The central hypothesis for this thesis is that antibody-targeted superparamagnetic iron oxide nanoparticles (SPIONs) can be used for diagnosis and therapy of cancer. The hypothesis is based on the knowledge that firstly, recombinant single chain Fv antibody fragments (scFv) are effective targeting reagents and second, SPIONs can substantially improve the sensitivity of magnetic resonance imaging (MRI). Furthermore, SPIONs can be induced to generate heat when subjected to an alternating magnetic field (AMF). The aim of the thesis was to test the cancer imaging and therapeutic potential scFvfunctionalised nanoparticles by: (1) Generating scFvs reactive with carcinoembryonic antigen (CEA) a cell surface tumour marker. (2) Developing conjugation methods to attach the scFv, in functional form, to SPIONs. (3) Evaluating the cellular interaction (targeting and specificity) of functionalised SPIONs and (4) Measuring the imaging and therapeutic heating effect of the targeted SPIONs. ScFvs reactive to CEA were generated in Pichia pastoris and conjugation chemistries optimised for attachment of purified scFv to SPION surface. Targeting efficacy of the scFv functionalised SPIONs was tested by ELISA, cellular uptake, confocal microscopy and MRI. Results demonstrated unequivocal CEA-specific cellular uptake and CEAspecific MRI, using SPIONs conjugated with Sm3E, a high affinity humanized anti- CEA scFv. Cellular interaction of the Sm3E-SPIONs was found to be influenced by size and surface properties; neutrally charged Sm3E functionalised dextran SPIONs localised preferentially to the outside of the cell membrane, whilst negatively charged Sm3E functionalised PEGylated SPIONs showed evidence of intracellular uptake. The SPIONs were shown to be effective generators of heat when exposed to AMF of 150V, 0.74A and 1MHz. AMF treatment of Sm3E-SPION targeted cells was found to induce expression of the stress protein HSP70 and lead to hyperthermic cell death in vitro. These results indicate that scFv-SPION conjugates have potential for selective tumour imaging and therapy.

Overexpression of the epidermal growth factor receptor (EGFR) is associated with resistance to chemotherapy and radiotherapy. EGFR involvement, in repair of radiation-induced DNA damage, is mediated by association with the catalytic subunit of DNA protein kinase (DNAPKcs). This study investigated the role of EGFR nuclear import, and its association with DNAPKcs, on DNA repair following treatment either with cisplatin or ionizing radiation (IR). EGFR- null murine NIH3T3 cells were transfected with wild type or with mutated EGFR (mutations found in human cancers L858R, EGFRvIII and mutations in the EGFR nuclear localization signal (NLS) sequence NLS123, LNLS123). Comet assay analysis, which measures unhooking of cisplatin crosslinks and repair of IR induced strand breaks, demonstrated that wtEGFR and EGFRvIII completely repair cisplatin and IR induced DNA damage. Immunoprecipitation studies show that repair is associated with the binding of both wtEGFR and EGFRvIII to DNAPKcs, which increases by 2- fold 18 hours following cisplatin treatment. Confocal analysis and proximity ligation assay indicated that this association takes place both in the cytoplasm and in the nucleus resulting in a significant increase of DNA-PK kinase activity. Intermediate levels of repair as shown by the L858R construct with impaired nuclear localization demonstrated that EGFR kinase activity is partially involved in repair but is not sufficient to determine EGFR nuclear expression. EGFR-NLS mutants showed impaired nuclear localization and impaired DNAPKcs association resulting in significant inhibition of DNA repair and downregulation of DNA-PK kinase activity. Our data suggest that EGFR nuclear localization is required for the modulation of cisplatin and IR induced DNA damage repair. The EGFR-DNAPKcs binding is triggered by cisplatin or IR and not by EGFR nuclear translocation per se. Understanding mechanisms regulating EGFR subcellular distribution in relation to DNA repair kinetics will be a critical determinant of improved molecular targeting and response to therapy.

