text: DSpace Collection: http://hdl.handle.net/2381/381 2017-05-30T01:30:59Z 2017-05-30T01:30:59Z Biomarkers for diagnosis and prognosis of heart failure using label free LC-MSE Auluck, Janica http://hdl.handle.net/2381/39733 2017-05-06T02:54:33Z 2017-05-05T13:29:35Z Title: Biomarkers for diagnosis and prognosis of heart failure using label free LC-MSE Authors: Auluck, Janica Abstract: In the UK 900,000 people suffer from heart failure, of which 30-40% die within 1 year of diagnosis. Heart failure is a prevalent disease worldwide and is associated with high rates of morbidity and mortality. Current biomarkers suffer from poor levels of accuracy and efficacy. Therefore, accurate, reproducible, and reliable diagnostic and prognostic biomarkers are needed. In this study, we have chosen mass spectrometry based proteomics to profile patient plasma to discover diagnostic and prognostic biomarkers of heart failure. This experimental method allows simultaneous qualitative and quantitative analysis. Bioinformatic analysis of the protein profiles to detect protein changes has been undertaken to identify potential markers of disease. Upon method development, plasma protein profiles from one hundred acute heart failure patients were obtained using a Waters Synapt G2 QTOF mass spectrometer post plasma enrichment (ProteominerTM, Bio-Rad) and 2D-RP-RP fractionation. Samples were analysed using a HDLC-MSE experiment and run in triplicate. Statistical comparisons of the protein profiles were made using PLGS v2.5.2 and progenesis LC-MS to identify potential candidates for biomarkers. Using a label free 2D HDLC-MSE experiment we have found that differences in protein expression of acute heart failure patient profiles exist. Seven candidate proteins have been identified and are shown to be involved in many different physiological processes that play a role in the pathophysiology of heart failure. ELISA analysis of the seven identified markers has identified SAP as a strong predictor of adverse patient outcome in acute heart failure. Using multivariate analysis, SAP has been found to be an independent prognostic marker in acute heart failure patients. Further studies are needed to verify SAP as a biomarker in a larger patient cohort, and measure SAP alongside current prognostic markers. A mechanistic study to identify the role of SAP in heart failure pathology needs to be undertaken. 2017-05-05T13:29:35Z Translating curcumin into clinical practice for treatment of metastatic colorectal cancer: the CUFOX trial Iwuji, Chinenye Oluchi Obiageri http://hdl.handle.net/2381/39727 2017-05-04T02:26:47Z 2017-05-03T14:02:43Z Title: Translating curcumin into clinical practice for treatment of metastatic colorectal cancer: the CUFOX trial Authors: Iwuji, Chinenye Oluchi Obiageri Abstract: Palliative treatment of metastatic colorectal cancer provides an overall median survival rate of approximately 21 months, with response rates of less than 60%. Curcumin is a low molecular weight polyphenol derived from the spice turmeric that inhibits carcinogenesis in vitro and in vivo via multi-targeted mechanisms. A clinical study was established investigating the safety and feasibility of administering curcumin with standard oxaliplatin-based chemotherapy in patients with metastatic colorectal cancer and in parallel biomarker analysis was conducted to identify potential biomarkers of efficacy and toxicity. Methods: Phase I was a dose escalation phase using the traditional escalation rule (3+3+3) design to establish the maximum target dose of curcumin. Phase IIa was an open-labelled, two-armed, randomised controlled feasibility trial. Patients received standard oxaliplatin and 5-FU chemotherapy with or without the maximum target dose of curcumin established in Phase I. Biomarker studies were conducted involving measurement of miR-122, curcumin/curcuminoids and DNA platination in patient plasma samples, and proteomic analysis of treated explant media from patient-derived colorectal liver metastasis. Results: Phase I dose escalation was successfully completed with thirteen patients receiving curcumin plus standard oxaliplatin-based chemotherapy up to the target dose of 2 grams daily with no significant issues identified with toxicity or feasibility. Eighteen patients had been recruited into Phase IIa at the time of this report with no notable safety concerns. Changes in miRNA and curcumin/curcuminoid levels were successfully measured in patient plasma samples. Explant culture analysis showed proteins involved in apoptosis, angiogenesis and inflammation/immune response were selectively upregulated following treatment with CUFOX. Conclusion: Addition of curcumin to standard FOLFOX chemotherapy up to a dose of 2 grams daily has shown good tolerability, feasibility and no safety concerns across Phase I and IIa of this study. Potential biomarkers for future investigation have been identified. 2017-05-03T14:02:43Z Effect of Supplementary Omega-3 Fatty Acids on the Biochemical, Radiological and Clinical Outcome of Patients with Metastatic Oesophago-Gastric Cancer Receiving Palliative Chemotherapy Eltweri, Amar Mohamed Elhadi http://hdl.handle.net/2381/39723 2017-05-03T02:27:38Z 2017-05-02T15:39:11Z Title: Effect of Supplementary Omega-3 Fatty Acids on the Biochemical, Radiological and Clinical Outcome of Patients with Metastatic Oesophago-Gastric Cancer Receiving Palliative Chemotherapy Authors: Eltweri, Amar Mohamed Elhadi Abstract: Background: Omega-3 polyunsaturated fatty acids (PUFAs) have reported anti-cancer effects. Few studies have assessed oesophago-gastric cancer, either in vitro or in vivo. Aims: To evaluate whether addition of omega-3 PUFAs (Omegaven) to palliative chemotherapy would influence the clinical and biochemical outcome of patients with oesophago-gastric cancer, and to assess the response of oesophageal adenocarcinoma cell lines to omega-3 PUFAs compared to oxaliplatin. Methods: Participants in a phase II trial received palliative chemotherapy and Omegaven infusion. Clinical and radiological outcome were assessed. Comparison was made to historical patients who received chemotherapy alone. Serum cytokine concentrations and uptake of PUFAs in plasma and red cell membrane were assessed in trial patients. The in vitro response of cell lines to omega-3 PUFAs or oxaliplatin were evaluated. Results: Participants who received chemotherapy and Omegaven had a higher radiological response rate than those who received chemotherapy alone ((overall response: 73% (95% CI 51 to 95) vs 43% (95% CI 25 to 61), p=0.05; partial response 73% (95% CI 51 to 95) vs 39% (95% CI 21 to 57), p=0.03)). . Grade 3/4 toxicity was observed less frequently in those who received Omegaven (thromboembolism, gastrointestinal side-effects). This translated into fewer hospital admissions. There were significant reductions in the concentrations of TNF-α (p<0.0001, 95% CI -0.0121 to -0.0046) & VEGF (p=0.002, 95% CI -0.0161 to -0.0034) following each treatment. Participants with low baseline IL-6 & TNF-α expression had a superior survival. Each infusion of Omegaven resulted in a short lived increase in plasma EPA and DHA. The serial infusions of Omegaven caused a sustained increase in the EPA content of the red cell membrane. DHA, EPA and oxaliplatin had an anti-proliferative effect on the oesophageal cancer cell lines at all concentrations. Omegaven had a more concentration dependent anti-proliferative effect. Reduction in VEGF expression was the most consistently observed cytokine effect. The anti-proliferative effect was associated with reduction in anti-apoptotic protein and an increase in pro-apoptotic protein. Conclusions: Infusion of omega-3 PUFAs resulted in a more favourable chemotherapy side-effect profile and an improved radiological response rate. Treatment resulted in a reduction in serum pro-inflammatory cytokines. Repeated Omegaven infusion resulted in a gradual and sustained uptake of EPA in the red cell membrane. The anti-proliferative effect of omega-3 PUFAs on oesophageal cancer was demonstrated in vitro. 2017-05-02T15:39:11Z An Exploration into the Role of the Endocannabinoid System in Endometrial Receptivity Melford, Sarah Emily http://hdl.handle.net/2381/39713 2017-05-03T02:27:13Z 2017-05-02T11:27:27Z Title: An Exploration into the Role of the Endocannabinoid System in Endometrial Receptivity Authors: Melford, Sarah Emily Abstract: While it is clear that the control of implantation is multifactorial, one emerging component that appears to be important in the success or failure of embryo implantation is the endocannabinoid system. The primary ligand of the endocannabinoid system (ECS) is anandamide (AEA) which is synthesised by a N-acylphosphatidylethanolamine-specific phospholipase D (NAPE-PLD) and degraded by fatty acid amide hydrolase (FAAH). A careful balance in the activities of NAPE-PLD and FAAH is required to ensure the appropriate levels of AEA are available during implantation. The purpose of this thesis was to explore further the role of the ECS, specifically its role in uterine receptivity using both in-vivo and in-vitro models. In-vivo models were used to study the expression of the ECS by measuring plasma concentrations of AEA, along with OEA and PEA (two other ligands of the ECS). A statistically significant change in plasma PEA concentrations was noted when comparing urine pregnancy test results. No significant differences in either AEA or OEA plasma concentrations were demonstrated. In-vitro models were used to investigate the interactions between galectin 3, integrin β3 and the ECS. The results show that while galectin 3 did not have any effect on the ECS, up-regulation of the expression of integrin β3 in receptive endometrial cells both increased the expression of FAAH and decreased the expression on NAPE-PLD in a dose-dependent manner. However, no effect was demonstrated in non-receptive endometrial cells, suggesting that integrin β3 expression, in collaboration with the ECS, plays an important role in endometrial receptivity. Overall, this work gives us further insight into the immensely complex processes involved in ensuring the endometrium is receptive to an embryo. Most importantly, it has suggested a link between the expression of integrin β3 and the enzymes of the ECS that is absent in the non-receptive endometrium. 2017-05-02T11:27:27Z Targeting PI3K pathway to enhance cisplatin cytotoxicity in lung cancer models Liao, Wenjing http://hdl.handle.net/2381/39671 2017-04-20T02:20:31Z 2017-04-19T15:12:45Z Title: Targeting PI3K pathway to enhance cisplatin cytotoxicity in lung cancer models Authors: Liao, Wenjing Abstract: Lung cancer is a leading cause of cancer deaths and is commonly diagnosed in both males and females. The cause of lung cancer is mainly attributable to cigarette smoking and aging, accounting for more than half of all lung cancer deaths. Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer (about 80%) compared to small cell lung cancer (SCLC) and it is normally subclassified into squamous cell carcinoma, adenocarcinoma and large cell carcinoma. Cisplatin-based treatments are the main first-line regimens for any stage of NSCLC, although platinum has over a 40-year history of use in clinical practice. Recently, molecularly targeted agents have been developed for improvement of treatment outcomes in patients with advanced lung cancers, such as EGFR-and ALK-targeted drugs. However, the acquired resistance to these agents eventually impairs sensitivity to treatment. Studies have shown that PI3K/Akt signaling activation is associated with resistance to the targeted treatments, and highlights a role for targeting PI3K/Akt signaling to restore sensitivity to targeted therapies. Moreover, Akt inhibition has been shown to promote sensitivity to cisplatin treatment. In this project, both cisplatin and various agents which target Akt directly or indirectly, were tested on 2D NSCLC cell cultures for assessment of potencies. Single-agent treatment showed that the mutant P53 cell line (H596) showed the greatest sensitivity to cisplatin treatment compared to two WT P53 cell lines (A549 and H460). This was similarly observed in a patient-derived tissue explant model which showed that P53 mutant cases were more sensitive to cisplatin treatment than those WT P53 cases. In 2D adherent cell culture and 3D organotypic co-culture of cancer cells with fibroblasts, Akt activity was activated in response to cisplatin treatment, which was similarly observed in one explant case. Moreover, decreased Akt phosphorylation was significantly correlated with increased PARP cleavage in WT P53 cases only. Both PI3K- and Akt-targeted treatments were shown to be highly potent on 2D culture and 3D spheroid models compared to other cisplatin-based regimens. Further analysis of molecular biomarkers by In-Cell western assay corroborated this, with induction of caspase-3-dependent cell death significantly higher when cisplatin treatment was combined with PI3K- or Akt-targeting drugs. The increase in apoptosis was concurrently observed with decreases in pAkt levels. P53 and PTEN levels were not induced by combination treatments compared to cisplatin alone, suggesting that functional P53 might not be required for induction of cell death. 