text: DSpace Community: http://hdl.handle.net/2381/162 2015-07-05T09:29:51Z 2015-07-05T09:29:51Z Associations of Sedentary Time with Fat Distribution in a High-Risk Population. Henson, Joseph Edwardson, Charlotte L. Morgan, Bruno Horsfield, Mark A. Bodicoat, Danielle H. Biddle, S. J. Gorely, T. Nimmo, M. A. McCann, Gerry P. Khunti, Kamlesh Davies, Melanie J. Yates, Thomas http://hdl.handle.net/2381/32505 2015-07-04T02:01:37Z 2015-07-03T11:41:14Z Title: Associations of Sedentary Time with Fat Distribution in a High-Risk Population. Authors: Henson, Joseph; Edwardson, Charlotte L.; Morgan, Bruno; Horsfield, Mark A.; Bodicoat, Danielle H.; Biddle, S. J.; Gorely, T.; Nimmo, M. A.; McCann, Gerry P.; Khunti, Kamlesh; Davies, Melanie J.; Yates, Thomas Abstract: Purpose. The effect of sedentary behaviour on regional fat deposition, independent of physical activity remains equivocal. We examined the cross-sectional associations between objectively measured sedentary time and markers of regional fat distribution (heart, liver, visceral, subcutaneous and total body fat) in a population at a high risk of type 2 diabetes mellitus (T2DM). Methods. Participants were recruited from primary care to two diabetes prevention programmes. Sedentary time (<25 counts per 15 seconds) was measured using Actigraph GT3X accelerometers. Heart, liver, visceral, subcutaneous and total body fat were quantified using magnetic resonance images (MRI). Fat volumes were calculated by multiplying the crosssectional areas of the fat-containing pixels by the slice thickness. The liver fat percentage was measured using a representative region of interest created in the right lobe of the liver avoiding the main portal veins. Linear regression models examined the association of sedentary time with markers of regional fat deposition. Results. Sixty-six participants (age = 47.9±16.2 years; male = 50.0%) were included. Following adjustment for several covariates, including glycaemia, whole body fat and moderate-to-vigorous physical activity (MVPA), each 30 minutes of sedentary time was associated with 15.7cm3 higher heart fat (p=0.008), 1.2% higher liver fat (p=0.026) and 183.7cm3 higher visceral fat (p=0.039). Conclusion. This study provides new evidence suggesting that objectively measured sedentary behaviour may have an independent association upon heart, liver and visceral fat in individuals at a high risk of T2DM. 2015-07-03T11:41:14Z Urinary Benzene Biomarkers and DNA Methylation in Bulgarian Petrochemical Workers: Study Findings and Comparison of Linear and Beta Regression Models Seow, W. J. Baccarelli, A. A. Pesatori, A. C. Albetti, B. Bollati, V. Bertazzi, P. A. Dimont, E. Farmer, P. B. Ettinger, A. S. Bolognesi, C. Roggieri, P. Panev, T. I. Georgieva, T. Merlo, D. F. http://hdl.handle.net/2381/32487 2015-07-03T02:00:28Z 2015-07-02T11:48:04Z Title: Urinary Benzene Biomarkers and DNA Methylation in Bulgarian Petrochemical Workers: Study Findings and Comparison of Linear and Beta Regression Models Authors: Seow, W. J.; Baccarelli, A. A.; Pesatori, A. C.; Albetti, B.; Bollati, V.; Bertazzi, P. A.; Dimont, E.; Farmer, P. B.; Ettinger, A. S.; Bolognesi, C.; Roggieri, P.; Panev, T. I.; Georgieva, T.; Merlo, D. F. Abstract: Chronic occupational exposure to benzene is associated with an increased risk of hematological malignancies such as acute myeloid leukemia (AML), but the underlying mechanisms are still unclear. The main objective of this study was to investigate the association between benzene exposure and DNA methylation, both in repeated elements and candidate genes, in a population of 158 Bulgarian petrochemical workers and 50 unexposed office workers. Exposure assessment included personal monitoring of airborne benzene at work and urinary biomarkers of benzene metabolism (S-phenylmercapturic acid [SPMA] and trans,trans-muconic acid [t,t-MA]) at the end of the work-shift. The median levels of airborne benzene, SPMA and t,t-MA in workers were 0.46 ppm, 15.5 μg/L and 711 μg/L respectively, and exposure levels were significantly lower in the controls. Repeated-element DNA methylation was measured in Alu and LINE-1, and gene-specific methylation in MAGE and p15. DNA methylation levels were not significantly different between exposed workers and controls (P>0.05). Both ordinary least squares (OLS) and beta-regression models were used to estimate benzene-methylation associations. Beta-regression showed better model specification, as reflected in improved coefficient of determination (pseudo R) and Akaike's information criterion (AIC). In beta-regression, we found statistically significant reductions in LINE-1 (-0.15%, P<0.01) and p15 (-0.096%, P<0.01) mean methylation levels with each interquartile range (IQR) increase