Oesophageal adenocarcinoma (OAC), a cancer with dismal prognosis, has been increasing rapidly in incidence over the last 30 years, nowhere more so than in the UK. Intriguingly, it is a disease predominantly among white males, but there is a paucity of data from England. In performing a range of epidemiological studies, it has been confirmed that OAC has risen five-fold in the West Midlands, UK, five times more common among men, and predominantly a disease among Caucasians. A reduced incidence of OAC was identified among subjects with prostate cancer, suggesting a protective effect of anti-androgen therapy. Examination of a general practice database revealed a negative association with aspirin, non-steroidal anti-inflammatories and statins with OAC, and a positive association with inhaled steroids, increasing number of drugs with a side effect of reducing the lower oesophageal sphincter, and drugs used for asthma/COPD. Finally, a region wide case-control study, confirmed the positive association seen with increasing body mass index, waist circumference, smoking and reflux symptoms, with negative associations seen with a diet high in fruit and vegetables. This work has identified potentially modifiable risk factors that may be employed to reduce the incidence of oesophageal adenocarcinoma, and better help stratify those most likely to benefit from endoscopic surveillance.

Human papillomavirus (HPV) E2 is essential for transcriptional regulation of viral oncoprotein expression and the replication and persistence of episomal HPV genomes. Episomal persistence is mediated by tethering of viral genomes to host cell chromosomes during mitosis. Previous work demonstrated that interaction of E2 with the cellular DNA helicase ChlR1 is necessary for viral genome tethering. Therefore, disruption of this interaction is a potential therapeutic target for persistent HPV infections. To investigate the use of fragment-based drug discovery in the development of novel inhibitors of the E2-ChlR1 interaction, a fragment library was screened to identify those that bind E2 and several hits were identified. Concurrently, the interaction between HPV16 E2 and ChlR1 was characterised and shown to be a direct protein-protein interaction. The binding sites within E2 and ChlR1 were mapped and this information was used to identify a mutant E2 protein unable to bind ChlR1 (E2-Y131A). E2-Y131A was functionally characterised. HPV16 genomes encoding E2 wild type and Y131A were transfected into primary human keratinocytes to study the differentiation-dependent virus life cycle. Mutant genomes failed to establish genome maintenance, providing strong evidence that the interaction between HPV16 E2 and ChlR1 is necessary for the persistence of HPV infection.

Introduction: Paediatric low grade brain tumours present diagnostic and prognostic challenges, providing a need for better non-invasive imaging characterization. The value of \(^1\)H Magnetic Resonance Spectroscopy (MRS) performed on 5 scanners in the diagnosis and prognostication of an extensive bi-centre cohort of low-grade gliomas is investigated. Methods: Single voxel MRS was performed routinely in children with brain tumours at the Birmingham Children’s Hospital and Queen’s Medical Centre. Histopathological features were semi-quantified and in vitro \(^1\)H NMR used to study pilocytic astrocytoma cell lines. Magnetic Resonance Spectroscopic Imaging (MRSI) and texture analysis of MR images were performed. Results: MRS detects differences between subgroups of low grade brain tumours in children and between tumours of the same histology. High myo-inositol and glycerophosphocholine and low phosphocholine are markers of good prognosis. Histological correlates for MRS metabolites have been identified and paediatric pilocytic astrocytoma cell lines (‘typical’, metastatic and recurrence) have been discriminated. The value of MRSI in answering clinical questions has been demonstrated. Texture analysis achieved high accuracy in the diagnosis of paediatric posterior fossa tumours. Conclusion: Advanced MR techniques have a significant role in the study of paediatric brain tumours, and promising results from MRS, MRSI and texture analysis are reported here.

Heterogeneous blood vessels are created by angiogenesis and vasculogenesis in colorectal cancer (CRC). Assessing endothelial activities had promising applications. However no marker has translated to clinical care. Hence a multifactorial assessment or ‘endotheliome’, including angiogenic activity, the ‘angiome’, was proposed. I tested that circulating cellular and plasma biomarkers determined outcome(s). Flow cytometry quantified circulating endothelial cells (CECs, displaced from blood vessels) and endothelial progenitor cells (EPCs, for vasculogenesis). Plasma markers measured by ELISA were: von Willebrand factor (vWf, for endothelial damage/turnover), soluble E-selectin (adhesion in tumour migration), vascular endothelial growth factor (VEGF) and angiogenin (for the ‘angiogenic switch’). All markers were prospectively quantified in 154 CRC participants before treatment and compared to non-cancer controls. They were tested against the tumour’s histopathology and repeated after surgery +/- adjuvant therapy. CECs and EPCs were highest in CRC and correlated to VEGF only. Angiogenin was diagnostic of CRC and vWf predicted metastasis. All markers fell after surgery but inconsistently after adjuvant treatment. Lower CD34+CD45- cells identified responders to anti-angiogenic therapy. Models incorporating CEC, EPC, angiogenin and CRC stage predicted progression within 2 years better than CRC stage alone. In summary, the endotheliome and angiome are determinants of outcomes and may aid decisions on therapeutic strategies.