2017-04-19T15:12:45Z Molecular analysis of circulating cell-free DNA in lung cancer Trigg, Ricky Mark http://hdl.handle.net/2381/39394 2017-03-07T03:20:28Z 2017-03-06T11:24:41Z Title: Molecular analysis of circulating cell-free DNA in lung cancer Authors: Trigg, Ricky Mark Abstract: Two-thirds of patients with non-small cell lung cancer (NSCLC) are diagnosed with incurable disease, reflecting the need for early detection. However, research in this area has been hampered by the inability to identify individuals with early-stage tumours or preneoplastic lesions that may progress to malignancy. In this thesis, the Kras+/LSL-G12D mouse was used to model lung preneoplasia, permitting analysis of circulating-free DNA (cfDNA) in comparison with tumour burden. Using end-point polymerase chain reaction (PCR), tumour-derived cfDNA (ctDNA) was detected in 3/7 Kras+/LSL-G12D mice with lung preneoplasia but no evidence of malignant transformation. Moreover, total levels of cfDNA were elevated in Kras+/LSL-G12D mice (mean, 66.6 ng/mL) relative to Kras+/+ mice (mean, 2.5 ng/mL) (P = 0.006). To permit a quantitative analysis of cfDNA, digital and quantitative real-time PCR strategies were then developed to detect KrasLSL-G12D and KrasLox-G12D. Using these assays, analysis of the sensitivity of ctDNA in Kras+/LSL-G12D mice may inform its diagnostic potential in human NSCLC. In a separate project, somatic copy number alteration (SCNA) in cfDNA was investigated in 143 patients with advanced NSCLC. Analysis of SCNA in five commonly amplified genes (EGFR, FGFR1, HER2, MET and PIK3CA) by qPCR and ddPCR identified gene amplification in 18 patients. Next-generation sequencing identified mutations in 28 patients, of which only 2 patients also had evidence of SCNA. Interestingly, patients with SCNA in cfDNA had significantly poorer overall survival (OS) than patients without (HR, 1.78; 95% CI, 1.05 – 3.02; P = 0.03). This significance was improved further when SCNA and mutation data were combined; patients with one or both types of alteration had significantly poorer progression-free survival (PFS) (HR, 1.50; 95% CI, 1.01 – 2.21; P = 0.02) and OS (HR, 1.88; 95% CI, 1.24 – 2.84; P = 0.003) than patients without. This is the first time SCNA in cfDNA has demonstrated predictive potential in any solid cancer type. 2017-03-06T11:24:41Z Investigation of Endocannabinoid System Signalling Pathways and Their Regulations in Endometrial Carcinoma Ayakannu, Thangesweran http://hdl.handle.net/2381/39387 2017-02-28T03:44:58Z 2017-02-27T12:22:07Z Title: Investigation of Endocannabinoid System Signalling Pathways and Their Regulations in Endometrial Carcinoma Authors: Ayakannu, Thangesweran Abstract: In the UK, endometrial cancer (EC) is the 4th most common cancer and its incidence is rapidly rising. This study aimed to elucidate the role of the endocannabinoid system (ECS) in the pathogenesis of EC. Plasma and endometrial tissues levels of anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) quantified by UHPLC from atrophic and EC group. Using the qRT-PCR and immunohistochemistry (IHC), CB1, CB2, FAAH, NAPE-PLD, GPR55 and TRPV1, mRNAs and protein levels was evaluated, respectively. The effects of in-vitro exposure of Ishikawa cell-line to AEA, OEA, PEA and capsaicin were evaluated. Plasma and tissue AEA levels were significantly (p<0.05) higher in EC than control, as were PEA levels. Logistic regression raised the area under the ROC curve (AUC) from 0.781 for plasma AEA, 0.857 for PEA and 0.543 for OEA to a combined AUC of 0.933 for EC diagnosis. The transcript level of FAAH was 75% lower in EC and NAPE-PLD levels were more variable. Histomorphometric analysis of FAAH and NAPE-PLD staining complements the transcript data. CB1 and CB2 mRNA were significantly decreased by 90% (p< 0·0004) and 80% (p< 0·0001), respectively, compared with control and these was supported by the IHC. In the EC, GPR55 mRNA were significantly raised (p<0.0020) compared with control and its protein expressions were markedly stained in EC tissues. TRPV1 receptor transcript levels were significantly reduced (p<0.0054) in EC compared with controls and markedly decreased in EC by IHC. Cancer cell growth in-vitro was decreased by the endocannabinoids in a pseudo dose-dependent and time-dependent manner. The endocannabinoids might prevent cancer cell growth by inhibiting cell proliferation or by activating apoptosis. This idea was tested using BAX/Bcl-2 and Ki-67 expression and found to be due to decrease cell proliferation. It is evident that there is an apparent perturbation of ECS at tissue and plasma levels and this could be used as diagnostic and prognostic markers with potential therapeutic target for the prevention EC. 2017-02-27T12:22:07Z 'An Investigation of the Infant Cerebrodual Venous System in Relation to the Source of Subdural Haemorrhage in Paediatric Head Injury' Cheshire, Emma C. http://hdl.handle.net/2381/39225 2017-01-21T03:57:15Z 2017-01-20T10:37:17Z Title: 'An Investigation of the Infant Cerebrodual Venous System in Relation to the Source of Subdural Haemorrhage in Paediatric Head Injury' Authors: Cheshire, Emma C. Abstract: A common finding in babies who suffer a head injury is subdural haemorrhage (SDH). When an infant presents with SDH, alongside other signs of injury (e.g. retinal haemorrhages, encephalopathy, bone fractures and bruising), the majority of relevant medical professionals would be concerned that the baby could have been subjected to abusive head trauma (AHT). However, the events that lead to a SDH and the source of bleeding have been debated. The most widely assumed source of bleeding is the rupture of bridging veins which extend from the surface of the brain to the dural membrane. There is limited knowledge in the current literature of the infant's cerebrodural venous system. Autopsy observations at the beginning of this project have subjectively indicated that infant bridging veins are relatively small and delicate. The aim of this project was to attempt to overcome some of the practical and organisational difficulties related to research in the area of paediatric head injury, and to fill the identified gap in the current medical literature. A novel approach to the post-mortem removal of the infant calvarial bones was developed. This made it possible to document and anatomically map the locations, size and numbers of these vessels and also enhanced the ability to observe pathological features of head injury, free from post-mortem artefact. Optical clearing techniques were used alongside optical coherence tomography to image the dural membrane and within the dural venous sinuses. Infant bridging veins were found to be more numerous than previously described, and in locations which have not previously been reported. A proportion of bridging veins were also relatively small and delicate (˂ 0.3mm). These findings may be important in building a more accurate picture of the mechanisms and forces that result in SDH. The new techniques described in this thesis have expanded the scope for future autopsy-based research into AHT. 2017-01-20T10:37:17Z Oncogene driver mutations and copy number variation as markers for predicting drug resistance and disease outcome in NSCLC Jaber, Almahdi http://hdl.handle.net/2381/39168 2017-01-17T03:23:02Z 2017-01-16T16:55:56Z Title: Oncogene driver mutations and copy number variation as markers for predicting drug resistance and disease outcome in NSCLC Authors: Jaber, Almahdi Abstract: Introduction: Worldwide, lung cancer is the leading cause of death. Non-small cell lung cancer (NSCLC) patients with KRAS mutations have a poor prognosis and show drug resistance, with MCL-1 implicated in this mechanism via evasion of apoptosis. This study is aimed at evaluating the relationship between key oncogene driver mutations, MCL-1 regulation and MCL-1-induced drug resistance. Methods: We analysed 39 adenocarcinomas (ADCs), 38 squamous cell carcinomas (SCCs), two large cell lung carcinomas (LCCs) and cell lines. A PNA-LNA-based TaqMan qPCR approach was performed for eight KRAS, one BRAF, three PIK3CA and one EGFR mutations. MCL-1, PIK3CA and SOX2 were analysed for copy number variation (CNV). Immunohistochemistry (IHC) and in situ hybridization (ISH) was performed for protein and mRNA, respectively. Alamar blue assay was used to evaluate drug responses. Results: KRAS mutations were detected in 36% of ADCs and 0% of SCCs, whereas PIK3CA mutation was most common in SCC (15.7%) and found in 5% of ADCs. MCL-1, PIK3CA and SOX2 amplification were detected in 13%, 74% and 86.6% of SCCs respectively, and were detected in 2.5%, 25.9% and 13.3% of ADCs respectively. MCL-1-amplified tumours demonstrated high expression of MCL-1 mRNA compared to non-amplified (p < 0.05). Cases with high phospho-ERK and phospho-AKT also showed high expression of MCL-1 mRNA. No growth inhibition was detected in cell lines treated with 5 μM of SH-4-54 inhibitor; however, 10 μM caused 99.9% growth inhibition. Ex vivo experiments mimicking the in vivo tumour environment demonstrated that NSCLCs are sensitive to cisplatin, one of which was MCL-1amplified. Conclusion: We have shown that KRAS mutation is common in ADC, whereas PIK3CA is more common in SCC. Importantly, we have highlighted that CNV of MCL-1 may be an important driver in resistance to chemotherapy. The JAK/STAT pathway was the key regulator for MCL-1 transcription. Resistance to cisplatin was observed in the KRAS mutant cell, MCL-1-amplified cell, cells harbouring MCL-1 gain and Chr1 polysomy but not the SOX2-amplified cell. 2017-01-16T16:55:56Z Circulating nucleic acids as biomarkers of breast cancer Rghebi, Basma http://hdl.handle.net/2381/39137 2017-01-14T04:08:06Z 2017-01-13T15:23:47Z Title: Circulating nucleic acids as biomarkers of breast cancer Authors: Rghebi, Basma Abstract: Background: Breast cancer is the commonest cancer and the second cause of cancer death among women. Two circulating biomarkers microRNAs (miRNAs) and circulating cell-free tumour DNA (cf-DNA) are currently under active investigation as a liquid biopsy for their utility in screening and management of breast cancer. Aims: The aim was to profile circulating miRNAs in plasma of breast cancer and compare to healthy controls to determine whether there were any miRNAs that differentially expressed in cancer. A second aim was to compare different methods to detect PIK3CA and TP53 mutations in breast cancer tissues DNA and cf-DNA. Methods: The expression level of 384 miRNAs in pooled plasma of breast cancer was compared to healthy controls using TaqMan microRNA array, followed by validation of specific miRNAs in single cancer and control plasma, using qPCR. Multiple different methods (qPCR, HRMC, ddPCR and Sanger sequencing) were compared for detection of PIK3CA or TP53 mutations in breast cancer tissue and matched plasma (cf-DNA). Results: 49 miRNAs (12.8%) showed significant expression change between pooled breast cancer plasma and controls. 11 of these were upregulated (P<0.05) in individual cancer plasma but there was wide variation in expression in controls. Overall, the ddPCR technology had higher sensitivity in detecting PIK3CA (0.1%) or TP53 (0.01%) mutations in cell line and breast cancer tissue than that achieved with other methods and overall 18 of 26 samples had a mutation in one or other gene. Conclusions: There was expression difference of a number of miRNAs in plasma of breast cancer compared to healthy controls, which needs further validation in other cohorts. The ddPCR was able to detect PIK3CA or TP53 mutations in breast cancer tissues with higher sensitivity than the other methods tested. 2017-01-13T15:23:47Z Distinguishing heart failure with preserved ejection fraction from heart failure with reduced ejection fraction using proteomics techniques Mbasu, Richard Juma http://hdl.handle.net/2381/39013 2016-12-21T03:32:00Z 2016-12-20T11:49:40Z Title: Distinguishing heart failure with preserved ejection fraction from heart failure with reduced ejection fraction using proteomics techniques Authors: Mbasu, Richard Juma Abstract: Heart failure is the second leading cause of morbidity and mortality in the world after cancer. In the UK, over 500,000 people are living with heart failure of which 30-40% die within 1 year of diagnosis. Some biomarkers for diagnosis and prognosis of heart failure have been established. However, they suffer from poor levels of accuracy and efficacy and their roles in clinical use is poorly understood. Thus, new biomarkers are needed. In this research, mass spectrometry based proteomics was used to profile patients plasma for clinical biomarker discovery due to its ability to perform both quantitative and qualitative protein profiling on clinical samples. Ninety samples from control, heart failure with preserved ejection fraction and heart failure with reduced ejection fraction subjects were used. Plasma protein profiling was performed using an optimised UPLC-IMS-DIA-MSE label free quantitation method. Bioinformatics analysis was used to analyse the changes observed in the protein profiles to identify potential biomarkers of heart failure. A novel method, termed mixed mode matrix was used for pilot study prior to main study with lipid removal agent. Samples were analysed using Waters Synapt G2S HDMS QToF mass spectrometer in triplicate on positive mode electrospray ionisation. Statistical comparisons of protein profiles was carried out using Progenesis LC-MS prior to data mining using SPSS, RapidMiner and SIMCA 14 to identify potential biomarkers. Thirty proteins were identified as potential biomarkers and shown to be involved in various pathophysiological processes leading to heart failure. ASL which plays role in nitrogen oxide production in the epithelium was upregulated in heart failure cohort. Conversely, GPX3 which scavenges free radicals in blood preventing apoptosis and necrosis of cells was downregulated in heart failure cohort. These two proteins were proposed as potential biomarkers for heart failure with preserved ejection fraction. Future studies to validate these biomarkers with the developed targeted LC-MS based MRM assay is needed. 2016-12-20T11:49:40Z Cell-free plasma markers of breast cancer in young women and women at high risk Al Mull, Nuha Abdulwahid M. http://hdl.handle.net/2381/39012 2016-12-21T03:31:49Z 2016-12-20T11:43:06Z Title: Cell-free plasma markers of breast cancer in young women and women at high risk Authors: Al Mull, Nuha Abdulwahid M. Abstract: Breast cancer in younger women can have an aggressive course. In pre-menopausal women (<50 years in age), mammography has limited value due to higher breast density. Therefore, there is a need for early and accurate detection to improve patient outcomes. The aim of this thesis was to investigate specific microRNAs (miRNAs) and copy number variations (CNVs) in plasma cell free DNA (cfDNA) as circulating biomarkers in the blood of women who have developed breast cancer at a young age (<50 years) and compare results between breast cancer patients and healthy controls. Candidate miRNAs were selected from TaqMan array card data and quantitative realtime polymerase chain reaction (qPCR) was used to investigate miRNA profiles in patient samples and controls. For plasma cfDNA CNV was compared at key chromosome intervals/genes and results compared with matched normal lymphocytes (as a germline DNA control) using both qPCR and droplet digital PCR (ddPCR) techniques. Both markers were compared in a cohort of women from a family history clinic (97) including 53 women from either BRCA1 or BRCA2 family and 44 women with a family and/or personal history of breast cancer as well as 12 healthy controls. MiRNA profiling showed that five miRNAs (mir-26a, mir-27b, mir-130b, mir-324-3p, and mir-181a) might be useful in monitoring women at high-risk due to BRCA mutation, on follow-up after breast cancer surgery. Similarly CNV was identified in cfDNA in five genes (MYC, CDKN2A, CCND1, HER2, and DMXL2) in women who had had previous surgery for BRCA1/ BRCA2 breast cancer, but not in BRCA1/BRCA2 carriers or healthy controls. The circulating biomarkers (miRNAs and gene specific amplification) identified in the plasma may have use in monitoring women with breast cancer due to inherited BRCA mutation after surgery and adjuvant therapy. 