D-alanine is often lost during cell wall maturation, either as a result of the crosslinking reaction, in which the penultimate D-alanine is attached to the side-chain of a neighbouring L-lysine or meso-diaminopimelic acid by an isopeptide bond, or as a consequence of the activities of DD-carboxypeptidases, and results in a tetrapeptide. The tetrapeptide can then be trimmed further to form a tripeptide by the action of LD-carboxypeptidases. Although many DD-carboxypeptidases have been well characterised, the majority of LD-carboxypeptidases that have been studied are active only against peptidoglycan fragments and so cannot be responsible for producing the tripeptides found in the cell wall. Of the LD-carboxypeptidases active against the mature cell wall, DacB (Streptococcus pneumoniae), Csd6 (Helicobacter pylori) and Pgp2 (Campylobacter jejuni), each has been shown to be essential in maintaining cell morphology. It should be noted, however, that neither Csd6 nor Pgp2 share any sequence similarity with DacB and belong to different peptidase families. This thesis concerns the structural and biochemical characterisation of DacB, herein renamed to LdcB (LD-carboxypeptidase B). The crystal structures of the apo form of LdcB from both S. pneumoniae and Bacillus subtilis were solved, revealing a single domain, globular protein with 2 sub-domains forming a V-shaped cleft in which the active site is located. LdcB binds one zinc ion per monomer, located at the bottom of the active site, and is a member of the LAS (lysostaphin, D-Ala-D-Ala peptidases, sonic hedgehog) family of metalloproteins. Additionally, the activity of LdcB as an LDcarboxypeptidase was confirmed and the crystal structure of LdcB from S. pneumoniae ii was solved in complex with a product mimic, M-Tri-Lys(D-Asn), revealing the molecular basis for peptidoglycan recognition in this family of enzymes. Finally, the affinity of LdcB for zinc and copper has been determined and it has been shown that catalysis is not inhibited by the substitution of zinc by copper or cobalt.

Epidemiological and experimental evidence suggests that non-digestible carbohydrates (NDCs) e.g. resistant starch (RS) are protective against colorectal cancer (CRC), resulting primarily from the production of the short-chain fatty acid (SCFA) butyrate. The WNT signalling pathway is central to the maintenance of homeostasis within the large bowel through regulation of physiological processes and is frequently aberrantly activated in CRCs. Butyrate has been shown to positively modulate WNT signalling, affecting functional outcomes such as apoptosis and proliferation and, therefore, protecting against CRC. To investigate the molecular mechanisms through which NDCs may reduce CRC risk in humans, we undertook a double-blind, randomised, controlled trial (The DISC Study) in which 75 healthy participants were supplemented with two NDCs: RS (Hi-maize® 260) and polydextrose (PD) (Litesse® Ultra™) for 50 days using a 2x2 factorial design. Colorectal mucosal biopsies were collected before and at the end of intervention and used to quantify expression of WNT pathway-related genes and of BAX and BCL-2 (two regulators of apoptosis) and to assess colonic crypt cell proliferation. WNT signalling pathway genes were also investigated in an additional 38 participants at higher risk of CRC because of quiescent ulcerative colitis or a history of adenomatous polyps. RS supplementation significantly reduced expression of CTNNB1 (p=0.045), encoding β-catenin (a key component of the WNT pathway), and c-MYC (p=0.037), suggesting a reduction in WNT signalling in these participants. RS and/or PD significantly reduced expression of two antagonists of WNT signalling, SFRP1 and SFRP2, suggesting an increase in WNT activity. RS and PD did not affect SFRP1 methylation or expression of miRNAs that may target this gene. RS increased total colonic crypt cell proliferation (p=0.030) but did not alter the proportion of mitotic cells in the top half of the crypt (a marker of crypt health). Participants at higher risk of CRC had increased expression of c-JUN (p=0.046) and WNT11 (p=0.040) and increased SFRP1 methylation. The BAX to BCL-2 ratio was lower in higher-risk participants, suggesting a reduction in BCL-2 ii family-mediated apoptosis. Unexpectedly, crypt cell proliferation was also reduced in higher-risk participants (p=0.009). These findings suggest that the increased cell proliferation with RS supplementation may have resulted from induction of the WNT pathway. However, these effects were not mediated via alterations in SFRP1 methylation or expression of miRNAs. This study has also shown that WNT signalling is aberrantly active in the macroscopically-normal mucosa of people at higher risk of CRC.