Actin-binding proteins play well established roles in the regulation of actin dynamics and assembly of F-actin based structures involved in cell motility and adhesion. The Phosphatase and actin regulator (Phactr) family of proteins each contain four G-actin binding RPEL motifs and has been found to bind protein phosphatase 1 (PP1) via their C-terminal domain. Their function is not well established and it has been unclear whether G-actin can be their regulator. Members of the Phactr family are highly expressed in the nervous system and in some metastatic cancers. The RPEL domain was previously shown to confer Rho-regulated nuclear shuttling and activation of Serum Response Factor (SRF) coactivator myocardin – related transcription factor A (MRTF-A, also known as MAL/MKL1). MRTF-A is cytoplasmic in unstimulated cells and accumulates in the nucleus upon activation of Rho-actin signalling. In this thesis I show that activation of Rho-actin signalling by serum stimulation induces nuclear accumulation of Phactr1, but not other Phactr family members (Phactr2-4). Actin binding by the three Phactr1 C-terminal RPEL motifs is required for Phactr1 cytoplasmic localisation in resting cells. Phactr1 nuclear accumulation is Importin α−β-dependent. I also reveal that G-actin and Importin α−β bind competitively to nuclear import signals associated with the N- and C-terminal RPEL motifs in Phactr1. All four motifs are required for the inhibition of serum-induced Phactr1 nuclear accumulation by elevated G-actin. G-actin and PP1 bind competitively to the Phactr1 C-terminal region, and expression of Phactr1 C-terminal RPEL mutants that cannot bind G-actin induces actomyosin foci dependent on PP1 binding. In CHL-1 metastatic melanoma cells, Phactr1 exhibits actin-regulated subcellular localisation and is required for stress fibre assembly, motility, and invasiveness. These data support a role for Phactr1 in actomyosin assembly and suggest that Phactr1 G-actin sensing allows its coordination with F-actin availability.

The self renewal of haematopoietic stem cells is a complex, tightly regulated process. One of the key players in this mechanism BMI1, which belongs to a family of proteins called the polycomb group (PcG) proteins. These form two distinct complexes, that act as chromatin modifiers to regulate gene expression. In mammals, there are several paralogues of BMI1, however their role in haematopoiesis is unclear. The expression of a panel of PcG genes was assessed in umbilical cord blood HSCs and progenitors. Several PcG genes were found to be more highly expressed in the HSCenriched CD34+ CD38- population compared to the progenitor subpopulations of cord blood cells. PCGF2, which shares high homology with BMI1 but has been reported to have a contrasting function, was chosen for further investigation. The effects of knockdown of PCGF2 were compared to those of BMI1 knockdown, using lentiviral delivery of shRNA in cord blood cells. Loss of either BMI1 or PCGF2 resulted in reduced colony formation and a reduction in the frequency of longterm culture initiating cells (LTC-IC). In primary and secondary transplantation in NSG mice, human cells with down regulated PCGF2 had reduced engraftment capacity compared to control cells. As PCGF2, like BMI1, appeared to be required by human HSCs, the question was raised as to whether the two act via the same or independent pathways. This was addressed in two ways. Global gene expression profiling of transduced cells demonstrated that different pathways are altered upon knockdown of either BMI1 or PCGF2. These results indicate that these two proteins play complementary roles. However it seems that one can also substitute for the other as overexpression of PCGF2 rescued the phenotype of BMI1 knockdown in vivo although not in vitro. It is likely that PCGF2 shares a role with BMI1 but also has unique functions. The individual contributions of the PcG proteins are likely to be complex and inter-related and warrant further investigation.