2016-12-20T11:43:06Z Preclinical evaluation of Meriva (a formulation of curcumin) as a putative agent for chemoprevention of lung cancer Mahale, Jagdish http://hdl.handle.net/2381/38655 2016-11-22T03:24:16Z 2016-11-21T15:32:30Z Title: Preclinical evaluation of Meriva (a formulation of curcumin) as a putative agent for chemoprevention of lung cancer Authors: Mahale, Jagdish Abstract: Curcumin, a naturally occurring dietary polyphenol, has been investigated for several years for its role in chemoprevention of cancer. There are emerging evidences that curcumin may have a potential role in prevention and treatment of lung cancer. Curcumin in its natural form is poorly absorbed, extensively metabolised and rapidly excreted. Meriva, a phospholipid formulation of curcumin, promises to enhance its bioavaibility. The work described in this thesis investigates whether Meriva offers pharmacokinetic advantage over unformulated curcumin, elucidate a possible mechanism of action for curcumin and determine efficacy of Meriva in vivo using xenograft model of lung cancer. Meriva offered superior curcumin and curcumin metabolites levels in mice plasma and lungs at two different dose levels (high and low). The levels were 5-16 fold higher for Meriva as compared to unformulated curcumin. A HPLC-UV method developed and validated for simultaneous quantification of curcumin and metabolites was found to reliable and reproducible. In vitro, a 3D organotypic model was used to demonstrate important role stromal components, namely fibroblasts, play in cell invasion and metastasis. Curcumin treatment of organotypic cultures significantly inhibited tumour cell invasion. Curcumin also modulated a number of key proteins in HGF/MET signalling axis. Nude mice were administered either control diet or diet supplemented with 0.226% w/w Meriva one week after A549:MRC5 (1:5) cell inoculation. Meriva significantly inhibited tumour growth versus control (p<0.05). The expression of p53, Ki-67 and cleaved caspase-3 which were significantly altered following Meriva intervention. These results together provide evidence that Meriva at a clinical relevant dose regime may be investigated in prevention and treatment of lung cancer. 2016-11-21T15:32:30Z The role of Epithelial Mesenchymal Transition Transcription Factors on DNA Damage Response Tse, Gina Chan http://hdl.handle.net/2381/38292 2016-11-01T03:23:30Z 2016-10-31T11:49:35Z Title: The role of Epithelial Mesenchymal Transition Transcription Factors on DNA Damage Response Authors: Tse, Gina Chan Abstract: The role of epithelial mesenchymal transition (EMT) has been identified to influence many processes associated with cancer development; malignant transformation, invasion, resistance to therapeutics to name but a few. Despite this, little is known about the relationship between EMT and DNA damage response (DDR). This study aims to investigate how the EMT programs activated in different cancer cell backgrounds influence DDR. The understanding of this interrelationship will help in designing new therapies to combat forms of cancer in which EMT plays a role. Microarray data obtained in three different EMT cell models show a strong influence of EMT transcription factor (TF) expression on DDR genes. The role of DDR in the genome integrity allowed us to suggest that EMT is a determinant of genome instability. A novel link between ZEB2, cyclin D1 and DNA Ligase 1 has been identified, and we propose the expression of ZEB2 increases the concentration of unrepaired single strand breaks within cells. Further investigation reveals EMT-TFs reduce HR gene expression, suggesting that activation of EMT pathways may represent the mechanism underlying the formation of BRCAness phenotype in sporadic cancers. BRCAness predicts PARP inhibitor sensitivity; and this project identifies two cell models in which activation of EMT programs confers sensitivity to the PARP inhibitor olaparib. However, the role of EMT-TF in conferring sensitivity to PARP inhibition is not conclusive. Overall, this project has identified two potential sources of genomic instability imposed by the deficiencies in single strand and double strand break repair pathways, both enforced by EMT. This project concludes EMT does play role in DDR; and further investigation will be required to elucidate the exact mechanisms and therapeutic perspectives of these observations. 2016-10-31T11:49:35Z The endometrial-myometrial interface (emi) in the aetiopathophysiology of adenomyosis uteri Kalathy, Vijayakumar http://hdl.handle.net/2381/38256 2016-10-27T02:49:01Z 2016-10-26T14:31:46Z Title: The endometrial-myometrial interface (emi) in the aetiopathophysiology of adenomyosis uteri Authors: Kalathy, Vijayakumar Abstract: Adenomyosis is a uterine disease where ectopic, non-neoplastic endometrium is histologically observed within the myometrium. The research presented herein examines the hypothesis that uterine adenomyosis is caused by abnormal behaviour of the cells at the endometrial-myometrial interface (EMI) through the actions of nerve growth factors (NGF), their receptors, the caveolin proteins and wnt signalling pathways during estradiol (E2) or tamoxifen (TMX) stimulation. In a 3-dimensional coculture model, the invasion depth of endometrial stromal cells from affected uteri was greater than that of unaffected uteri. Furthermore, invasion depth of unaffected and affected stromal cells increased by an average of 41.3% and 64.6%, respectively in the presence of E2 and 73.3% and 73.5%, respectively in the presence of TMX, indicating an inherent predisposition of the stromal cell for myometrial invasion and the enhancing effects of both E2 and TMX. Immunohistochemical analysis of NGF expression indicated a significant 2-4 fold increase in adenomyosis with the transcript level (measured by qRT-PCR) showing decreased expression in normal myocytes (0.72 fold) in response to E2 and increased expression in both normal (1.08 fold) and adenomyotic myocytes (1.20 fold) in response to TMX. Similarly, caveolin 1 protein expression was increased in the adenomyotic group, whilst transcripts for the caveolin 1a (0.70 fold) and caveolin 1b (0.82 fold) isoforms were reduced by E2 in normal myocytes. Conversely, TMX increased caveolin 1a (1.4 fold) and caveolin 1b (1.32 fold) expression in the adenomyotic myocytes. The data for the caveolin 2 data mirrored that of caveolin 1 in that caveolin 2a and 2b protein expression showed increased expression in the adenomyotic group, whilst the transcript levels of the caveolin isoforms 2a (0.65 fold) and 2b (0.79 fold) were reduced by E2 in normal myocytes, while upregulated by TMX in adenomyosis group (1.57 and 2.00 fold, respectively). Wnt5a expression at both the transcript and protein level was decreased in adenomyosis implicating the loss of wnt5a in adenomyosis progression. Furthermore, decidualisation experiments of isolated stromal cells from normal and adenomyotic uteri suggested no difference in the timing to decidualisation, with no significant difference in cell morphology, IGFBP-1 or prolactin expression, which strongly suggests that disordered stromal differentiation is not the main causal event in the pathogenesis of adenomyosis. Overall, the results from this research supported the key hypothesis of disordered cellular function and gene expression at the uterine endometrial-myometrial interface in adenomyosis. 2016-10-26T14:31:46Z Determining mechanisms of sensitivity and resistance to HSP90 inhibition in non-small cell lung cancer Law, Edward William Preston http://hdl.handle.net/2381/38121 2016-10-05T02:20:40Z 2016-10-04T09:17:58Z Title: Determining mechanisms of sensitivity and resistance to HSP90 inhibition in non-small cell lung cancer Authors: Law, Edward William Preston Abstract: BACKGROUND: HSP90 is a molecular chaperone which supports the maturation of numerous client proteins, many of which are involved in oncogenic signaling pathways such as PI3K/AKT, MAPK and JAK/STAT. HSP90 is an appealing target for small molecule inhibitors due to its ability to simultaneously downregulate multiple oncogenic conduits. Results in the clinic have been mixed and biomarkers of response to HSP90 inhibitors for patient stratification remain obscure. RESULTS: 1. EML4-ALK is a HSP90 client which degrades in response to HSP90 inhibition. Variants of EML4-ALK exist with disparate structures and stabilities. This chapter demonstrates differential stability of EML4-ALK variants and cell line sensitivity in response to HSP90 inhibition. A RT-PCR test to identify variants from FFPE tumour tissue is developed for tissues from the CHIARA trial to link variant status and response to ganetespib. 2. Characterisation of a cell line resistant to ganetespib, and verification that high UGT1A levels prevent ganetespib from inhibiting HSP90 in a NSCLC context. 3. Array-based analysis of genome-wide copy number alterations in the GALAXY-1 trial, which examined ganetespib plus docetaxel (G-D) in the second line setting against docetaxel alone (D), following platinum-based chemotherapy in NSCLC patients. Three CNAs were associated with sensitivity to ganetespib plus docetaxel: loss in cytoband 18q23, and gains in 11q13.3 and 16q22.3. Patients with multiple CNAs may represent a group of patients who perform badly on docetaxel but respond much better with the addition of ganetespib. SUMMARY: Several putative biomarkers which are predictive of response to ganetespib are identified in this study. The relationship between EML4-ALK variant stability and ganetespib response may shed light on HSP90 client selection. Genome wide CNA analysis associated with randomized clinical trial survival data has identified putative biomarkers of efficacy to ganetespib plus docetaxel. Description: Some images have been removed from the electronic version of this thesis due to copyright restrictions. The unabridged version can be consulted, on request, an the University of Leicester library. 2016-10-04T09:17:58Z MicroRNAs are novel biomarkers for the detection of colorectal neoplasia and high risk Dukes' B cancers Aslam, Muhammad Imran http://hdl.handle.net/2381/37943 2016-08-16T02:32:00Z 2016-08-15T10:24:47Z Title: MicroRNAs are novel biomarkers for the detection of colorectal neoplasia and high risk Dukes' B cancers Authors: Aslam, Muhammad Imran Abstract: Aims: This study aimed to identify which circulating miRNAs can be used for the early detection of colorectal cancers (CRCs) and to assess the utility of tissue miRNAs combined with common gene mutations, to predict the development of metastasis in patients with Dukes' B CRCs. Methods: microRNA (miRNA) expression profiling was performed for total RNA extracted from plasma samples (colonoscopy negative controls=11, adenomas=9, carcinomas=12) and formalin-fixed paraffin embedded (FFPE) matched paired cancerous with adjacent normal tissue (n=20, 5 cases from each group of Dukes' A, Dukes' B with metastasis during 5 year follow up, Dukes' B without metastasis during 5 year follow up, and Dukes' C) using Taqman® MicroRNA Array, Megaplex™ RT and pre-amplification primers Human Pool A v.2.1 and Pool B v.2.0. Discriminatory miRNAs identified from plasma and tissue expression profiles were validated further on cohorts of plasma (n=190) and FFPE tissues (n=72). Three common gene mutations (KRAS, BRAF and PIK3CA) were analysed in DNA extracted from FFPE cancer tissue. miRNA expression analysis was applied to circulating exosomes to quantify CRC-related exosomal miRNAs. Results: Receiver operating characteristics analysis showed miR-135b was associated with an area under the curve value of 0.82 (95% CI: 0.71-0.92), with 80% sensitivity and 84% specificity for the detection of adenomas and carcinomas. miR-135b was also detectable in immunoaffinity-isolated plasma exosomes from patients with CRC. No significant differences were noted for mutation status and the development of metastasis. Expression levels of miR-135b and miR-15b were significantly associated with Dukes' B cancers tissue and the development of metastasis. Conclusions: miR-135b is a novel diagnostic and prognostic marker. Its expression levels in blood and tissue can be used for the early detection of CRCs and to predict the development of metastasis in Dukes' B cancers. 2016-08-15T10:24:47Z Maxillo-Facial Surgical Considerations in the Management of Obstructive Sleep Apnoea Islam, Shofiq http://hdl.handle.net/2381/37932 2016-08-10T02:23:02Z 2016-08-09T10:59:46Z Title: Maxillo-Facial Surgical Considerations in the Management of Obstructive Sleep Apnoea Authors: Islam, Shofiq Abstract: Obstructive sleep apnoea (OSA) represents an important public health issue affecting approximately 4% of the UK population. Maxillo-mandibular advancement (MMA) can be considered the most successful surgical procedure for the treatment of OSA. Despite the evidence of the efficacy of MMA, there have been no previously published studies from the UK in this area. This research was designed to investigate several key aspects relevant to the application of MMA in OSA. The results from these studies shed light on both the effectiveness and role of maxillofacial orthognathic procedures in the treatment of selected patients with OSA who require alternative treatment options to first-line therapies. The potential applications of technical modifications to maxillofacial procedures in order to improve outcome is also discussed. The research addresses the important consideration of optimising patient selection for MMA procedures through the utilisation of clinical tools, as well as considering important individual patient factors. In particular, these studies have demonstrated that the Malampatti airway classification does not have a predictive role in outcome following MMA, in contrast to other surgical modalities used to treat OSA. The Kushida morphometric model which incorporates cranio-facial dysmorphism, does not appear to correlate with surgical outcomes, but in spite of this, it retains a diagnostic value to maxillofacial surgeons treating OSA patients. These research findings have shown that baseline OSA severity as well as duration of continuous positive airway pressure (CPAP) use prior to MMA surgery, would appear to significantly correlate with a reduction in subjective outcome measures, however, this association was not seen with objective outcome measures. The lack of consistent correlation between these outcome variables perhaps highlights a complexity that has not been reflected in previously published surgical literature. The important surgical consideration of patient acceptance of a permanently altered facial profile was investigated, with the findings demonstrating that the majority of patients subjectively rated their postoperative facial appearance positively following MMA. This was found to be independent of overall surgical outcome. Additionally, we examined cardiovascular risk factor modification after MMA demonstrating that this surgery has a potent beneficial effect on blood pressure reduction, particularly in those with established hypertension. This body of research significantly contributes to the evidence base in support of this branch of maxillofacial surgery which is currently not widely practiced in the UK. The research demonstrates that surgery can be safely and efficaciously applied to treat selected patients with MMA procedures as an alternative treatment modality in OSA, when other first-line treatments are not tolerated or successful. Description: Thesis by Published Works. Due to copyright restrictions the published works have been removed from the electronic copy of this thesis. 