The Holy Grail of cancer research is to find effective treatments that can be easily delivered to diseased cells with minimal collateral damage to healthy tissue. In this context, recent developments in nanoparticle technology have aroused considerable interest with the promise of multifunctional vectors for both diagnostic and treatment of cancer. Recently, new emphasis has been placed on hybrid nanoparticle (NP) systems, where combinations of different types of nanostructured materials are used to create multimodal systems that exhibit the combined beneficial properties of the component modules. In particular, nanorattles, which are NPs with a core-shell structure containing a distinctive void separating the core material from the shell, constitute promising launch platforms for many biomedical applications. Current hybrid NP systems tend to concentrate on adding extra properties by increasing the number of modules and therefore, system complexity. However, added complexity in itself does not guarantee higher effectiveness. Therefore, in this thesis, a more holistic approach is proposed whereby simplicity, efficiency and usefulness of the design are not overlooked. The work presented here describes a gold-silica rattle-type particle, the Quantum Rattle (QR), made of a hollow mesoporous silica shell (HS) hosting two classes of hydrophobic gold nanostructures: gold quantum dots (AuQDs) and gold nanoparticles (AuNPs). The HS stabilises the gold nanostructures, making them dispersible in water and thereby enables biomedical applications. It also allows passive targeting for the QR via the enhanced permeability and retention (EPR) effect. The AuQDs absorb and emit light in the near-infrared (NIR) biological window where blood and soft tissue are relatively transparent (650 nm - 900 nm). With their NIR photonics, the AuQDs mediate both photothermal therapy (PPT) and live infrared imaging. Finally, the hydrophobic AuNPs optimise the system’s drug carrying performance by increasing the payload’s loading efficiency as well as controlling its release profile. This thesis exhibits the first evidence of the intrinsic and efficient therapeutic and diagnostic potential of this new class of hybrid NP system and discusses how these results could have a significant impact on the growing field of nanosystems used for cancer treatment.

Introduction: Cholangiocarcinoma (CC) is a malignancy of the biliary tract. It has a dismal prognosis and complete surgical resection offers the only chance of cure. The aim of this study was to identify prognostic DNA/microRNA signatures, and to identify key targets and pathways in CC to improve treatment. Methods: We performed a retrospective study to assess the role of surgery and adjuvant therapy on the survival outcomes of patients with CC based on the experience of two institutions. We investigated the molecular pathogenesis of CC assessing DNA copy number alterations and differential miRNA expression. We used array comparative genomic hybridization (CGH) (1Mb BAC array-CGH, and 180K Oligonucleotide array-CGH) on 71 UK and 24 Thai cases CC. We performed microRNA-arrays (Agilent Human miRNA slides V3) on 34 CC and 10 normal cholangiocyte samples. Results: Survival analysis showed a statistically significant difference in survival between those resected and those receiving medical management only. Thai CC cases exhibited a lower proportion of CNA compared to UK cases. A common UK alteration was seen at 17q12, the region encoding ErbB-2. The copy number gain at 17q12 was validated using CISH and IHC for ErbB-2 expression, revealing heterogeneous expression. Copy number gain of chromosome 8q24.21-24.3 was significantly related to survival. Median survival was 14.4 months vs 28.3 months with and without the gain (p = 0.016). Thirty-eight miRNAs showed significantly different expression, including several microRNAs implicated in other malignancies, with predicted gene targets including the p53 signaling pathway and the TGF-beta signaling pathway. We identified a 4-microRNA signature that correlated with overall survival. With a median survival of 15.7 months vs 35.6 months: p = 0.00016. Conclusion: This study illustrates the genetic variability of CC, highlights several potential therapeutic targets, and identified a DNA and miRNA signature that correlated with prognosis.