The objective of this project is to contribute to the development of therapeutic ultrasound as a surgical technique for the "non-invasive" treatment of discrete liver tumours. The design, focusing and calibration of high intensity sound sources is discussed, and equipment used to treat specimens ranging in weight from a few grams up to several hundred kilograms, is described. The dependence of damage (lesion) dimensions in excised liver samples (at an ambient temperature of 37°C) on the ultrasonic free-field spatial peak intensity (between 1350 and 5400 Wcm⁻²) and the depth of the focal plane beneath the tissue surface (between 10 and 30 mm) was investigated over a range of exposure times (1<t<40 seconds). Thus, the reproducibility and predictability of the position, shape and size of ultrasonically induced lesions could be assessed. In addition, a simple theoretical model of thermally induced ultrasound damage was developed to predict the effects of ultrasonic parameters and tissue properties, such as thermal diffusivity and blood perfusion, on lesion diameter. The validity of the model was tested extensively, in unperfused porcine liver. Other experiments were performed in vivo to investigate both a host's response to ultrasonic liver damage, and the feasibility of placing lesions side by side (in arrays), in an attempt to destroy all the cells within a volume much larger than the focal region. After preliminary studies in excised tissue, both normal and tumour bearing livers were exposed. The clear visual and histological differences between treated and normal liver parenchyma, and between tumour cells before and after treatment are also reported. The possibility of imaging ultrasound lesions was investigated in excised liver using diagnostic ultrasound, and in vivo by obtaining magnetic resonance images of both liver and tumour, before and, at various times up to 8 weeks, after ultrasound exposure. Finally, the potential to produce localized ultrasound damage non-invasive1y in vivo, using pulse-echo ultrasound imaging to plan a treatment, was explored, and although extensive dosimetric studies are required before liver tumours can be treated clinically, much of the necessary physics of this high intensity focused ultrasound technique has been completed.

Neuroblastoma is a cancer of the sympathetic nervous system derived from neural crest cells that fail to differentiate during development. Neuroblastoma tumours and cell lines are heterogeneous, comprised of ‘neuroblastic’ N-type cells, precursors to a neuronal neural crest cell lineage and ‘substrate-adherent’ S-type cells, precursors to a non-neuronal neural crest cell lineage. Retinoids, such as retinoic acid (RA), cause both N- and S-type cells to switch from proliferation to differentiation. This underlies the use of RA in the treatment of neuroblastoma disease. The aim of this study was to investigate the role of Ca2+ signalling in the process of differentiation. N- and S-type cell populations were enriched from the SH-SY5Y neuroblastoma cell line to allow characterisation of Ca2+ signalling and differentiation within the two cell phenotypes. The RA-induced switch from proliferation to differentiation was accompanied by a down-regulation in store-operated Ca2+ entry (SOCE) in N-type cells but not in S-type cells. In N-type cells expression of the ER Ca2+ sensor protein STIM1 and the channel protein Orai1 also became down-regulated, whilst expression of the channel protein TRPC1 became up-regulated. Knockdown of STIM1 and Orai1 in proliferating N-type cells down-regulated SOCE. Knockdown of Orai1, but not STIM1, induced differentiation and also enhanced differentiation induced by RA. Overexpression of STIM1 and Orai1 in RA-differentiated cells restored SOCE and reduced the extent of differentiation. Knockdown of TRPC1 had no effect on SOCE or differentiation in proliferating N-type cells but reduced the extent of SOCE down-regulation and differentiation induced by RA. These observations suggest that Orai1 may be a negative regulator of differentiation in N-type cells whereas STIM1 down-regulation may be required to maintain the differentiated state. TRPC1 expression may be required for a fully functional differentiated phenotype. These proteins could represent putative drug targets in the multi-modal treatment of neuroblastoma disease.