2016-08-09T10:59:46Z Single Nucleotide Polymorphisms of Mannan Binding Lectin and Complications of Chronic Liver Disease Lo, Robert Su Chun http://hdl.handle.net/2381/37781 2016-06-18T03:14:05Z 2016-06-17T09:42:18Z Title: Single Nucleotide Polymorphisms of Mannan Binding Lectin and Complications of Chronic Liver Disease Authors: Lo, Robert Su Chun Abstract: Background: Mannan-binding lectin (MBL) is an innate immune system pattern recognition protein that kills a wide range of pathogenic microbes through complement activation. Infection is a major complication in patients with chronic liver disease (CLD). Infection is also thought to lead to variceal bleeds. These complications cause significant mortality and morbidity. MBL single nucleotide polymorphisms (SNPs) may result in an inability to protect against pathogens which may contribute to the development of infection including spontaneous bacterial peritonitis (SBP). Association between MBL SNPs and SBP/variceal bleed is unclear. The aim of this thesis is to comprehensively assess the association of MBL SNPs and SBP/variceal bleed, and its role as a prognostic marker in liver related death. Methods: A case control study was performed. CLD patients with a prior history of complications (SBP/variceal bleed) were compared to those without. All patients were followed up subsequently in a longitudinal study to assess MBL SNPs as a risk factor for liver related death. Lastly, an open labelled proof of concept study was performed to determine the effect of 4 weeks probiotics therapy on bacterial translocation, systemic inflammation and the severity of liver disease. Results: No significant relationships were found between MBL SNPs and baseline inflammation or bacterial translocation. MBL SNPs was not a risk factor for the occurrence of SBP/variceal bleed in both the univariate and multivariate analaysis. In a Kaplan-Meier survival anaylsis, MBL SNPs was not associated with increased liver related death. MBL SNPs was also not an independent predictor in a multivariate Cox-regression analysis. The introduction of 4 weeks of probiotics therapy did not alter the baseline inflammation, bacterial translocation and severity of liver disease significantly. Conclusion: The results of this thesis suggest that MBL SNPs do not associate with the occurence of CLD complications (SBP/variceal bleed), and that MBL SNPs is not a prognostic marker for liver related death. 2016-06-17T09:42:18Z Transcriptional and Translational control of the expression of the EMT transcription factor ZEB2 in cancer cells Alghamdi, Youssef Saeed H. http://hdl.handle.net/2381/37698 2016-06-09T02:17:00Z 2016-06-08T08:56:59Z Title: Transcriptional and Translational control of the expression of the EMT transcription factor ZEB2 in cancer cells Authors: Alghamdi, Youssef Saeed H. Abstract: Epithelial-mesenchymal transition (EMT) is a crucial process involved in the metastasis and invasion of cancer cells. EMT promotes migration, invasion and separation of individual cells from the primary cancer enabling them to access the vascular system and promoting tumour dissemination. In different types of human cancer, invading cells recapitulate elements of embryonic EMT pathways to extravasate into the blood stream and form metastases at distant sites. Several pleiotropically activated transcription factors, categorised as master regulators of EMT (EMT-TFs), acting downstream of EMT pathways have a central role in cancer. EMT-TFs include Zn finger transcription factors of SNAIL (SNAIL1 and SNAIL2) and ZEB (ZEB1 and ZEB2) families, basic helix-loop-helix (bHLH) proteins E47, TWIST1, TWIST2, a forkhead transcription factor FOXC2, and a few other relatively less studied proteins. Though ZEB family members, ZEB1 and ZEB2, are both efficient executers of EMT programs in human cancer and their roles in tumourigensis might be different. In particular, ZEB2, but not ZEB1 exhibited tumour-suppressive features in malignant melanoma and, possibly, hepatocellular carcinoma. Regulation of ZEB proteins expression occurs at different levels. The activity of three putative transcriptional enhancer controlling ZEB2 gene expression were analysed, and found that RE-4 regulatory element known to control ZEB2 expression in human embryonic stem cells (hESC) is also active in malignant melanoma cells. However, in contrast with hESC, in melanoma cells ZEB2 expression is not regulated by core pluripotency factors. Instead, in these cells, the activity of RE-4 element is affected by ZEB1- or ZEB2- induced EMT programs. By studying post-transcriptional level of ZEB regulation, a novel mechanism that limits ZEB2 protein synthesis was described in this study. A protein motif adjacent to the smad-binding domain within ZEB2 protein induces ribosome stalling and compromises translation. The activity of this motif is dependent on triplets of rare codons, Leu(UUA)-Gly(GGU)-Val(GUA). Introducing these stretches in the homologous region of ZEB1 has no effect on protein expression. By using retroviral expression of pBABE-ZEB2-mut, it was shown that these stretches might contribute to EMT. The data suggest that rare codons play a regulatory role in the context of appropriate protein structures and we speculate that pools of tRNA available for protein translation influence configuration of EMT programs in cancer cells. 2016-06-08T08:56:59Z Assessment of UVR-Induced DNA Damage and Repair in Nuclear Genome versus Mitochondrial Genome Alrumaihi, Faris Abdulrahman I. http://hdl.handle.net/2381/37614 2016-05-21T03:39:23Z 2016-05-20T09:26:50Z Title: Assessment of UVR-Induced DNA Damage and Repair in Nuclear Genome versus Mitochondrial Genome Authors: Alrumaihi, Faris Abdulrahman I. Abstract: DNA is a key molecular-target for the deleterious effects of ultraviolet radiation (UVR). Cells contain two types of DNA: nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) and UVR induces various types of damage in the both DNAs, notably CPDs and 8-oxodG. The aim of this thesis is to examine UVR induced DNA damage formation and repair in nDNA and mtDNA and to determine which is the most important genomic target with respect to cell killing in vitro using HaCaT calls as models of human skin. The cell viability data showed that UVB induces significant cell death, which increased over 48 h. SSR-exposure also showed significant levels of cell death after 24 h but with evidence of significant survival after 48 h. Alkaline modified comet assay data showed that CPDs and 8-oxodG were significantly produced in HaCaT cells exposed to UVB and SSR, with CPDs being formed in a greater yields and there being no significant repair of CPDs over 48 h post-exposure to UVB. However, HaCaT cells irradiated with SSR showed significant repair of both CPD and 8-oxodG over 48 h. QPCR data showed that UVB and SSR induced similar profiles of damage in both nDNA and mtDNA; despite the induced damage levels being higher with UVB. The data also showed that nDNA is the main target for UVR in HaCaT cells exposed to UVB and SSR. The UVB-induced QPCR-detectable DNA damage in nDNA and mtDNA was not fully repaired, with a significant level of DNA damage remaining at 48 h, however, there was significant repair of the induced-damage in nDNA post-exposure to SSR (correlating with survival/re-growth), whereas the damage to mtDNA was not fully repaired. The greater lethality of UVB is probably due to more the damage induced and poorer repair (notably of CPD) in nuclear DNA following UVB exposure. Whereas the proficient repair of SSR-induced CPD in nDNA probably dictates survival following SSR exposure – as there was still a notable level of residual damage in mtDNA post-SSR exposure. However, nDNA is the main target for UVR causing DNA damage and may lead to mutations, which increase the risk of skin cancer development. 2016-05-20T09:26:50Z A study of the distribution of DNA damage, repair and epigenetic changes following exposure to ultraviolet radiation Alhegaili, Alaa Saud http://hdl.handle.net/2381/37416 2016-04-22T02:06:44Z 2016-04-21T09:36:42Z Title: A study of the distribution of DNA damage, repair and epigenetic changes following exposure to ultraviolet radiation Authors: Alhegaili, Alaa Saud Abstract: The distribution of DNA damage and repair has been reported to occur heterogeneously. Commonly available techniques, such as comet assay, allow for the measurement of DNA damage at the global, whole-genome level; but this does not reflect important events that happen at the gene/sequence-specific level. In this study, an assay based on a combination of DNA Immunoprecipitation plus next-generation sequencing (DIP-Seq) was developed to assess the induction and repair of DNA damage induced by solar-simulated radiation (SSR) at the genome-wide, sequence-specific level. The global induction and repair of DNA damage including cyclobutane thymine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) lesions following irradiation with UVB and SSR was assessed via an enzyme-modified comet assay. The results showed that significant levels of DNA damage were induced and that these damages were repaired within 48 h post exposure to SSR. Furthermore, ELISA was used to quantify the global level of CPDs following UVR exposure. Additionally DIPqPCR, as a prelude to DIP-Seq was performed to assess the induction of CPD in response to UVB, and the results again showed a good dose response. Then the DIP-Seq was performed to assess CPD induced by SSR in isolated and cellular DNA. CPD repair assessed by DIP-seq showed gene-dependent highly variable removal of CPDs with high resolution. Also, the effect of SSR irradiation on the levels of histone modifications, H3K9 acetylation and H3K4 trimethylation in HaCaT cells, was quantified by chromatin immunoprecipitation (ChIP) followed by qPCR. Western blot analysis was used to confirm the ChIP assay results, and to assess the global level of histone modification in HaCaT cells after UVR irradiation. Overall, the findings of this project revealed a good agreement between both the global and sequence-specific measurements of SSR-induced DNA damage and its repair, and also between global and sequence-specific measurements of the epigenetic modifications assessed. 2016-04-21T09:36:42Z Proteomic Approaches for Identifying New Mechanisms of Action and Pharmacodynamic Biomarkers of Resveratrol in Colorectal Cancer Kovoor, Christina J. http://hdl.handle.net/2381/37248 2016-04-12T02:00:37Z 2016-04-11T13:44:02Z Title: Proteomic Approaches for Identifying New Mechanisms of Action and Pharmacodynamic Biomarkers of Resveratrol in Colorectal Cancer Authors: Kovoor, Christina J. Abstract: The dietary constituent resveratrol (trans-3,5,4'-trihydroxystilbene) has received a great deal of scientific attention and publicity due to its broad ranging pharmacological effects in preclinical systems, including cancer chemoprevention. Depending on the disease studied and the models used there have been a huge number of proposed 'targets' of resveratrol. Nevertheless, the key targets which may mediate its anticancer activity in humans have not yet been identified. To fully understand how resveratrol exerts its effects it is important to elucidate the direct molecular interactions and how they affect function and downstream networks in healthy and diseased cells. An affinity purification platform was developed to identify direct protein binding partners of resveratrol using resveratrol-derivatised affinity beads to identify the interacting proteins from human-derived colon adenocarcinoma (HCA7) or adenoma cells (AAC1), followed by LC-MS/MS. Resveratrol affinity beads were designed and synthesised. Proteins that were reliably pulled from biological and technical repeats and were not present in the controls were designated hits and considered for validation via Western blot prior to functional assay development to ascertain the consequence of the resveratrol-protein interaction. Following biological repeats, 980 proteins were identified by this method of which 29 proteins were validated and achieved 97% correlation between the two detection techniques. In a separate approach, plasma proteomics of ApcMin mice was conducted to compare the effect of high-fat diet with/without clinically relevant resveratrol doses. The proteins whose expression was significantly altered between the treatment groups were shortlisted and cross-referenced with the resveratrol-binders from the pull-down list to identify interactomes. In conclusion, the protein pull-down assay combined with plasma proteomics described here may help discover mechanism-related protein biomarkers that can be used to monitor the efficacy of multi-targeted cancer chemopreventive agents in chemoprevention trials and begin to shed light on the direct mechanisms of action of resveratrol. 2016-04-11T13:44:02Z Pilot study of hepatic microwave ablation using an extracorporeally perfused porcine liver model Ong, Seok Ling http://hdl.handle.net/2381/36067 2015-12-15T03:10:24Z 2015-12-14T10:45:00Z Title: Pilot study of hepatic microwave ablation using an extracorporeally perfused porcine liver model Authors: Ong, Seok Ling Abstract: Introduction: Preclinical research into many aspects of microwave hepatic ablation has been traditionally conducted in in vivo studies. These are costly, can only be performed in laboratories with animal facilities and are associated with significant ethical issues. The main aim of this project was to study microwave ablation using an extracorporeally perfused porcine liver model to confirm that it accurately mimicked an in vivo model and could be used to investigate ablative techniques. Methods: Seventeen livers harvested from domestic pigs were subjected to extracorporeal normothermic autologous haemoperfusion followed by dissection of the hepatic artery and its branches to establish an effective procedure of liver procurement for subsequent experiments. A further seventeen livers in three experimental groups (control, microwave and Pringle groups) were perfused for six hours with tissue and blood samples collected at various time points for histological, biochemical and cytokine analyses. Seven livers in the control group did not undergo any ablation. Five livers in the microwave group underwent microwave ablations at three power settings after the first hour of perfusion for a fixed duration of two minutes per ablation. Five livers in the Pringle group underwent microwave ablation at the same power settings and for the same duration as the microwave group but the ablations were carried out with temporary cessation of blood flow to the liver. Results: Dissection of the hepatic artery and its bifurcation identified all the common variants and understanding these is crucial for successful liver perfusion. The livers were perfused at flows and pressures mimicking physiological levels with biochemical and blood gas analyses indicating normal function across all three experimental groups. The volume of the lesions increased with power of ablation in both the microwave and Pringle groups (p<0.001 and p=0.003 respectively), with no significant differences in overall volume between the groups (p=0.275) but a greater transition zone volume in the microwave group (p=0.012). Apoptotic activities in the transition zone lessened towards the periphery of the ablation