in SPMA. This study showed statistically significant but weak associations of LINE-1 and p15 hypomethylation with SPMA in Bulgarian petrochemical workers. We showed that beta-regression is more appropriate than OLS regression for fitting methylation data. © 2012 Seow et al. 2015-07-02T11:48:04Z Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression Barbone, D. Cheung, P. Battula, S. Broaddus, V. C. Busacca, S. Fennell, D. A. Gray, S. G. Longley, D. B. Bueno, R. Sugarbaker, D. J. http://hdl.handle.net/2381/32479 2015-07-03T02:00:30Z 2015-07-02T09:23:35Z Title: Vorinostat Eliminates Multicellular Resistance of Mesothelioma 3D Spheroids via Restoration of Noxa Expression Authors: Barbone, D.; Cheung, P.; Battula, S.; Broaddus, V. C.; Busacca, S.; Fennell, D. A.; Gray, S. G.; Longley, D. B.; Bueno, R.; Sugarbaker, D. J. Abstract: When grown in 3D cultures as spheroids, mesothelioma cells acquire a multicellular resistance to apoptosis that resembles that of solid tumors. We have previously found that resistance to the proteasome inhibitor bortezomib in 3D can be explained by a lack of upregulation of Noxa, the pro-apoptotic BH3 sensitizer that acts via displacement of the Bak/Bax-activator BH3-only protein, Bim. We hypothesized that the histone deacetylase inhibitor vorinostat might reverse this block to Noxa upregulation in 3D. Indeed, we found that vorinostat effectively restored upregulation of Noxa protein and message and abolished multicellular resistance to bortezomib in the 3D spheroids. The ability of vorinostat to reverse resistance was ablated by knockdown of Noxa or Bim, confirming the essential role of the Noxa/Bim axis in the response to vorinostat. Addition of vorinostat similarly increased the apoptotic response to bortezomib in another 3D model, the tumor fragment spheroid, which is grown from human mesothelioma ex vivo. In addition to its benefit when used with bortezomib, vorinostat also enhanced the response to cisplatin plus pemetrexed, as shown in both 3D models. Our results using clinically relevant 3D models show that the manipulation of the core apoptotic repertoire may improve the chemosensitivity of mesothelioma. Whereas neither vorinostat nor bortezomib alone has been clinically effective in mesothelioma, vorinostat may undermine chemoresistance to bortezomib and to other therapies thereby providing a rationale for combinatorial strategies. © 2012 Barbone et al. 2015-07-02T09:23:35Z Analysis and Design of Randomised Clinical Trials Involving Competing Risks Endpoints Tai, B-C. Wee, J. Machin, D. http://hdl.handle.net/2381/32461 2015-07-02T02:00:37Z 2015-07-01T11:03:13Z Title: Analysis and Design of Randomised Clinical Trials Involving Competing Risks Endpoints Authors: Tai, B-C.; Wee, J.; Machin, D. Abstract: Background: In randomised clinical trials involving time-to-event outcomes, the failures concerned may be events of an entirely different nature and as such define a classical competing risks framework. In designing and analysing clinical trials involving such endpoints, it is important to account for the competing events, and evaluate how each contributes to the overall failure. An appropriate choice of statistical model is important for adequate determination of sample size. Methods: We describe how competing events may be summarised in such trials using cumulative incidence functions and Gray's test. The statistical modelling of competing events using proportional cause-specific and subdistribution hazard functions, and the corresponding procedures for sample size estimation are outlined. These are illustrated using data from a randomised clinical trial (SQNP01) of patients with advanced (non-metastatic) nasopharyngeal cancer. Results: In this trial, treatment has no effect on the competing event of loco-regional recurrence. Thus the effects of treatment on the hazard of distant metastasis were similar via both the cause-specific (unadjusted csHR = 0.43, 95% CI 0.25 - 0.72) and subdistribution (unadjusted subHR 0.43; 95% CI 0.25 - 0.76) hazard analyses, in favour of concurrent chemo-radiotherapy followed by adjuvant chemotherapy. Adjusting for nodal status and tumour size did not alter the results. The results of the logrank test (p = 0.002) comparing the cause-specific hazards and the Gray's test (p = 0.003) comparing the cumulative incidences also led to the same conclusion. However, the subdistribution hazard analysis requires many more subjects than the cause-specific hazard analysis to detect the same magnitude of effect. Conclusions: The cause-specific hazard analysis is appropriate for analysing competing risks outcomes when treatment has no effect on the cause-specific hazard of the competing event. It requires fewer subjects than the subdistribution hazard analysis for a similar effect size. However, if the main and competing events are influenced in opposing directions by an intervention, a subdistribution hazard analysis may be warranted. 