The aim of modern cancer chemotherapy is to develop targeted drugs designed to exploit pharmacological differences between tumour cells and healthy tissues. One focus of this effort has been the identification of protein kinases that are expressed at elevated levels or in mutated forms, indicating a reliance of the tumour on specific kinase function. Nek2 is a human serine/threonine protein kinase related to the fungal protein NIMA, a critical mediator of mitosis. Interestingly, Nek2 is found to be upregulated in a variety of tumour cell lines derived from breast, cervical and prostate carcinomas, as well as lymphomas. Human Nek2 is implicated in the regulation of the centrosome and formation of a bipolar spindle, a framework that is vital for correct separation of sister chromatids during mitosis. It is proposed that Nek2 may complex with, and phosphorylate, proteins accumulated at the centrosome, possibly playing a role in intercentriolar linker cleavage during the centrosome cycle. Abnormalities in centrosome number and function are common in many cancers, indicating that loss of centrosome cycle regulation may be a major contributing factor in tumour progression. Overexpression of Nek2 may result in premature centrosome disjunction, and deregulation of this tightly controlled mitotic machinery leads to chromatid segregation errors, aneuploidy and chromosomal instability, common genetic abnormalities observed in tumour cells. This indicates a role for abnormal Nek2 function in tumourigenesis, and Nek2 depletion in a number of tumour cell lines has been shown to cause growth suppression and apoptosis. Nek2 is thus a potentially attractive cancer therapeutic target for small-molecule kinase inhibitors. Previous studies identified substituted purine derivatives as modest inhibitors of Nek2, leading to the discovery of two distinct inhibitor classes, exhibiting ATP-competitive and irreversible inhibition of the kinase, respectively. Purine-based compounds bearing substituents at the 8-position have emerged as modest competitive inhibitors of Nek2 that occupy the kinase ATP-domain through an unusual binding orientation (45; IC = 51.8 M). Additional possible interactions within the ATP- 50 binding site available to inhibitors of this class were explored, with the objective of developing tight-binding type II reversible inhibitors of Nek2. Structure-activity relationship studies resulted in a 10-fold improvement in activity over the initial hit compounds and a substantial improvement in drug-like properties (e.g. 129; IC = 5.1 M)). However, all 50 efforts to improve the potency of this series were unsuccessful. 6-Ethynylpurines have been identified as irreversible inhibitors of Nek2 through covalent modification of an active-site cysteine residue. The initial hit compound (147; IC = 0.14 50 M) was found to be a potent and selective inhibitor of the kinase in vitro, but with poor cellular activity attributed to limited permeability. Extensive structural modification of the 2- arylamino side-chain of this series afforded cell permeable analogues with improved potency, both in vitro and in vivo (e.g. 177; IC = 0.062 ± 0.01 M). 50 Biochemical studies using 177 suggested that inhibition of Nek2 resulted in an increase in mitotic abnormalities and a delay in mitotic progression, despite poor cellular growth inhibition being observed in initial tumour cell lines. Further cellular growth inhibition and cytotoxicity studies with selected compounds identified several sensitive tumour cell lines. However, kinase-inactive control compounds essentially devoid of Nek-inhibitory activity (e.g. 425; IC > 100 M) retained growth-inhibitory activity, indicating an alternative locus 50 of activity for the 6-ethynylpurine chemotype.

This study was designed to investigate psychological adjustment to breast cancer in relation to social support, and family cohesion and adaptability. A sample of forty one women, admitted to hospital with breast cancer for surgery, were given an assessment package six to eight weeks after hospital discharge. The package consisted of the Mental Adjustment to Cancer Scale (MAC), the Family Adaptability and Cohesion Evaluation Scales(FACES) and the Michigan Social Support Scale for breast cancer patients. An identical package was posted to the patients after six months. Three models were tested corresponding to different levels of consistency with a causal interpretation of a relationship between social support and psychological adjustment. The results indicated that psychological morbidity was high at both ti me points. Social support from a doctor, nurse specialist, friend, and spouse were each found to be correlated with at least one psychological adjustment sub- scale at time one. The strongest relationship emerged for social support from the nurse specialist and the ''fighting spirit&quot; sub- scale of the MAC. None of the family scales were found to be related to psychological adjustment or social support. Discriminant function analysis was performed to investigate variables which discriminated caseness at time one and time two. Social support from a doctor emerged as the most significant variable discriminating cases from non-cases at time one . At time two negative support was the most significant variable. The results were discussed in relation to previous research and a service development emerging from the study was described.