in both microwave and Pringle groups (p<0.001 for both groups). There were significant elevations in the levels of circulating interleukin-6 and interleukin-8 over time with or without microwave ablation (p<0.001 for both cytokines in both groups). Conclusion: An extracorporeally perfused porcine liver model has a role in the preclinical study of microwave ablation. The model can be reliably used to study the histological and physical profile of microwave lesions. It also permits the study of the heat sink effect of ablation. Overall, it has a considerable potential to reduce the number of in vivo preclinical studies required. However, the ability of this isolated organ (which has a relatively short perfusion lifespan) to study the systemic effect of a microwave ablation is limited. Further experiments involving other hepatic ablative techniques should be conducted using this model to fully establish its role as a preclinical liver ablation research tool. 2015-12-14T10:45:00Z Redox modulation of oxidatively induced DNA damage by ascorbate enhances melanoma cancer cell DNA damage formation & cell killing Najeeb, Hishyar Azo http://hdl.handle.net/2381/36021 2015-12-09T03:08:42Z 2015-12-08T14:53:13Z Title: Redox modulation of oxidatively induced DNA damage by ascorbate enhances melanoma cancer cell DNA damage formation & cell killing Authors: Najeeb, Hishyar Azo Abstract: Malignant melanoma (MM) is the 5th most common cancer in the UK and the most lethal form of skin malignancies. Its incidence has more than quadrupled and mortality more than doubled over the last three decades. With a five-year survival rate for patients with advanced melanoma of ≤ 20%, there is an obvious need for a better treatment approach. Heightened genome-instability in cancer-cells suggests a model-scenario for their selective killing via the therapeutic delivery of defined levels of further genomic damage. To interrogate/exploit this model-scenario, it was proposed to investigate intracellular ascorbate's redox-modulation of oxidatively-induced DNA damage in MM cancer-cells, to selectively enhance both DNA damage and cell-killing. Alkaline comet assay (ACA) data reveals MM-cells to have higher endogenous DNA damage levels than "normal" skin cells. This in turn correlates with MM-cells having higher intracellular ROS and lower catalase activity. ACA data also shows MM-cells to be more sensitive towards the induced-damaging effects of H2O2 than "normal" skin cells, and that ascorbate further enhances this effect in MM-cells. This effect was also noted in primary melanoma cancer cells exposed to H2O2 and other oxidants (e.g. Elesclomol). A proposed model for the enhancement of H2O2-induced oxidatively-damaged DNA by ascorbate suggests that an increased local production of hydroxyl radicals (●OH) at the DNA may lead to relatively greater increases in complex lesions (i.e. DSBs) relative to single/'isolated' lesions (i.e. SSBs). However, correlated measures of DSBs vs. SSBs reveal only a proportional increase in DSBs relative to SSBs with ascorbate. Further data shows that ascorbate enhances oxidative-induced cell death in MM-cells and that in HaCaT cells the effect was slightly protective. Together, these results suggest that ascorbate enhances DNA damage/cell-killing through modulation of oxidative stress in MM-cells. This could increase the possibility of using ascorbate plus novel oxidant therapies to treat metastatic melanomas. 2015-12-08T14:53:13Z A novel approach to enhancing the effectiveness of chemotherapy: combining curcumin with FOLFOX chemotherapy for metastatic colorectal cancer Irving, Glen Robert Bell http://hdl.handle.net/2381/35981 2015-12-03T03:09:22Z 2015-12-02T16:05:09Z Title: A novel approach to enhancing the effectiveness of chemotherapy: combining curcumin with FOLFOX chemotherapy for metastatic colorectal cancer Authors: Irving, Glen Robert Bell Abstract: FOLFOX (5FU/leucovorin/oxaliplatin) is standard first-line therapy for metastatic colorectal cancer (mCRC), with response rates of 50%. Factors limiting the use of oxaliplatin include chemo-resistance and severe dose-related peripheral neuropathy. New interventional strategies are required which can improve disease outcomes by increasing drug efficacy whilst avoiding toxicity. Curcumin is a constituent of turmeric. Mechanisms of chemopreventive action, extensively investigated in vitro and in vivo, provide compelling evidence that curcumin exhibits efficacy in preventing neoplastic conditions and can act synergistically with chemotherapy. Laboratory endpoints must be tested clinically. In preparation for a clinical trial combining curcumin with FOLFOX, 3 lines of investigation have been followed. In vitro analysis of CRC cell lines treated with the investigative agents has explored mechanisms of chemo-resistance and the efficacy of curcumin therein. The acceptability of oral curcumin to patients has been assessed, supported by sensitive Ultra Performance Liquid Chromatography (UPLC) evaluation of curcumin and its metabolites in biomatrices. Parallel methods of biomarker profiling of plasma and urine from patients with CRC by NMR, LC-MS/MS and HD-MS have been tested for their suitability as adjuncts to trials studying mCRC. The resultant clinical method, guided by these components, devised for the assessment of curcumin and FOLFOX has been presented alongside early trial data. In vitro, curcumin was observed to overcome oxaliplatin-resistance and increase efficacy. Mechanisms of action have been proposed and sub-populations of stem-like cells suggested as potential treatment targets. Safety and tolerability of a suitable curcumin regimen have been shown clinically. Sensitive analysis of plasma, urine and tissue curcuminoids has been achieved, and prolonged mucosal exposure revealed. The proteomic and metabonomic profiles of patients with CRC are distinct from controls and could feasibly be employed as surrogate endpoints to trials. A dose-escalation study has endorsed an acceptable regimen for an on-going randomised trial investigating curcumin with FOLFOX ("CUFOX"). 2015-12-02T16:05:09Z Pre-clinical development of oxidative DNA adducts as biomarkers for the chemoprevention of prostate cancer. Pathak, Sanjeev. http://hdl.handle.net/2381/35622 2015-11-20T04:27:58Z 2015-11-19T09:15:08Z Title: Pre-clinical development of oxidative DNA adducts as biomarkers for the chemoprevention of prostate cancer. Authors: Pathak, Sanjeev. Abstract: Reactive oxygen species (ROS) are mechanistically implicated in the development and progression of prostate cancer. Intracellular ROS may cause oxidative DNA damage, resulting in the formation of adducts, such as 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N-2 malondialdehyde-2'-deoxyguanosine (M1dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. It has previously been shown in vitro that testosterone and dihydrotestosterone (DHT) increase ROS levels in the androgen-sensitive, human prostate cancer cell line, LNCaP. We tested two hypotheses using human prostate cancer cells grown in vitro and in a xenograft nude mouse model. 1) Levels of oxidative DNA adducts increase in LNCaP cells treated with DHT. 2) Flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. The levels of M1dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography-tandem mass spectrometry, respectively. M1dG and 8-oxo-dG adduct levels were significantly higher than control levels in LNCaP cells exposed to physiological levels of DHT. This effect was also concentration-dependent. Pre-treatment with flutamide completely prevented increases in oxidative DNA adducts. In the athymic nude mouse LNCaP xenograft, tumour levels of M1dG were decreased by 46% in flutamide-treated animals compared with controls. Flutamide also inhibited the growth of LNCaP tumours and this correlated with a reduction in serum Prostate Specific Antigen (PSA) levels. The changes in oxidative DNA adduct levels in vitro and in vivo suggest that oxidative DNA adducts may serve as potential biomarkers of the efficacy of androgen manipulation in chemoprevention trials. Furthermore, a secondary finding of the study suggests that flutamide may be considered as a chemopreventive agent for prostate cancer. These adducts may serve as biomarkers of such a trial. 2015-11-19T09:15:08Z The use of radiosensitising agents in the treatment of muscle-invasive bladder cancer. Colquhoun, Alexandra J. http://hdl.handle.net/2381/35621 2015-11-20T04:27:55Z 2015-11-19T09:15:07Z Title: The use of radiosensitising agents in the treatment of muscle-invasive bladder cancer. Authors: Colquhoun, Alexandra J. Abstract: Radical radiotherapy is the principal bladder-preserving treatment for muscle-invasive bladder cancer. 70% of muscle-invasive bladder cancers express epidermal growth factor receptor (EGFR), which is associated with poor prognosis. Recent evidence suggests that ionising radiation (IR) stimulates EGFR causing activation of cytoprotective signalling cascades, thereby mediating radioresistance. Tumour tissue from 110 patients who received radical radiotherapy for muscle-invasive bladder cancer was immunohistochemically stained for EGFR. Multivariate analysis assessed the prognostic utility of tumour EGFR status in predicting outcome following radical radiotherapy. The effect of IR on EGFR activation, and also on signalling pathways downstream of EGFR was assessed using Western blotting. Potential inhibition of radiation-induced activation of EGFR was assessed using the two small molecule tyrosine kinase inhibitors (TKI) Iressa and Tarceva. Finally we assessed the activity of TKI as a single agent treatment and in combination with IR, in vitro and in vivo. 72% of tumours stained positively for EGFR and 69% of these exhibited positive response to radiotherapy. Positive response to radiotherapy correlated significantly with negative EGFR status (X2 test; p=0.05). Response to radiotherapy at 3-months, local recurrence, metastatic spread and presence of ureteric obstruction were independent prognostic factors for diminished bladder cancer-specific survival. IR activated EGFR and Iressa partially inhibited this activation. Radiation-induced activation of EGFR activated MAPK and Akt but not STATl. Activation of the MAPK but not Akt was partially inhibited by Iressa. Bladder cancer cells treated with combined TKI and IR exhibited significantly greater growth inhibition in comparison to treatment with either IR or TKI alone both in vitro and in vivo (p=0.001-0.04). IR activates EGFR in bladder cancer cells and this activation is partially inhibited by TKI. Treatment of bladder cancer cells using combined TKI and IR results in significantly greater growth inhibition both in vitro and in vivo. Given the high frequency of EGFR expression by bladder tumours and its importance in local tumour control there is now justification to translate this work into a clinical trial. 2015-11-19T09:15:07Z Investigation into the hepatotoxicity of the novel anti-cancer drug ecteinascidin-743 (ET-743). Donald, Sarah. http://hdl.handle.net/2381/35620 2015-11-20T04:27:53Z 2015-11-19T09:15:06Z Title: Investigation into the hepatotoxicity of the novel anti-cancer drug ecteinascidin-743 (ET-743). Authors: Donald, Sarah. Abstract: Ecteinascidin-743 (ET-743) is a novel marine-derived anti-cancer drug with demonstrated anti-tumour activity in clinical trials against sarcoma, breast and ovarian carcinoma. Reversible transaminitis and subclinical cholangitis have frequently been described in patients who receive ET-743. To facilitate understanding of this adverse effect and help design suitable therapeutic rescue strategies, the hepatic effects of ET-743, administered as a single i.v. dose of 40 ?.g/kg, were characterised in female Wistar rats by histopathology, electron microscopy, hepatic and plasma biochemistry and DNA microarray analysis. Plasma levels of bilirubin were elevated up to 7-fold over those in untreated rats and activities of alkaline phosphatase and aspartate aminotransferase in plasma were slightly elevated. Livers displayed degeneration and necrosis of bile duct epithelia associated with mild inflammation followed by fibrosis. DNA microarray analysis of livers from ET-743-treated rats showed a significant increase in the expression of ATP binding cassette transport genes abcbla and abcblb and cell cycle genes cdc2a and ccndl. The cell cycle gene expression changes mirrored ET-743- induced increases in liver weight and Ki-67 labelling of liver nuclei. The results suggest that the toxicity exerted by ET-743 in the rat liver is a consequence of biliary damage and is accompanied by a wave of mitogenic activity. Pre-treatment with dexamethasone (10 mg/kg p.o.) 24 hours prior to ET-743 abrogated the biochemical and histopathological manifestations of ET-743-induced liver changes without interfering with the anti-tumour activity. Dexamethasone pre-treatment decreased hepatic levels of ET-743 dramatically compared to those obtained after administration of ET-743 alone. Pre-treatment with dexamethasone effectively protected against ET-743-mediated hepatic damage by decreasing hepatic exposure to ET-743, probably linked to induction of certain cytochrome P450 enzymes. Pre-treatment with cytochrome P450 inducers ?-naphthoflavone, phenobarbitone or indole-3-carbinol also reduced ET-743-mediated hepatotoxicity. In conclusion, the results strongly advocate the clinical evaluation of pretreatment with dexamethasone in patients who receive ET-743 to ameliorate its unwanted effects in the liver. 2015-11-19T09:15:06Z Identification of potential mechanisms of action of 3ʹ,4ʹ,5ʹ-trimethoxyflavonol in the inhibition of prostate cancer Hill, Cordella Uleta Fiona Kelly http://hdl.handle.net/2381/33357 2015-10-23T02:01:11Z 2015-10-22T14:36:41Z Title: Identification of potential mechanisms of action of 3ʹ,4ʹ,5ʹ-trimethoxyflavonol in the inhibition of prostate cancer Authors: Hill, Cordella Uleta Fiona Kelly Abstract: 3ʹ,4ʹ,5ʹ-Trimethoxyflavonol (TMFol), a synthetic analogue of the naturally occurring flavonols quercetin and fisetin, has demonstrated putative anti-cancer activity in the prostate. The mechanisms of action that are engaged are largely unknown. Therefore, the work presented in this thesis investigated the mechanisms used by TMFol to compromise cell proliferation in prostate cell lines 22Rv1, PC-3 and PNT2. Furthermore, it investigated the effect of TMFol on prostate cancer development and progression in two separate transgenic models, PBCre4p53floxRbflox and PBCre4Ptenflox mice. Apoptosis and cell cycle distribution were analysed by flow cytometry, while biochemical assays, Western blots, histological evaluations or qPCR were used to analyse senescence, changes in prostate metabolism and the effect of TMFol on mice. TMFol inhibited cell proliferation in vitro without inducing apoptosis. However the reduction in tumour growth in 22Rv1 xenografts, that were fed a 0.2 % TMFol diet, was associated with a significant increase in cleaved caspase-3 staining, an indicator of apoptosis. There was S phase arrest in the PC-3 and PNT2 cells, while there was an increase in senescence-associated β-galactosidase activity in both 22Rv1 and PC-3 cells. TMFol also caused a significant increase in the protein expression of p21 and p53 in 22Rv1 cells. TMFol significantly inhibited the expression of acetyl-CoA carboxylase at the protein and mRNA levels and the expression and activity of mitochondrial aconitase, which could result in the inhibition of fatty acid synthesis and citrate oxidation, respectively. PBCre4p53floxRbflox and PBCre4Ptenflox mice fed a 0.2 % TMFol diet did not display any evidence of tumours in the prostate, but there was no significant difference in the PIN development between the mice on the control diet and those on the TMFol diet. Taken together, TMFol has the potential to induce cell cycle arrest, apoptosis and senescence and to inhibit fatty acid synthesis and citrate oxidation in prostate cancer cells. 