2015-07-01T11:03:13Z ATP release and P2Y receptor signalling are essential for keratinocyte galvanotaxis Riding, Aimie Pullar, Christine E. http://hdl.handle.net/2381/32383 2015-06-19T02:00:19Z 2015-06-18T13:23:43Z Title: ATP release and P2Y receptor signalling are essential for keratinocyte galvanotaxis Authors: Riding, Aimie; Pullar, Christine E. Abstract: Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes towards the cathode when an EF of physiological strength (100–150 mV/mm) is applied in vitro, but the "sensor" by which keratinocytes sense the EF remains elusive. Here we use a customised chamber design to facilitate the application ofa direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation ofboth connexin hemichannels and purinergic receptorsto determine their role inEF-mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2(3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP-medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activatinga subset of purinergic P2Y receptors, locally, to initiate the directional migration of keratinocytes towards the cathodein vitro, the center of the woundin vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. This article is protected by copyright. All rights reserved 2015-06-18T13:23:43Z 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 Future of Pediatric and Perinatal Post-mortem Imaging Gorincour, G. Sarda-Quarello, L. Pierre-Eloi, L. Brough, Alison Rutty, Guy N. http://hdl.handle.net/2381/32221 2015-05-08T02:00:53Z 2015-05-07T13:40:11Z Title: The Future of Pediatric and Perinatal Post-mortem Imaging Authors: Gorincour, G.; Sarda-Quarello, L.; Pierre-Eloi, L.; Brough, Alison; Rutty, Guy N. Abstract: The field and applications of postmortem imaging are exponentially growing. Its potential to identify the cause of death in trauma and ballistic cases is now properly documented, as well as its use in drug mule identification. In pediatric and perinatal practice, large significant series are less available, except for MRI and central nervous system analysis where scientific evidence is now robust. In this review, after a short historical review and analysis of current problems and challenges, we will try to depict the way we see the future of this subspecialty of postmortem cross-sectional imaging, including all specific situations: terminations of pregnancy, intrauterine death, sudden unexpected infant death and identification issues. 2015-05-07T13:40:11Z Combining curcumin (C3-complex, Sabinsa) with standard care FOLFOX chemotherapy in patients with inoperable colorectal cancer (CUFOX): study protocol for a randomised control trial. Irivng, Glen R.. B. Iwuji, Chinenye O. O. Morgan, Bruno Berry, D. P. Steward, William P. Thomas, Anne Howells, Lynne M. http://hdl.handle.net/2381/32019 2015-04-21T02:00:20Z 2015-04-20T14:03:54Z Title: Combining curcumin (C3-complex, Sabinsa) with standard care FOLFOX chemotherapy in patients with inoperable colorectal cancer (CUFOX): study protocol for a randomised control trial. Authors: Irivng, Glen R.. B.; Iwuji, Chinenye O. O.; Morgan, Bruno; Berry, D. P.; Steward, William P.; Thomas, Anne; Howells, Lynne M. Abstract: Background: The need for low toxicity adjuncts to standard care chemotherapy in inoperable colorectal cancer, with potential to improve outcomes and decrease the side-effect burden, is well recognised. Addition of the low toxicity diet-derived agent, curcumin (the active ingredient of turmeric), to standard oxaliplatin-based therapy has shown promise in numerous pre-clinical studies. Methods/Design: This study is the first to combine daily oral curcumin with standard care FOLFOX-based (5-fluorouracil, folinic acid and oxaliplatin) chemotherapy in colorectal cancer patients with inoperable liver metastases: the CUFOX trial. CUFOX comprises a Phase 1 dose-escalation study (3 + 3 + 3 design) to determine an acceptable target dose of curcumin with which to safely proceed to a Phase IIa open-labelled randomised controlled trial. Thirty three participants with histological or cytological confirmation of inoperable colorectal cancer will then be randomised to oxaliplatin-based chemotherapy with the addition of daily oral curcumin at the target dose determined in Phase I, or to standard care oxaliplatin-based chemotherapy alone (recruiting at a ratio of 2:1). Discussion: Primary outcome measures will be the determination of a target dose which is both safe and tolerable for long-term administration to individuals in receipt of first-line oxaliplatin-based chemotherapy