Epithelial Misplacement (EM) is a benign phenomenon that occurs within polyps most commonly associated with the sigmoid colon. It is brought about because of the colons convulsive nature and this forces a polyps surface epithelium into its submucosa and also causes bleeding. This is problematic as the Bowel Cancer Screening Programme (BCSP) uses positive Faecal Occult Blood (FOB) test results to identify patients that require pathological review. As EM polyps bleed, they get selected for assessment and this results in them being sectioned and stained. In these cross sections, submucosal glandular tissue will be found that looks like it has formed due to metastatic mechanisms. This can lead to ambiguous diagnoses that will cause some patients to undergo unnecessary surgery. It is postulated that this can be prevented if the continuity of the EM samples could be measured. This is because only in the EM cases will the submucosal epithelial tissue remain in continuity with the surface. To test this, volumes representative of 9 samples of cancer and 13 cases of EM were segmented and their number of 26 three dimensional (3D) connected components were recorded. These were used with the 99% confidence limits of the two tailed Mann Whitney U Statistic and tested the null hypothesis that the cancer cases were as connected as the EM samples. In this instance, no significant differences were found and so the benefit of measuring the connectivity of these pathologies is questionable. It was because of this that Immunohistochemical (IHC) alternatives were considered. It was found that Collagen IV antibody staining correctly differentiated nine samples of EM from ten cases of cancer. The Mann Whitney U Statistic found this to be highly significant, p < 0.001, and future investigations should concentrate on automating this analysis. Although, Collagen IV provided a good classification it relied upon the subjective assessment of a pathologist. Therefore, the use of epithelial specific IR spectra was also investigated and this enabled the eleven EM and nine cancer cases that were investigated to be accurately classified 80% of the time upon cross validation. The collection of epithelial specific spectra relied upon a novel digital staining technique that has much application within future research. This study demonstrates that the intermodal registration of complementary modalities is of benefit to the disease classification problem. This technique has potential to be used in the correct identification of EM but more work is required.

Evidence exists to suggest that diets high in omega-3 fatty acids may be protective against the development of colorectal cancer with in vitro and pre-clinical models. Additionally, patients with a sporadic or genetic (eg familial adenomatous polyposis (FAP)) predisposition to developing colorectal adenomatous polyps exhibit altered cell kinetics in their colorectal mucosa as compared to normal subjects. The mam body of this thesis encompasses two clinical, randomised, placebo controlled trials. The first, EPA/POL/02 assessed whether subjects with a history of sporadic colorectal adenomata had any beneficial modulation of their colorectal mucosal cytokinetics following six months treatment of eicosapentaenoic acid (EPA) in free-fatty acid form (EPA-FFA), at doses of 19/day or 2g/day, when compared to placebo. One hundred and fifty two subjects were randomised in a 1: 1: 1 ratio. At 6 months, cell proliferation was significantly reduced in the 2g/d group. There were no significant changes in apoptosis scores across the groups. There were also marked increases in the omega-3 fatty acid profiles in this group. The second study, EPA/POL/03 assessed whether 6 months treatment with 2g/day of EPA-FFA exhibited chemopreventative activity in subjects with familial adenomatous polyposis (FAP) compared to placebo. Fifty five patients with F AP and an intact rectal remnant were randomised to treatment (n=28) or placebo (n=27). Treatment was assessed by changes in number and size of polyps within an assessable area of the rectum, together with global rectal burden, as measured by video endoscopy records. Treatment with 2g/day for 6 months was associated with a significant reduction in number, size and overall rectal polyp burden. Mucosal levels of EPA were increased in the treatment arm which was safe and well tolerated. EPA-FF A therefore holds promise as a chemopreventative agent against colorectal cancer.