2015-10-22T14:36:41Z Investigating the effects of curcumin and resveratrol on pancreatic cancer stem cells Karim, Karzan Khowaraham http://hdl.handle.net/2381/33155 2015-10-02T02:01:14Z 2015-10-01T14:35:59Z Title: Investigating the effects of curcumin and resveratrol on pancreatic cancer stem cells Authors: Karim, Karzan Khowaraham Abstract: Anti-proliferative and cancer stem-cell targeting abilities of curcumin and resveratrol individually have been shown in different cancers. This project aimed to assess the activity of these compounds, alone and in combination in pancreatic cancer cell lines (PCCLs) and stellate cells. Anti-proliferation assays were performed for curcumin and resveratrol alone and in combination, combined with end point markers of activity including apoptosis and cell cycle arrest. Pancreatic cancer stem cell populations were defined using the cell surface markers CD44, CD24, ESA, CD133, ALDH-1 activity or sphere forming ability, and finally Nanog expression was assessed. The intracellular uptake of curcumin and its metabolites was analysed by HPLC. The PCCLs were more sensitive to curcumin than resveratrol, and combinations of these compounds showed anti-proliferative efficacy through apoptosis and cell cycle arrest at low, clinically achievable concentrations (CACs) in 2 out of 4 cell lines. Capan-1 cells exhibited the highest sensitivity to curcumin, which was able to enhance the effectiveness of resveratrol treatments in targeting cancer stem-like populations. Spheroid growth was significantly inhibited by curcumin and resveratrol combinations in Capan-1 cells, correlating with decreased ALDH1 activity and Nanog expression. In human pancreatic cancer tissue, various stem-like populations were identified based on expression of ALDH1 or CD24+/CD44+, which may provide a suitable target in vivo. Capan-1 cells metabolised curcumin to detectable amounts of curcumin glucuronide. However, curcumin metabolites did not show any significant activity at CACs. Curcumin alone may have activity against pancreatic cancer stem cells, and enhances efficacy at low concentrations when in combination with resveratrol. Capan-1 cells are able to internalise curcumin, and this cell line exhibited the greatest sensitivity to treatment. Overall, the results suggest that curcumin and resveratrol warrant further investigation as combination therapies for targeting cancer stem-like cells and stellate cells responsible for the dense stroma observed in pancreatic cancer. 2015-10-01T14:35:59Z The role of endocannabinoids and their receptors on the regulation of bladder function and detrusor overactivity Bakali, Evangelia http://hdl.handle.net/2381/33068 2015-09-22T02:01:01Z 2015-09-21T12:31:16Z Title: The role of endocannabinoids and their receptors on the regulation of bladder function and detrusor overactivity Authors: Bakali, Evangelia Abstract: The endocannabinoid system (ECS) has been identified in the urinary bladder of humans and rats and is purported to play a modulatory role in detrusor overactivity (DO). Clinical studies demonstrated that cannabis improves urgency in patients with neurogenic DO. However, how endocannabinoids are regulated in vivo as well as the pathophysiological relevance of deregulation of the system in the aetiopathogenesis of DO remain unexplored. This thesis explores the clinical relevance of the ECS in the aetiopathogenesis of DO, by evaluating the effects of cannabinoid agonists on bladder contractions in vitro and in vivo. Cannabinoid receptor (CBR) expression was localised in human and rat bladders. In vitro, ACEA (CB1 agonist) had both pre- and postsynaptic effects on bladder contraction, while GP1a (CB2 agonist) only had postsynaptic effect. CB1 receptor was co-localised with ChAT (choline acetyltransferase enzyme) in human detrusor but not in the urothelium, while CB2 co-localised with ChAT in both urothelium and detrusor. qRT-PCR showed that CB1 receptor was significantly upregulated by 2.8-fold in the urothelium of DO patients and downregulated by 3.2-fold in the detrusor of DO patients compared to normal. Rat cystometry experiments demonstrated CP55,940 (CBR non-selective agonist) significantly increased micturition interval (MI) and bladder capacity (BC) by 52±5.5% and induced a 25 ± 2.9% decrease in maximal pressure (MP). Addition of CP55,940 to rats with induced bladder irritation, showed an increase in MI by 78% ± 16.7% and a 22% ± 5.8% reduction in MP. In conclusion, cannabinoid receptors are involved in normal micturition at both peripheral and CNS sites, data supported by in vitro studies where CB1 reduced neuronal activity and where both cannabinoid receptors modulated bladder contractility. CB1 receptors expressed in afferent fibre endings in the urothelium could influence bladder function. CB1 receptor agonists may be useful for future treatment of DO as there is altered CB1 receptor expression in these bladders. 2015-09-21T12:31:16Z Molecular analysis of circulating cell-free DNA in breast cancer patients Blighe, Kevin Anthony http://hdl.handle.net/2381/32369 2015-06-12T02:00:24Z 2015-06-11T09:00:48Z Title: Molecular analysis of circulating cell-free DNA in breast cancer patients Authors: Blighe, Kevin Anthony Abstract: Identifying disease-related variants is a primary aim of human genetics. In breast cancer, loss of heterozygosity at specific loci was previously demonstrated in paired tumour and circulating plasma cell-free DNA (cfDNA) samples. However, alterations unique to cfDNA were also found in all cases, suggesting disease progression. These results prompted the characterisation of the circulating breast cancer genome in more detail in this thesis, to test the hypothesis that cfDNA acts as a surrogate tumour marker. This was achieved using Affymetrix SNP 6.0 technology and bioinformatics to map SNP and copy number variation (CNV), comparing cfDNA with matched normal lymphocyte and tumour DNA in 65 breast cancer patients and 8 healthy female controls. Results in this thesis show that comparison of cfDNA SNP genotypes can distinguish between primary breast cancer patients and healthy controls (p<0.0001), and between pre-surgical breast cancer patients and those who already had surgery/treatment (p=0.0016). A significant difference (p=0.0006) was also found between cfDNA samples taken an average of 3 years apart in women on follow-up, again suggesting progression. In addition, CNV amplification was observed in matched tumour and cfDNA at numerous loci on different chromosome arms. Many of these tumour-specific CNVs contributed significantly to disease through logistic regression analysis and remained detectable in cfDNA up to 12 years after diagnosis despite no other evidence of disease. This finding strongly infers breast cancer dormancy in the majority of patients on follow-up. In addition, candidate CNVs were validated by real-time qPCR and additional bioinformatics revealed key SNP and CNV signatures of breast cancer patients. If validated in other patient series, the results could alter the diagnostic and prognostic 2015-06-11T09:00:48Z Treatment of metastatic colorectal cancer : A role for Curcumin? James, Mark Ian http://hdl.handle.net/2381/32351 2015-06-05T02:00:40Z 2015-06-04T10:17:16Z Title: Treatment of metastatic colorectal cancer : A role for Curcumin? Authors: James, Mark Ian Abstract: Colorectal cancer remains the third largest cause of mortality from cancer related death, with approximately 90% of these deaths attributed to metastatic spread of the disease. There is a need to improve chemotherapy, with the dietary polyphenol curcumin, derived from turmeric, representing a potential candidate as it possess very few side effects and it has shown efficacy in mouse models. Recent advances in our understanding of tumour development have highlighted the existence of tumour initiating cells (TIC), which possess clonogenic potential, are essential for tumour growth, and represent an important therapeutic target. This study sought to determine whether curcumin in combination with oxaliplatin+5-Fluorouracil (OX+5-FU) represented a better combination for targeting TICs, using a variety of models consisting of cells derived directly from colorectal liver metastasis (CRLM). ALDHHigh activity, CD133 and CD26 were all found to mark a spheroid forming population, a method that tests for clonogenicity and selects for TICs. Curcumin significantly reduced the number of spheroids compared to DMSO, and enhanced efficacy of OX+5-FU. The only marker to decrease after treatment was ALDHHigh activity, which was also positively associated with spheroid growth. CD26 expressing cells were identified as a possible chemo-resistant population, however, this population remained unaffected by curcumin. Ex vivo analysis using explant cultures demonstrated that curcumin could significantly decrease ki67 and increase cleaved capase-3 expression, notably enhancing the effects of oxaliplatin in a proportion of patients. A pilot study was undertaken using non-obese diabetic/severe combined immunodeficient (NOD-SCID) mice to reflect a clinical regimen, and assess in vivo whether curcumin could enhance efficacy of OX+5-FU, but the results advocate the use of higher doses as little effect was seen. Overall this body of work contributes to knowledge on propagating CRLM TICs, their expression of known TIC markers, and response to curcumin. 2015-06-04T10:17:16Z Managing the Morbidity Related to Flap Reconstruction in Major Maxillofacial Oncology Surgery Avery, Christopher Martin Edward http://hdl.handle.net/2381/32343 2015-06-03T02:00:30Z 2015-06-02T15:35:50Z Title: Managing the Morbidity Related to Flap Reconstruction in Major Maxillofacial Oncology Surgery Authors: Avery, Christopher Martin Edward Abstract: There is substantial systemic and local morbidity associated with complex reconstruction of the maxillofacial (face, mouth, jaws and neck) region and the distant donor site of the free flap used to reconstruct the defect. This morbidity may be alleviated by careful patient preparation, selection of operation, improving wound care and new surgical procedures. Many aspects of morbidity are not readily amenable to investigation because of a multitude of confounding clinical factors. Improvements in the quality of care may be obtained by careful evaluation of outcomes and comparison with the literature. This body of work represents a reflection on my surgical practice, animal experimental studies and computer modelling techniques which have reduced morbidity and improved my ability to counsel patients. Major elements include the techniques of suprafascial dissection of the soft tissue radial flap and prophylactic internal fixation of the osteocutaneous radial donor site which have become increasingly accepted within the United Kingdom and overseas. The impact of differing flap choice, particularly in the medically compromised group, has been considered. The quality of the clinical documentation has been improved. Biomechanical issues raised by the clinical studies were investigated in laboratory work using the sheep tibia model of the human radius. A computer based model of the sheep tibia, created using a finite element analysis technique, was validated against the preceding biomechanical studies. This simulation was used to investigate the most effective design of osteotomy cuts and type of plate for reinforcement of a straight osteotomised bone, such as the radius, or segmental defects of the mandible. Interest in my work is reflected in the high rate of presentations at the Annual Scientific Meetings of the British Association of Oral & Maxillofacial Surgery combined with the joint second highest rate of successful conversion to publication in the United Kingdom. Description: Thesis Submitted for the Degree of Doctor of Medicine by Published Work University of Leicester 2015-06-02T15:35:50Z Modulation of micrornas in pancreatic cancer by the dietary agent curcumin Lewis, Lara Christina http://hdl.handle.net/2381/32242 2015-05-09T02:04:02Z 2015-05-08T10:59:28Z Title: Modulation of micrornas in pancreatic cancer by the dietary agent curcumin Authors: Lewis, Lara Christina Abstract: Pancreatic tumourigenesis is a furtive disease characterised by its lack of palpable symptoms and the inconsistency in expression of its various diagnostic markers. The early detection of pancreatic cancer is therefore problematic, with most patients presenting with advanced disease resulting in limited treatment options. MicroRNAs post-transcriptionally regulate gene expression and their aberrant expression has been linked to disease progression in various cancers including pancreatic. In vitro studies have shown that by manipulating microRNA expression, it is possible to inhibit proliferation, suppress metastasis and induce drug sensitivity in pancreatic cancer cells. The phytochemical curcumin, which is under investigation in combination treatment trials for pancreatic cancer, is believed to alter microRNA expression. The work presented in this thesis aimed to identify novel, aberrantly expressed microRNAs which could potentially be used as diagnostic markers for pancreatic cancer, and to ascertain if curcumin could modulate their expression. Additionally, possible mechanisms by which curcumin may regulate the expression of key microRNAs in pancreatic cancer cells when compared to non-transformed cells were also examined. This thesis details the identification of differentially expressed microRNAs using microarray analysis. Cell viability and growth were assessed in order to determine a dose and a treatment time with the capacity to elicit a biological response in pancreatic cancer cell lines. Validation of changes observed in microRNA expression using the microarrays was carried out using RT-qPCR. The results demonstrated that, despite being unable to validate the differential expression of various microRNAs identified by microarray analysis, curcumin did appear to modulate the expression of a number of miRNAs known to be deregulated in pancreatic cancer. 