Background: Ovarian cancer is the most lethal gynaecological malignancy, accounting for an estimated 140,000 deaths per year worldwide. Five year survival rates have not increased significantly in the last 10 years and the acquisition of resistance to chemotherapy remains a significant barrier to improving patient survival. Isogenic cell line models of in vivo acquired resistance to chemotherapy were used to examine cellular responses to cisplatin and identify differences between sensitive and resistant pairs that might be exploited to sensitise cells to treatment. Results: Microarray analysis of the isogenic paired sensitive/resistant high grade serous ovarian cell lines PEO1 and PEO4 revealed IL6 expression is induced by cisplatin exposure. This result was replicated by QRT-PCR and validated in the additional isogenic pair PEA1/PEA2. Western blotting demonstrated the lack of a correlation between IL6 expression and phosphorylation of either Y1007/1008 JAK2 or Y705 STAT3 levels, suggesting IL6 is not driving the constitutive activation of these proteins. Cells did however display dose dependant changes in STAT3, JAK2 and ERBB2 activation in response to cisplatin that differed between sensitive and resistant isogenic pairs. Both sensitive clones increased their activation of JAK2 and ERBB2 when exposed to low dose, 2μM, cisplatin but reversed these increases at higher concentrations. Resistant clones, PEO4 and PEA2, experienced no low dose increases in ERBB2 JAK2 or STAT3 activation instead reducing the activation of these proteins with greater sensitivity to cisplatin dose. Common to all cell lines was a high degree of correlation in the levels of activated JAK2 and ERBB2. Interfering with cisplatin dependent STAT3 deactivation using IL6 treatment was able to both sensitise cells and reduce cisplatin IC50, suggesting a functional role for STAT3 in both response, and acquired resistance, to cisplatin. Overexpression and knockdown of STAT3 demonstrated it promotes proliferation and the expression of cyclin D1 and BCL xL/S. STAT3 knockdown increased cisplatin resistance as quantified by IC50 whereas STAT3 overexpression was able to potentiate cisplatin induced apoptosis and decrease cisplatin IC50. Similarly the overexpression and knockdown of JAK2 demonstrated it also promotes proliferation, in part by regulating the activity of STAT3. The inhibition of JAK2 activity also increased resistance to cisplatin; small molecule inhibition of JAK2 both lowered background levels of apoptosis as well as attenuating cisplatin induced apoptosis. In common with STAT3 ablation JAK2 knockdown also increased cisplatin IC50. Surprisingly knockdown, overexpression and inhibition of JAK2 were all associated with changes in the activation of ERBB2. Knockdown and inhibition were associated with decreases in Y1248 phosphorylated ERBB2 whereas overexpression was associated with an increase, changes in activation appear to be driven by changes at the level of total protein. GP130 was investigated for due to its role in IL6 signalling and STAT3 activation, mRNA overexpressed was detected in the resistant pair of 2/3 isogenic cell lines. GP130 overexpression was associated with growth promotion and cisplatin resistance as revealed by siRNA knockdowns, which had no effect in cisplatin sensitive non overexpressing cells lines. Knockdown also revealed different pathways to constitutive STAT3 activation, in each high grade serous line assessed there was no effect on pSTAT3 levels, which were almost completely abolished in SKOV3. RNAi of GP130 was also associated with an increase in ERK1/2 activation which was recapitulated by JAK2 knockdown, inhibition as well as cisplatin and doxorubicin exposure. Treatment with a MEK1/2 inhibitor was able to reverse cisplatin and doxorubicin dependent activation and attenuate cytotoxic induced apoptosis. MEK1/2 inhibition was associated with an increase in JAK2, pSTAT3 and pERBB2 which was capable of reversing cisplatin dependent down regulation of these protein, highlighting a mutual feedback mechanism between the GP130/JAK2 and MAPK pathways. Central Conclusion Transcriptional regulation of JAK2 in response to cisplatin exposure drives differential behaviour of paired isogenic cell lines. Greater sensitivity of cisplatin resistant cells lines, in their deactivation of STAT3 and ERBB2 is regulated by their greater extent of JAK2 downregulation upon cisplatin exposure. Downregulation of JAK2 and its commensurate reduction in STAT3 activation were associated with reduced proliferation rates, rendering cells in a state refractory to cisplatin toxicity. This may be due to either or both of reducing the accumulation of DNA double stranded breaks associated with cell division and allowing more time for the repair of single stranded DNA adducts before cell division. While STAT3 has been suggested as a target for adjuvant chemotherapy, data presented here suggests that in combination with cisplatin STAT3 abrogation might actually reduce the effectiveness of treatment.