Nasopharyngeal carcinoma (NPC) is endemic in Southern China and South East Asia, causally linked to Epstein-Barr virus (EBV) infection, and frequently shows dysregulation in a number of stem cell maintenance signalling pathways. This thesis has endeavoured to investigate the status of one of these pathways; the Hedgehog (HH) signalling pathway, in NPC tumours, and reveals the novel finding that EBV is able to active the HH signalling pathway through autocrine induction of the SHH ligand in the C666.1 authentic EBV-positive NPC-derived cell line and latently infected epithelial carcinoma cell lines. This study demonstrates that constitutive engagement of the HH pathway in EBV-infected epithelial cells in vitro induces the expression of a number of stemness-associated genes and imposes stem-like characteristics. Using epithelial cells expressing individual EBV latent genes, this study also investigates the viral protein responsible for HH dysregulation demonstrating that EBNA1, LMP1 and LMP2A are all capable of inducing SHH ligand and activating the HH pathway, but only LMP1 and LMP2A are able to induce expression of stemness-associated marker genes. These findings not only identify a role for dysregulated HH signalling in NPC oncogenesis but also provide a novel rationale for therapeutic intervention.

The Anaphase Promoting Complex/Cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that regulates mitotic progression through targeting substrates for degradation by the 26S proteasome. In order to assess APC/C post-translational modification status, and identify novel APC/C substrates and regulators, a comprehensive analysis of the APC/C and APC/C-interacting proteins by mass spectrometry was undertaken. RNA polymerase I was identified as an APC/C-interacting complex, and the interaction was validated by reciprocal co-immunoprecipitation, GST pull-down and immunofluorescent confocal microscopy. Both RPA194 protein levels and RNA Polymerase I transcription were shown to be dependent upon APC/C activity. Ablation of APC/C function by RNAi interference increased RPA194 protein levels, and elevated RNA polymerase I activity significantly, as quantified by 5’-Fluorouridine incorporation into nascent pre-rRNA, and the increase in absolute levels of 45S, 28S and 18S rRNA transcripts, relative to non-silencing controls. A number of other potential APC/C substrates and regulators were identified by mass spectrometry. Many of these interacting proteins contained APC/C consensus degron motifs. The APC/C was also shown to be a major substrate for acetylation; a number of APC/C subunits were identified as being acetylated in vivo. In this regard, APC3 was shown to be a substrate for both CBP and p300 acetyltransferases.

Research in the field of neuroendocrine tumors (NETs) has increased over the last decade, including studies focused on biochemical markers (biomarkers) of the disease. There is also growing interest in how NETs impact patients' quality of life (QOL). Consequently, there is a paucity of information about whether the expression of the specific disease biomarkers affects QOL as well as whether the primary tumor site impacts QOL. Using the explanatory model of health promotion and quality of life in chronic disabling conditions as the theoretical framework and data collected with the Norfolk QOL-NET instrument, this study's purpose was to fill that gap in knowledge through research questions addressing the relationship between the primary tumor site and NET patients' total QOL score as well as the effect of specific NET biomarkers on NET patients' total QOL score. Data were analyzed using descriptive statistics, one-way analysis of variance (ANOVA), regression analysis, and post hoc tests to determine significance. Results from an ANOVA showed that abnormal NET biomarkers affected total QOL (p = 0.011). In the analyses of whether the independent biomarker variables affected the dependent total QOL variable, only the result for Serotonin Normal was significant (p = 0.002). The presence of abnormal biomarker measurements also affected two of the Norfolk QOL-NET domains significantly, gastrointestinal and physical functioning (p = 0.005 and p = 0.030, respectively). By understanding the relationship between NETs and patient QOL, the potential positive social change implications are helping NET patients assess the severity of their condition, determining what affects their well-being, and using this information to help monitor their treatment/progress.

The main strategy for treating solid cancers is based on the very early diagnosis of a malignant tumor, and in general the smaller the tumor, the greater the likelihood of successful treatment. Magnetic Resonance Imaging (MRI), based on the nuclear magnetic resonance phenomenon, provides the possibility of detecting early malignant tumors with the assistance of appropriate contrast agents. Hence, researchers continue to develop novel magnetic materials to achieve this aim. Superparamagnetic nanoparticles have become the focus of these studies because their superparamagnetic, biocompatible and hydrophilic properties would be revealed after modifying the particle surface by suitable surfactants. Considerable research in this area has provided valuable insights; however, suitable magnetic materials that can fulfill all the requirements of MRI application are still under investigation. Surface modification of superparamagnetic nanoparticles towards their use as MRI contrast agents has been the topic for many researchers, but implementation into fully functional in vivo procedures still remains as a challenging task. In the present study, high quality monocrystalline iron oxide nanoparticles have been synthesised and surface-modified with carboxymethylated dextran as well as polyethylene glycol (PEG). Dextran and PEG macromolecules with low and high carboxyl contents were synthesized and grafted onto dopamine-iron oxide nanoparticles. Furthermore, the coating procedure was optimised to prevent aggregation among the nanoparticles. Dextran-coated and PEG-coated nanostructures were characterised by using X- ray Photoelectron Spectroscopy (XPS), Fourier Transformer Infrared Spectroscopy (FTIR), Transmission Electron Microscopy (TEM) and Dynamic Light Scattering (DLS). Consequently, mono-dispersed dextran coated nanoparticles were obtained with an approximate hydrodynamic diameter of 50 nm. The resulting coated nanoparticles exhibited the nanostructures with an excellent colloidal stability in physiological environment even at high salt concentration. The resistance to non-specific protein adsorption was investigated in an in vitro model. Both dextran-coated and PEG-coated nanoparticles displayed low non-specific adsorption. However, the free carboxyl groups could be activated to covalently immobilize proteins. / Thesis (M.Eng.Sc.)--School of Chemical Engineering, 2006.