2015-05-08T10:59:28Z The expression of an EMT regulator ZEB2 is controlled by a new destabilisation motif Wan Makhtar, Wan Ratmaazila http://hdl.handle.net/2381/32014 2015-04-18T02:03:18Z 2015-04-17T11:06:42Z Title: The expression of an EMT regulator ZEB2 is controlled by a new destabilisation motif Authors: Wan Makhtar, Wan Ratmaazila Abstract: Epithelial-mesenchymal transitions (EMT) are genetic re-differentiation programs which generate motile and invasive cells during normal embryonic development and cancer metastasis. ZEB1 and ZEB2 are two-handed zinc finger transcription repressors which belong to the Zinc finger E-box (ZEB) protein family. These proteins inhibit transcription of E-cadherin gene and induce EMT in vitro. However, the regulation of ZEB genes is still incompletely understood. Given an important role for both proteins in EMT in normal development and cancer metastasis, better understanding of molecular mechanisms controlling their expression is an important challenge. Intriguingly, a comparative expression analysis of ZEB1 and ZEB2 proteins in several cancer cell lines indicated that ZEB1 protein was expressed at much higher level than ZEB2 mostly in mesenchymal carcinoma cells. On the other hand, these cell lines contained high levels of ZEB2 and ZEB1 mRNA. Therefore, we suggested that an additional specific control mechanism limiting ZEB2 protein synthesis exists; and we addressed this issue in greater detail. We showed that a protein motif adjacent to the smad-binding domain within ZEB2 protein induced ribosome stalling and compromised ZEB2 translation. The activity of this motif was dependent on triplets of rare codons, Leu(UUA)-Gly(GGU)-Val(GUA). However, introducing these rare codons in the ZEB1 region had no effect on ZEB1 protein expression. In conclusion, my study suggests that rare codon play regulatory role in the context of appropriate protein structures and may influence configuration of EMT programs in cancer cells. 2015-04-17T11:06:42Z Structural analysis of the complex formed between the ansamycin antibiotic rifabutin and the BCL6 BTB-POZ domain Evans, Sian Elizabeth http://hdl.handle.net/2381/31984 2015-04-16T02:01:00Z 2015-04-15T11:15:30Z Title: Structural analysis of the complex formed between the ansamycin antibiotic rifabutin and the BCL6 BTB-POZ domain Authors: Evans, Sian Elizabeth Abstract: BCL6 is a zinc finger transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~30-40% of the aggressive disease diffuse large B-cell lymphoma. BCL6 accomplishes its effects through the recruitment of co-repressors to the BTB-POZ domain of BCL6, which is a critical interaction for both a normal immune response and lymphomagenesis. Peptides or small molecule inhibitors, which prevent the association of the SMRT co-repressor with BCL6, abolish BCL6 function by attenuating its transcriptional repression. However, these agents are not yet suitable for clinical practice and there is a need to develop high-affinity and cell permeable BCL6 inhibitors. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, a natural product library was screened, and it was found that the ansamycin antibiotic, rifamycin SV, had the ability to inhibit BCL6 transcriptional repression. NMR spectroscopy confirmed a direct interaction between rifamycin SV and the BTBPOZ domain of BCL6. In addition to rifamycin SV, NMR was used to screen other members of the ansamycin family for potential binding to BCL6. The rifamycin SV derivative, rifabutin, was also shown to interact with the BCL6 BTB-POZ domain. A 2.3Å X-ray crystal structure of the BCL6-rifabutin complex revealed that rifabutin occupies a shallow pocket within the lateral groove, which is similar to that occupied by the SMRT binding peptide and 79-6, the previously described small molecule inhibitor. Further work employing artificial peptides showed the importance of interactions between specific residues of SMRT and the BCL6 BTB-POZ domain. The data presented in this thesis demonstrates a potentially druggable site on the BCL6 BTBPOZ domain and a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor. 2015-04-15T11:15:30Z Quantification of forces involved in stabbings Nolan, Gary http://hdl.handle.net/2381/31481 2015-01-24T02:01:47Z 2015-01-23T10:57:17Z Title: Quantification of forces involved in stabbings Authors: Nolan, Gary Abstract: Stabbing is the most common way of committing murder in the UK. Sharp implements are used in stabbing attacks with the intent to cause injury and death. The number of deaths that result from stabs is high, partly because major organs and major blood vessels are relatively close to the skin and easily penetrated. Knight in the late 1970s used a spring loaded device to make stab tests into cadavers. He concluded that skin was easily penetrated by sharp knives and that skin offered the most resistance to penetration, with underlying fat and muscle requiring less force to penetrate. Knight concluded that the sharpness of the tip was the most important factor in determining whether a knife penetrated. Various other investigators have since investigated the effect of skin and various skin simulants, concluding that most items of clothing are easily penetrated by a knife, and considered how the geometry of the knife affects it sharpness. This thesis has looked at the effect of clothing on forces required for stabbing and the use of glass bottles as impulsive weapons. It has been found that clothing does influence the force required for stabbing. For the glass bottles it was found that there was no simple method for predicting how sharp a particular bottle would be once it was fractured. A novel dynamometer has been developed to accurately mimic a stabbing event and record the forces generated. The data shows that there are considerable differences in the forces generated by males and females; discernable differences between the dominant and non-dominant hand and that stabbing, even with blunt weapons can cause considerable penetration in most cases. The data also shows that the traditional ranking of mild, moderate and severe used by forensic pathologists lacks meaning in interpreting stabbing events. 2015-01-23T10:57:17Z Assessing efficacy and molecular mechanisms of curcumin in targeting cancer stem-like cells in colorectal cancer Karmokar, Ankur http://hdl.handle.net/2381/31393 2016-06-09T08:39:03Z 2015-01-08T14:34:42Z Title: Assessing efficacy and molecular mechanisms of curcumin in targeting cancer stem-like cells in colorectal cancer Authors: Karmokar, Ankur Abstract: Curcumin inhibits the proliferation of chemotherapy-resistant cancer stem-like cells in cell lines but whether this contributes to its chemopreventive activity is unknown. This study aims to determine whether curcumin modulates the growth and expansion of colorectal stem-like cells in primary adenoma and carcinoma tissues, and in vivo using a patient-derived xenograft, then to elucidate a possible mechanism of action. Colorectal tissue obtained post-operatively (normal n=32, adenoma n=6, carcinoma n=40) was FACS profiled for markers of stem-like cells, aldehyde dehydrogenase (ALDH) activity and CD133 expression. The percentage of cells with ALDH[superscript high] activity was 11.8±1.8, 4.6±0.7 and 2.8±0.4 in adenoma, normal and carcinoma tissues, respectively. Equivalent values for CD133 expression were 1.2±0.6, 0.5±0.2 and 7.7±1.8%. To assess in vitro activity, single cells from adenomas and carcinomas (three patients each, in triplicate) were cultured as spheroids with clinically achievable curcumin concentrations. Curcumin significantly reduced adenoma and carcinoma sphere number, compared to controls for all patient samples, with a U-shaped dose-response in >50% of cases. Sphere size was also impaired at concentrations >1μM. Curcumin (0.2%) consumption in NOD/SCID mice injected (s.c) with cancer stem-like cells was associated with significant delay in time to palpable tumours, increased survival and reduced rate of tumour growth. There was also a ~60% reduction in proportion of ALDH[superscript high] cells in tumours from curcumin treated mice compared to controls (p<0.05). Curcumin (0.1, 1μM) treatment of Caco2 cells caused a significant decrease (p<0.01) in Nanog expression, an embryonic stem cell transcription factor, in cancer stem-like cells specifically. A protein pull-down assay confirmed the interaction between curcumin and Nanog. Curcumin reduced (p<0.01) Nanog phosphorylation in cancer stem-like cells which may destabilise the protein, leading to reduced levels. These results indicate that clinically achievable concentrations of curcumin target stem-like cells in colorectal adenomas and carcinomas, which may contribute to anti-cancer efficacy in humans. 2015-01-08T14:34:42Z The use of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to study tumour angiogenesis in the management of metastatic cancer Morgan, Bruno http://hdl.handle.net/2381/29875 2014-12-16T02:12:59Z 2014-12-15T10:34:03Z Title: The use of dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) to study tumour angiogenesis in the management of metastatic cancer Authors: Morgan, Bruno Abstract: Introduction: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) may act as a biomarker for successful cancer therapy. This thesis demonstrates an imaging technique developed to be simple and reproducible. PTK787/ZK222584 (PTK/ZK) is an inhibitor of vascular endothelial growth factor mediated angiogenesis. The pharmacodynamic effects of PTK/ZK were evaluated by assessing changes in contrast enhancement parameters of metastatic disease using DCE-MRI in advanced cancer patients treated in two dose escalating phase I studies using once and twice-daily dosing schedules. Patients and Methods: A theoretical analysis was performed to optimise sequence parameters. MRI phantom and clinical studies were performed to establish the validity and reproducibility of the technique. The clinical studies of PTK/ZK were performed with centralised DCE-MRI analysis. DCE-MRI was performed at baseline, day 2, and at the end of the 28-day cycle (EC1). Doses of oral PTK/ZK ranged from 50 to 2000 mg per day. Tumour permeability and vascularity were assessed by calculating the bi-directional transfer constant (Ktrans). Results: This technique can accurately measure R1 within the expected range of tumour enhancement. The reproducibility study showed a coefficient of variation (CoV) of 16% for Ktrans if tumours of diameter less than 3cm were excluded. A significant correlation was shown between changes in Ktrans and PTK/ZK oral dose, plasma levels and efficacy at both day 2 and ECI. Visual assessment of the DCE-MRI scans demonstrated similar results but with weaker correlations to dose and efficacy. Conclusion: The clinical findings suggest that DCE-MRI is sufficiently sensitive and reproducible to act as a useful biomarker for defining the pharmacological response and dose of angiogenesis inhibitors, such as PTK/ZK. 2014-12-15T10:34:03Z Comparative pre-clinical evaluation of resveratrol and 3,4,5,4'-tetramethoxystilbene (DMU212) as colorectal cancer chemopreventive agents Sale, Stewart http://hdl.handle.net/2381/29876 2014-12-16T02:13:00Z 2014-12-15T10:34:03Z Title: Comparative pre-clinical evaluation of resveratrol and 3,4,5,4'-tetramethoxystilbene (DMU212) as colorectal cancer chemopreventive agents Authors: Sale, Stewart Abstract: Recently a variety of analogues of resveratrol have been synthesised and investigated in in vivo assays. One analogue, 3,4,5,4'-tetramethoxystilbene (DMU212), showed preferential growth-inhibitory and pro-apoptotic properties in transformed cells, when compared with their untransformed counterparts. Cyclooxygenase enzymes are important mechanistic targets of resveratrol. As part of this study the pharmacokinetic properties of DMU212 were compared with those of resveratrol. Both agents were also compared in terms of abilities to prevent adenoma development in the ApcMin+ mouse, a model of human intestinal carcinogenesis, and to interfere with the expression and activity of COX in human-derived colon cells. Pharmakinetic data showed that resveratrol achieved significantly higher levels than DMU212 systematically, whilst DMU212 exhibited superior availability in the gastrointestinal tract. ApcMin+ mice received either compound with the diet (0.05, 0.2 or 0.5 %), and adenomas were counted after animals were killed. Resveratrol and DMU212 at 0.2 % decreased adenoma load by 27 and 24 % respectively. DMU212 is a better inhibitor of proliferation of human colon cancer cells than resveratrol. Incubation of HCA-7 colon cancer cells for 24-96 h with either compound (1-50 microM) decreased PGE2 production, but only resveratrol decreased COX-2 protein expression. Whilst resveratrol inhibited enzyme activity in purified COX preparations, DMU 212 failed to do so. The results suggest that chemical alteration of the resveratrol molecule to generate DMU212 does not alter its ability to decrease adenoma number in ApcMin+ mice or to interfere with PGE2 generation in cells. However, only resveratrol directly inhibits COX activity and expression. 2014-12-15T10:34:03Z The role of bone marrow mesenchymal stromal cells in the protection of B-cell chronic lymphocytic leukaemia from spontaneous and drug induced apoptosis Woolston, Caroline. http://hdl.handle.net/2381/29873 2014-12-16T02:12:57Z 2014-12-15T10:34:02Z Title: The role of bone marrow mesenchymal stromal cells in the protection of B-cell chronic lymphocytic leukaemia from spontaneous and drug induced apoptosis Authors: Woolston, Caroline. Abstract: An in vitro model to mimic in vivo conditions was developed using adherent BM Mesenchymal Stromal Cells (MSCs), isolated from the iliac crest of normal donors. These "fibroblast-like" MSCs provide a supporting layer for haemopoietic differentiation. They lack expression of haemopoietic differentiation antigens and lineage markers:- CD34, CD45, HLA-DR, CD31 and CD68 but express Vimentin, alphaSmooth Muscle Actin and Ab-1 (fibroblast marker). The model was used to determine if MSCs could protect B-CLL B cells from spontaneous apoptosis and prevent the effects of the therapeutic monoclonal antibodies Rituximab (CD20) and Campath-1H (CD52), the proteasomal inhibitor MG132 and BisIX, a protein kinase C inhibitor. Levels of apoptosis were determined by flow cytometry, using Annexin V-FITC/PI. MSCs abrogated spontaneous apoptosis of B-CLL B cells in direct contact with them, over 4 days (80-90% viability, n=30). Rituximab and Campath-1H induced apoptosis in the B-CLL B cells that could not be prevented by contact with MSCs (n=6). MG132 and BisIX caused substantial apoptosis in B-CLL B cells (0-15% viability) but MSCs could protect against lower concentrations (200nM) of both drugs (70-90% viability, n=6). MSCs' protection of B-CLL B cells from spontaneous apoptosis was through the upregulation of the expression of Mcl-1 and Xiap, but not Survivin. The cell-cell interactions include the beta1, beta2, beta3 and beta4 integrins but have variable effects on B-CLL cases when bound by blocking antibodies. Therefore MSCs can maintain B-CLL viability in vitro and influence chemotherapeutic strategies. The model provides a reliable method to evaluate drug cytotoxicity and Mcl-1 offers a novel therapeutic target. 