Molecular imaging is an ideal platform for non-invasive detection and assessment of cancer. In recent years, the targeted imaging of CXCR4, a chemokine receptor that has been associated with tumour metastasis, has become an area of intensive research. CXCR4 is a GPCR whose interaction with its natural ligand SDF-1α is essential for embryo development and haematopoiesis. Over-expression of this receptor is associated with aggressive types of cancer and potentially metastatic tumours. Several classes of CXCR4 inhibitors have been reported in the literature, including TN14003-, FC131- and IT1t-derivatives. The aim of the research presented herein was to develop CXCR4-targeted probes for positron emission tomography. To this end, we designed and synthesised 20 new potential tracers belonging to those three classes of CXCR4 inhibitors, for labelling with fluorine-18 or gallium-68. In vitro and in vivo evaluation of these radiotracer candidates allowed for further understanding of the structure-activity relationship and pharmacokinetic properties of these types of compounds. In particular, our gallium-68-labelled tracer based on the structure of TN14003 displayed the essential features for the identification of CXCR4-expressing tumours in a clinical setting.

The ability to image caspase activation provides a powerful tool for the evaluation of the oncologic therapeutic response via the apoptotic cascade. Research efforts have focused on the development of radiolabelled non-peptidyl caspase inhibitors to be utilised for imaging purposes. Isatin sulfonamides are of particular interest because of their specificity and potency for inhibiting the 'apoptotic executioner' caspase-3. However, imaging attempts using the radiolabelled lead isatin have shown low target-to-background radiotracer uptake. The research aim was to explore alternative series of caspase-3 inhibitors as radioligands with optimal properties for the PET imaging of apoptosis. The series based on the pyrimidoindolone, triazoleindolone and imidazoleindolone heterocycles are structurally similar to the isatin core heterocycle, with all containing the crucial ketone responsible for caspase inhibition via the formation of a thiohemiketal with an active site cysteine residue. Using computational modelling studies combined with UV-vis titrations, these heterocycles were shown to have the propensity to favourably form thiohemiketals and therefore potentially inhibit caspase-3. Initial target structures, modelled on the structure of the lead isatin, were subsequently designed for synthesis and biological characterisation. Pyrimidoindolone target was synthesised and radiolabelled using modified literature conditions. Biological results showed the pyrimidoindolone to have a similar biological profile to isatin. Four different synthetic strategies were investigated for the synthesis of the imidazoleindolone and triazoleindolone heterocycles including, the use isatin sulfonamide intermediates, an intramolecular SNAr cyclisation process, Friedel-Crafts cyclisation method and nucleophilic addition to a benzonitrile. Preparation of imidazoleindolone was achieved with the SNAr cyclisation route. Preparation of triazoleindolone proved challenging with synthesis of only the core heterocyclic precursors, hydrazone and ketal achieved using the SNAr cyclisation route. Acid-sensitivity of the ring system hindered deprotection reactions and led to ring destruction.

Breast cancer is the most common cancer among Chinese women living in the UK. However the literature suggests that Chinese women are less likely to attend breast screening than white British women. No studies have been conducted to explore reasons for low attendance among this specific population. The purpose of this thesis was to understand the psycho-social factors related to breast cancer prevention and screening among Chinese women in the UK, and then to inform a breast screening intervention design. Three studies were conducted. The first was a systematic review of interventions to increase breast screening among Chinese women living in Western countries. The second and third studies used focus groups to explore Chinese women’s beliefs about breast cancer prevention and screening practices among older and younger generations. Finally, Intervention Mapping was used to synthesise the findings of the focus groups with those of the systematic review to design an empirical and theoretical evidence based breast screening intervention directed at Chinese women who are non-adherent to the NHS Breast Screening Programme. The qualitative findings revealed that older participants held a more holistic view of health maintenance, and had less knowledge about breast cancer and its causes than younger participants. They showed positive attitudes to breast screening and most had responded to receiving a mammography invitation. Language was a key barrier to older participants using medical care and obtaining health-related information. Younger participants expressed high dissatisfaction with health care in UK and showed a strong ‘neo-fatalistic’ view of breast cancer prevention, believing the main cause of breast cancer to be genetic predisposition. The synthesis of findings suggest that healthcare providers need to take Chinese cultural and language concerns, but also the differences between generations, into account when designing and implementing breast screening services and educational programmes which target Chinese women.