for inoperable colorectal cancer. Secondary outcome measures will include observation of any changes in neuropathic side-effects of chemotherapy, improvement to progression-free or overall survival and identification of putative efficacy biomarkers in plasma. The results will be disseminated via presentation at national and international conferences, via publication in appropriate peer-reviewed journals and via the Cancer Research UK/Department of Health Experimental Cancer Medicine Centre Network. This trial has full ethical and institutional approval, and commenced recruitment in February 2012. Trial registration: ClinicalTrials.gov (NCT01490996, registered 7th December 2011), European Drug Regulating Authorities (EudraCT 2011-002289-19, registered 13th May 2011), UKCRN ID#10672. 2015-04-20T14:03:54Z Characterisation and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note. James, Mark I. Howells, Lynne M. Karmokar, Ankur Higgins, Jennifer A. Greaves, Peter Cai, Hong Dennison, Ashley Metcalfe, Matthew Garcea, Giuseppe Lloyd, David M. Berry, D. P. Steward, William P. Brown, Karen http://hdl.handle.net/2381/32016 2015-04-21T02:00:20Z 2015-04-20T13:19:41Z Title: Characterisation and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note. Authors: James, Mark I.; Howells, Lynne M.; Karmokar, Ankur; Higgins, Jennifer A.; Greaves, Peter; Cai, Hong; Dennison, Ashley; Metcalfe, Matthew; Garcea, Giuseppe; Lloyd, David M.; Berry, D. P.; Steward, William P.; Brown, Karen Abstract: Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained. 2015-04-20T13:19:41Z 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 Novel method for the high-throughput processing of slides for the comet assay Karbaschi, Mahsa Cooke, Marcus S. http://hdl.handle.net/2381/32007 2015-04-17T02:00:17Z 2015-04-16T13:23:39Z Title: Novel method for the high-throughput processing of slides for the comet assay Authors: Karbaschi, Mahsa; Cooke, Marcus S. Abstract: Single cell gel electrophoresis (the comet assay), continues to gain popularity as a means of assessing DNA damage. However, the assay's low sample throughput and laborious sample workup procedure are limiting factors to its application. "Scoring", or individually determining DNA damage levels in 50 cells per treatment, is time-consuming, but with the advent of high-throughput scoring, the limitation is now the ability to process significant numbers of comet slides. We have developed a novel method by which multiple slides may be manipulated, and undergo electrophoresis, in batches of 25 rather than individually and, importantly, retains the use of standard microscope comet slides, which are the assay convention. This decreases assay time by 60%, and benefits from an electrophoresis tank with a substantially smaller footprint, and more uniform orientation of gels during electrophoresis. Our high-throughput variant of the comet assay greatly increases the number of samples analysed, decreases assay time, number of individual slide manipulations, reagent requirements and risk of damage to slides. The compact nature of the electrophoresis tank is of particular benefit to laboratories where bench space is at a premium. This novel approach is a significant advance on the current comet assay procedure. 2015-04-16T13:23:39Z 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 Differential regulation of insulin-like growth factor binding protein-1 and -2 by insulin in the baboon (Papio anubis) endometrium Fleming, S. D. Fazleabas, A. T. Bell, Stephen C. http://hdl.handle.net/2381/31929 2015-03-28T03:00:21Z 2015-03-27T12:24:16Z Title: Differential regulation of insulin-like growth factor binding protein-1 and -2 by insulin in the baboon (Papio anubis) endometrium Authors: Fleming, S. D.; Fazleabas, A. T.; Bell, Stephen C. Abstract: Background The purpose of this study was to examine the effect of insulin on expression and synthesis of IGFBP-1 and IGFBP-2 in the baboon endometrium in vitro. Methods Baboon endometrial explants collected from cycling, ovariectomized, steroid-treated, simulated-pregnant and pregnant animals were cultured for 48 h in the presence or absence of insulin, with or without estradiol, progesterone and hCG. Results Insulin clearly inhibited IGFBP-1 production and mRNA expression in a time- and dose-dependent manner, whereas IGFBP-2 synthesis was not significantly affected. The inhibitory effects of insulin on IGFBP-1 were more evident in explants of non-pregnant tissue or tissue away from the implantation site. In the absence of insulin, synthesis of IGFBP-1 was induced in explants with low levels of de novo synthesis whereas IGFBP-2 synthesis was inhibited. This effect was potentiated by steroids and hCG in the explant cultures. Conclusion Insulin differentially regulates endometrial IGFBP-1 and IGFBP-2 secretion in the baboon. 