2014-12-15T10:34:02Z Radiosensitivity in bladder cancer cells Sherwood, Benedict T. http://hdl.handle.net/2381/29874 2014-12-16T02:12:59Z 2014-12-15T10:34:02Z Title: Radiosensitivity in bladder cancer cells Authors: Sherwood, Benedict T. Abstract: Potentially curative treatment options for patients with organ-confined transitional cell carcinoma (TCC) of the bladder (T1-4a/N0/M0) are radical cystectomy or radiotherapy (RT)-based 'bladder-preserving' regimens. A substantial number of patients who receive RT fail to respond (approximately 50%). Consequently, a greater understanding of the mechanisms of radioresistence is required, together with predictive information regarding the response of tumours to RT. Hypoxia and intrinsic cellular Radiosensitivity (IRS) are examined here, a factors that may influence the outcome of RT.;An immunohistochemical assay using hypoxia-related carbonic anhydrase IX (CA IX) was undertaken to determine the prognostic significance of hypoxia in bladder tumours treated with RT. A modified version of the alkaline comet assay (ACA) was used to examine differences in IRS between cells derived from TCC specimens. Nuclear factors that influence comet formation (and therefore radiosensitivity) were also examined, such as DNA double strand break (DSB) rates and differences in nuclear matrix protein (NMP) composition.;CA IX immunostaining did not provide prognostic information with respect to response to radical RT. ACA analysis indicated a wide range of responses between tumours. In TCC cell lines, DSB rates are not demonstrably different in cells of differentiated radiosensitivity, however, comparative analysis of nuclear proteins identified differences in their constitutive NMPs and repair enzymes.;These results do not provide evidence that hypoxia influences outcome after RT, but support the contention that ICR is important in dictating the response of bladder tumours to RT. Furthermore, in bladder cancer cell lines of differing radiosensitivity, differences in NMP and repair enzymes are identified. Further work is required to determine whether these are of prognostic importance. 2014-12-15T10:34:02Z Angiogenesis, cyclooxygenase-2 and matrix metalloproteinases in malignant mesothelioma Edwards, John G. http://hdl.handle.net/2381/29871 2014-12-16T02:12:55Z 2014-12-15T10:34:01Z Title: Angiogenesis, cyclooxygenase-2 and matrix metalloproteinases in malignant mesothelioma Authors: Edwards, John G. Abstract: Malignant Mesothelioma (MM) is a fatal tumour, related to prior asbestos exposure, of increasing incidence. Current treatment modalities may provide symptom palliation but survival benefits remain unclear. Angiogenesis is essential for tumour growth of greater than 1-2mm in diameter and is stimulated by hypoxia, which is reflected by tumour necrosis (TN). Angiogenesis can be assessed by the intratumoural microvessel density (MVD). Cyclooxygenase(COX)-2 plays a central role in the upregulation of angiogeneic growth factors, such as vascular endothelial growth factor and matrix metalloproteinases (MMPs). Epidermal Growth Factor Receptor (EGFR) is overexpressed in many solid tumours and participates both in COX-2 and MMP upregulation. These factors may be prognostic in solid tumours. A database of MM cases in Leicester from 1987 to 2001 was created. Clinical and pathological prognostic factors were derived. Angiogenesis, TN and EGFR were assessed in 171 MM cases by immunohistochemistry and/or microscopy. COX-2 and MMP expression were analysed prospectively by semi-quantitative Western blotting and gelatin zymography, respectively, in up to 47 snap-frozen samples. TN, COX-2 and MMPs were identified for the first time in MM. MVD, COX-2, and EGFR correlated with TN but not with each other. The prognostic significance of MVD and EGFR were confirmed in the largest series of MM studied. TN, COX-2 and MMP-2 were novel prognostic factors. MVD, TN, EGFR, COX-2 and MMP-2 each contributed both the CALGB and EORTC prognostic scoring systems in multivariate analyses. In addition to establishing new laboratory methods and the prognostic importance of these factors, this work has identified novel targeted therapies for MM. These include anti-angiogenic therapies, such as thalidomide, and COX-2, EGFR and MMP inhibition, all of which are now either under current or future investigation in clinical trials in Leicester and other international centres. 2014-12-15T10:34:01Z The molecular basis for the non-random manifestation of DNA phosphotriester adducts Le Pla, Rachel Caroline http://hdl.handle.net/2381/29872 2014-12-16T02:12:56Z 2014-12-15T10:34:01Z Title: The molecular basis for the non-random manifestation of DNA phosphotriester adducts Authors: Le Pla, Rachel Caroline Abstract: Previously, in this laboratory, it has been demonstrated that the manifestation of PTEs was non-random in vivo, but random in vitro with respect to normal base content. This was suggestive of either non-random formation in vivo or sequence specific repair (Guichard et al., 2000). In the present study, the [32P]-postlabelling protocol was further developed to allow for the more straight forward determination of nucleosides 5' to the PTEs. The molecular basis for the non-random manifestation of PTEs in vivo was studied using the improved assay. To determine the influence of cellular processes upon the manifestation of PTEs in liver DNA take from mice 5 hours to 56 days following a single dose of N-nitrosodiethylamine was analysed. Whilst there was an abrupt decline in the level of PTEs observed, possible indicative of active repair, there was no appreciable change in the frequency of nucleosides located 5' to PTEs between 5 hours and 56 days following treatment. It was demonstrated that the manifestation of PTEs in cells treated in vitro with diethylsulphate (DES) was non-random. To investigate the potential role of higher order packaging upon the manifestation of PTEs, nuclei and DNA were isolated from cells and treated in vitro with DES. The determination of the frequency of nucleosides found 5' to PTEs revealed the same non-random manifestation of PTEs observed in the whole cells treated with DES. From these studies it would appear that neither DNA packaging nor sequence specific repair play a significant role. Studies using synthetic oligonucleotides indicate that the manifestation of PTEs may be influenced by the primary structure of DNA (i.e. its sequence). In a separate study using specifically modified olignucleotides it was demonstrated that the postlabelling assay was able to detect tandem PTE damage. However, PTEs separated by a single normal nucleotide appeared as single lesions. 2014-12-15T10:34:01Z Characterisation of the voltage gated proton channel HVCN1 in B cells Bhamrah, Mandeep Kaur http://hdl.handle.net/2381/29869 2014-12-16T02:12:53Z 2014-12-15T10:34:00Z Title: Characterisation of the voltage gated proton channel HVCN1 in B cells Authors: Bhamrah, Mandeep Kaur Abstract: HVCN1 is a voltage-gated proton channel, detected in a proteomic screen profiling membrane proteins of Mantle Cell Lymphoma cells. Subsequent analysis showed that HVCN1 is expressed in naive mature resting B cells, such as cells of Mantle Zones in lymph nodes and circulating peripheral B cells. The role of voltage-proton channels in B cells is unknown and further characterized in this study. HVCN1 co-localizes with the BCR and markers of MIIC vesicles and translocates with the BCR upon internalization of receptor-antigen complexes. Mass spectrometry data confirmed key proteins involved in BCR signalling, such as IgM, CD79B (Igp), CD19 and CD22 were associated with HVCN1 at a resting and stimulated state of the cell, demonstrating HVCN1 functioned in the regulation of B-cell receptor signalling. BCR stimulation has shown to result in the production of ROS, which can regulate B-cell activation, through the oxidation of Protein Tyrosine Phosphatases. Primary B cells isolated from mice lacking HVCN1 demonstrated weak signalling following cross-linking of the BCR with anti-IgM, which was rescued in the presence of the Sodium Stibogluconate, an inhibitor of the CD22 associated phosphatase SHP-1. The catalytic activity of phosphatases was reduced by ROS and allowed phosphorylation of protein, which was regulated through a negative signalling feedback loop. Lack of HVCN1 dampened the phosphorylation of key signalling molecules SYK and AKT, involved in the activation and proliferation of B cells. Therefore, HVCN1 functions to modulate the balance of ROS production in the cell upon BCR activation, allowing the effective transmission of signalling in the cell. 2014-12-15T10:34:00Z Angiogenesis in resected colorectal liver metastases Sutton, Christopher Derek http://hdl.handle.net/2381/29870 2014-12-16T02:12:54Z 2014-12-15T10:34:00Z Title: Angiogenesis in resected colorectal liver metastases Authors: Sutton, Christopher Derek Abstract: Aims: To assess the prognostic significance of angiogenesis using microvessel density (MVD) and angiogenesis-modulating cytokines, in a consecutive series of patients undergoing liver resection for colorectal metastases. Methods: 5 microm sections from formalin-fixed, paraffin-embedded tissue blocks were immunohistochemically stained for microvessels, vascular endothelial growth factor (VEGF) thrombospondin-1 (TSP-1), thymidine phosphorylase (TP) and p53. MVD was measured using a computerised image analysis system. At the edge of the tumour, areas of highest vessel counts or hotspots, and the mean of contiguous x200 high power fields were counted. Within the tumour, hotspots and the random cumulative mean of vessel counts were analysed. The percentage expression of VEGF, TSP-1, TP and p53 was recorded. MVD and the cytokines were correlated using the test. The Kaplan-Meier method, the log rank test, and the Cox proportional hazard model were used to correlate MVD, the cytokines and clinico- pathological variables with patient survival. Results: 182 patients, age range 25-81 (mean 61) were included. On univariate analysis, bilobar disease (p=0.003), tumour edge hotspot (p= 0.005), lymphocytic TP (p=0.02), stromal TSP-1 (p=0.01) and perivascular TSP-1 (p=0.03) significantly correlated with poor prognosis. High expression of TP correlated with high MVD (p=0.04 for hotspots, p=0.001 for contiguous vessels). On multivariate analysis tumour edge hotspot, and bilobar disease were independent prognostic factors (p= 0.038, and p= 0.04 respectively). Conclusion: Thymidine phosphorylase is an important cytokine associated with high tumour edge microvessel density. Tumour edge hotspot and bilobar disease are independent prognostic markers of poor survival in patients who have undergone liver resection for colorectal liver metastases. 2014-12-15T10:34:00Z Alterations of tumour protein 53 (TP53) pathways in diffuse large B-cell lymphomas Magdi Abdel-Azim, Dalia http://hdl.handle.net/2381/29868 2014-12-16T02:12:51Z 2014-12-15T10:33:59Z Title: Alterations of tumour protein 53 (TP53) pathways in diffuse large B-cell lymphomas Authors: Magdi Abdel-Azim, Dalia Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common type of lymphoma. It has heterogeneous clinical features and varies markedly in response to treatment and in prognosis. Although the clinical variables widely used in clinical practice may be useful indicators in defining the underlying biological heterogeneity of DLCL, a search for molecular expression, such as TP53, would improve the prognostic predict ability and current understanding of the clinical behaviour of DLBCL. Seventy-two cases of DLBCL were assessed by immunohistochemistry using primary antibodies for TP53, p21, HDM2, Phospho-TP53, Bcl-2, Bcl-6 and MIB-1. There was a significant positive correlation between high proliferation index and TP53, phopho-TP53 and Bcl-6 expressions (p<0.005), while a significant negative correlation was found with Bcl-2 expression (p<0.005). Bcl-2 expression showed an inverse correlation with TP53, phospho-TP53, Bcl-6 (p<0.005). TP53 assessed using the DO-1 antibody showed a significant correlation with phopho-TP53 (p<0.005). p21 showed a similar correlation with HDM2 (p<0.005). TP53 mutations were assessed using PCR-SSCP analysis in 64 cases of DLBCL. The frequency of TP53 mutations in this study was 18.75%. Exon 7 harboured most mutations (30%). There was a significant correlation between low expression of TP53 and phospho- p53 assessed by immunohistochemistry and the presence of WT-p53, but not vice versa. Also, a significant correlation was found between missense TP53 mutations and TP53+/p21- phenotype (p<0.005). High proliferation index correlated with TP53 mutations (p<0.005). There was a strong association of the presence of TP53 mutations and low level of miR-34a (p<0.05). A strong correlation between miR-34a expression levels and TP53, p21 and HDM2 (p <0.05). In conclusion, although 80% of DLBCL cases posses WT-p53 gene , not of them are fully functional and they show phenotypic variabilty in their TP53 activity. Other mechanisms for inactivating TP53 must be responsible, such as alterations of the downstream or upstream regulators or targets that will mimic a TP53 aberration. 2014-12-15T10:33:59Z The role of anandamide in human folliculogenesis, implantation and early pregnacny El-Talatini, Mona R. T. http://hdl.handle.net/2381/29867 2014-12-16T02:12:50Z 2014-12-15T10:33:58Z Title: The role of anandamide in human folliculogenesis, implantation and early pregnacny Authors: El-Talatini, Mona R. T. Abstract: Plasma AEA levels were measured by ultra-performance liquid chromatography and mass-spectrometry throughout the menstrual cycle in healthy women both cross-sectionally and longitudinally. Plasma and follicular fluid (FF) AEA levels were also determined in women undergoing in-vitro fertilisation and embryo transfer (IVF-ET) and correlations between FF AEA and follicle size and oocyte maturity were undertaken. Plasma AEA levels were then measured in these women on the day of oocyte retrieval (OR), embryo transfer (ET) and at pregnancy test (PT) and comparisons made between pregnant and non-pregnant women and between those with viable pregnancies and those who had miscarried at 6 weeks. The relationship between plasma AEA and sex steroids in the stimulated cycles was also examined. AEA was found to be hormonally regulated, mainly by gonadotrophins and estradiol, and its highest levels were at ovulation and low at the implantation window in natural and stimulated cycles. The endocannabinoid system was localised in human ovary, FF AEA was associated with folliculogenesis, ovulation and oocyte maturity. ROC analysis indicated that a FF AEA concentration of 1.09 nM discriminated between mature and immature oocytes. In successful pregnancies a significant decline in plasma AEA levels from ovulation to the day of ET than a significant rise from the day of ET to the day of PT appeared to be important Plasma AEA levels were high at 4 and 5 weeks gestation and then significantly declined at 6 weeks gestation. These data may show the potential for using AEA as a biomarker test for oocyte assessment and to improve pregnancy outcomes in stimulated IVF/ICSI cycles. 2014-12-15T10:33:58Z