Since the first discovery of S100 members in 1965, their expressions have been affiliated with numerous biological functions in all cells of the body. However, in the recent years, S100A4, a member of this superfamily has emerged as the central target in generating new avenue for cancer therapy as its overexpression has been correlated with cancer patients’ mortality as well as established roles as motility and metastasis promoter. As it has no catalytic activity, S100A4 has to interact with its target proteins to regulate such effects. Up to date, more than 10 S100A4 target proteins have been identified but the mechanical process regulated by S100A4 to induce motility remains vague. In this work, we demonstrated that S100A4 overexpression resulted in actin filaments disorganisation, reduction in focal adhesions, instability of filopodia as well as exhibiting polarised morphology. However, such effects were not observed in truncated versions of S100A4 possibly highlighting the importance of C terminus of S100A4 target recognition. In order to assess some of the intracellular mechanisms that may be involved in promoting migrations, different strategies were used, including active pharmaceutical agents, inhibitors and knockdown experiments. Treatment of S100A4 overexpressing cells with blebbistatin and Y-27632, non muscle myosin IIA (NMMIIA) inhibitors, as well as knockdown of NMMIIA, resulted in motility enhancement and focal adhesions reduction proposing that NMMIIA assisted S100A4 in regulating cell motility but its presence is not essential. Further work done using Cos 7 cell lines, naturally lacking NMMIIA, further demonstrated that S100A4 is capable of regulating cell motility independent of NMMIIA, possibly through poor maturation of focal adhesion. Given that all these experiments highlighted the independency of NMMIIA towards migration, a protein that has been put at the forefront of S100A4-induced motility, we aimed to gather further understanding regarding the other molecular mechanisms that may be at play for motility. Using high throughput imaging (HCI), 3 compounds were identified to be capable of inhibiting S100A4-mediated migration. Although we have yet to investigate the underlying mechanism for their effects, these compounds have been shown to target membrane proteins and the externalisation of S100 proteins, for at least one of the compounds, leading us to speculate that preventing externalisation of S100A4 could potentially regulate cell motility.

Ovarian cancer is the leading cause of cancer death among all gynaecological cancers. Its aggressive nature is partly due to genetic heterogeneity and the lack of effective treatments strategies. Standard treatment involves cytoreductive surgery followed by chemotherapy using a platinum-based agent. Although initially, patients response well to this treatment, the majority will relapse and develop recurrent disease, predominantly due to the emergence of platinum resistance. Further understanding of the molecular changes which occur during ovarian ontogenesis and in the development of platinum resistance is essential to design new targeted drugs to improve patient prognosis. HOX gene are a family of homeodomain-containing transcription factors that determine cell and tissue identity in the early embryo and are found to be aberrantly expressed in cancer. HOX gene expression in ovarian cancer of different histological subtypes and in primary ovarian tumours were evaluated here. This is the first comprehensive study of HOX gene expression in a cohort of primary ovarian tumours, including statistical analysis of HOX gene expression profiles along with clinic-pathological data of each patient. HOX genes were found to be profoundly dysregulated in ovarian cancer cell lines and primary ovarian tumours, with very little to no expression found in normal ovarian and fallopian tube tissue. A 5-HOX gene signature which predicts poor overall survival in ovarian cancer patients was identified. Platinum resistant disease displayed an overall higher level of HOX gene expression, significantly HOXB4 and HOXB9. This dysregulated HOX expression reported could therefore act as a set of targets for therapeutic intervention. The novel peptide, HXR9, has been developed to block the interaction between HOX proteins and their co-factor, PBX, and therefore subsequent target gene expression. The efficacy of HXR9 treatment of ovarian cancer cells was explored. HXR9 treatment was shown to induce cell death via apoptosis in ovarian cancer cells shown by an increase in apoptotic cells identified by flow cytometric analysis, and increase in caspase-3 activity and the upregulation of pro-apoptotic gene cFos. Enhanced cell cytotoxicity was observed when combining HXR9 with cisplatin to treat platinum resistant cells, revealing a new therapeutic option for drug resistant disease that should be explored further for potential clinical trial investigation. A limiting factor towards the development of new treatment strategies is the lack of a reliable animal model of ovarian cancer. Common methods used to test new drugs involve in vitro investigation and the use of engineered animal models. However, these models do not represent the true heterogeneity and complexity of human ovarian tumours. Therefore, the use of the chicken chorioallontoic membrane (CAM) as a model of ovarian cancer for the testing of anti-cancer drugs was assessed. Cell lines grafted onto the CAM successfully and developing into micro-tumours. Cell cultured from ascites samples of ovarian cancer patients also grafted, but with a less success rate. Morphological and tumour retardation was detected after treatment with HXR9. This demonstrated the potential of this model to be developed for future personalised drug screening.