A novel approach to multiclass tumor classification using Artificial Neural Networks (ANNs) was introduced in a recent paper cite{Khan2001}. The method successfully classified and diagnosed small, round blue cell tumors (SRBCTs) of childhood into four distinct categories, neuroblastoma (NB), rhabdomyosarcoma (RMS), non-Hodgkin lymphoma (NHL) and the Ewing family of tumors (EWS), using cDNA gene expression profiles of samples that included both tumor biopsy material and cell lines. We report that using an approach similar to the one reported by Yeang et al cite{Yeang2001}, i.e. multiclass classification by combining outputs of binary classifiers, we achieved equal accuracy with much fewer features. We report the performances of 3 binary classifiers (k-nearest neighbors (kNN), weighted-voting (WV), and support vector machines (SVM)) with 3 feature selection techniques (Golub's Signal to Noise (SN) ratios cite{Golub99}, Fisher scores (FSc) and Mukherjee's SVM feature selection (SVMFS))cite{Sayan98}.

Durant molts anys la citogenètica ha estat l'únic mètode disponible per a analitzar els canvis genètics en els tumors. Malgrat això, els estudis cariotípics estan sovint obstaculitzats per problemes tècnics i metodològics. L'any 1992 es va descriure una nova estratègia, que es coneix com hibridació genòmica comparada (CGH), i que identifica en un sol experiment aquelles regions que s'hagin amplificat (indicadores d'oncogens), així com regions delecionades (indicadores de gens supressors de tumors). En aquesta tesi s'han estudiat 37 mostres de tumors de la família Ewing (ET), vuit xenografts de tumors pancreàtics i set metàstasis de tumors pancreàtics originades en els ratolins. En totes les mostres s'ha aplicat la CGH. En els ET s'han detectat guanys recurrents del braç llarg del cromosoma 1 (mínima regió comuna 1q21-q22), i guanys dels cromosomes 8 i 12, així com guany de 7q i 6p2,1-pter i pèrdua de 16q. Aquestes regions podrien contenir gens importants en el desenvolupament i/o progressió dels ET. Mitjançant Southern blot s'ha detectat l'amplificació de dos gens, FLG i SPRR 3, localitzats a 1q21. A més, mitjançant una anàlisi estadística s'ha observat que els guanys de 6p estaven associats a un pitjor pronòstic. També s'ha desenvolupat una nova estratègia, la CGH amb mescles de DNAs de diferents mostres del mateix tipus tumoral. Aquesta tècnica permet detectar en un únic experiment les alteracions presents a la majoria de les mostres. En el present estudi s'ha demostrat l'eficàcia d'aquest mètode amb nombroses mostres (14-28) de sis tipus tumorals diferents, que prèviament s'havien estudiat individualment. En els tumors pancreàtics s'han detectat guanys en els cromosomes 8 (8q24),15 (15q25-q26),16, 20q i 19q, i pèrdues en els cromosomes 18 (18q21), 6 (6q21 i 6q24-qter), 13 (13q21) i 10 (10q 14-pter), en ordre decreixent de freqüència. Els estudis de pèrdues al·lèliques a 10p14-pter no han permès una delimitació més exacta de la regió afectada, ja que tots els casos amb pèrdua per CGH han mostrat pèrdues al·lèliques per a tots els loci informatius. Les regions cromosòmiques 8q24 i 15q25-qter s'han estudiat amb més detall per hibridació in situ fluorescent i Southern blot. L'estudi dels oncogens MYC a 8q24 i FES i IGF1R a 15q25-qter ha mostrat, en general, un baix nivell d'amplificació. Podria ésser que solament unes poques còpies extra d'aquests gens fossin suficients per a tenir efectes en la tumorogènesi pancreàtica o bé que fossin altres gens localitzats en aquestes regions els que realment estarien amplificats i els quals jugarien un paper important en la carcinogènesi pancreàtica. D'altra banda, s'ha observat una forta relació clonal entre les metàstasis estudiades i els tumors originaris implantats en els ratolins. Totes les metàstasis tenien les mateixes alteracions que els implants i, a més, noves alteracions que podrien contenir gens relacionats amb la progressió metastàtica del carcinoma de pàncreas. La majoria d'aquestes alteracions addicionals ja estaven presents en els subclons de l'implant, però no en proporció suficient com per a ser detectades per CGH.