2015-03-27T12:24:16Z Characterisation and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note. Howells, Lynne M. James, Mark I. Karmokar, Ankur Higgins, Jennifer A. Greaves, Peter Cai, Hong Dennison, Ashley Metcalfe, Matthew Garcea, Giuseppe Lloyd, David M. Berry, David P. Steward, William P. Brown, Karen http://hdl.handle.net/2381/31905 2015-03-21T03:00:18Z 2015-03-20T08:52:57Z Title: Characterisation and propagation of tumor initiating cells derived from colorectal liver metastases: trials, tribulations and a cautionary note. Authors: Howells, Lynne M.; James, Mark I.; Karmokar, Ankur; Higgins, Jennifer A.; Greaves, Peter; Cai, Hong; Dennison, Ashley; Metcalfe, Matthew; Garcea, Giuseppe; Lloyd, David M.; Berry, David P.; Steward, William P.; Brown, Karen Abstract: Tumor initiating cells (TIC) are increasingly being put forward as a potential target for intervention within colorectal cancer. Whilst characterisation and outgrowth of these cells has been extensively undertaken in primary colorectal cancers, few data are available describing characteristics within the metastatic setting. Tissue was obtained from patients undergoing surgical resection for colorectal liver metastases, and processed into single cell suspension for assessment. Tumor initiating cells from liver metastases were characterised using combinations of EPCAM, Aldehyde dehydrogenase activity, CD133 and CD26. CD133 expression was significantly lower in patients who had received chemotherapy, but this was accounted for by a decrease observed in the male patient cohort only. ALDHhigh populations were rare (0.4 and 0.3% for EPCAM+/ALDHhigh/CD133- and EPCAM+/ALDHhigh/CD133+ populations respectively) and below the limits of detection in 28% of samples. Spheroid outgrowth of metastatic tumor cells across all samples could not be readily achieved using standard spheroid-formation techniques, thus requiring further method validation to reliably propagate cells from the majority of tissues. Spheroid formation was not enhanced using additional growth factors or fibroblast co-culture, but once cells were passaged through NOD-SCID mice, spheroid formation was observed in 82% samples, accompanied by a significant increase in CD26. Order of spheroid forming ability was ALDHhigh>CD133>CD26. Samples sorted by these markers each had the ability to reform ALDHhigh, CD133 and CD26 positive populations to a similar extent, suggestive of a high degree of plasticity for each population. Ex vivo TIC models are increasingly being utilised to assess efficacy of therapeutic interventions. It is therefore essential that such investigations use well-characterised models that are able to sustain TIC populations across a large patient cohort in order that the inherent heterogeneity observed in cancer populations is maintained. 2015-03-20T08:52:57Z The role of stromal fibroblasts in lung carcinogenesis: a target for chemoprevention? Howells, Lynne M. Mahale, Jagdish Smagurauskaite, Gintare Brown, Karen Thomas, Anne http://hdl.handle.net/2381/31904 2015-03-21T03:00:17Z 2015-03-20T08:45:04Z Title: The role of stromal fibroblasts in lung carcinogenesis: a target for chemoprevention? Authors: Howells, Lynne M.; Mahale, Jagdish; Smagurauskaite, Gintare; Brown, Karen; Thomas, Anne Abstract: The tumour microenvironment plays an essential role in the development and spread of cancers. Tumour cells interact with the surrounding extracellular matrix (ECM), embedded within which, are a variety of non-cancer cells including cells of the vasculature, immune system and fibroblasts. The essential role of fibroblasts in the cultivation and maintenance of an environment in which tumour cells are able to maintain their aggressive phenotypic traits is becoming increasingly well documented. Cancer-associated fibroblasts are able to secrete a vast array of ECM-modulating factors, meaning that they have potential for a functional role in every step of the carcinogenic process. In particular, they are likely to have a role in early tumour-initiating inflammatory events, and so may provide a potential target for chemopreventive intervention. This review summarises the known interactions between lung tumour cells and surrounding reactive fibroblasts, highlighting the need to further investigate cancer-associated fibroblasts as therapeutic targets in lung cancer chemoprevention strategies. Description: This is the accepted version of the following article: Mahale, J., Smagurauskaite, G., Brown, K., Thomas, A. and Howells, L. M. (2015), The role of stromal fibroblasts in lung carcinogenesis: A target for chemoprevention?. Int. J. Cancer. doi: 10.1002/ijc.29447, which has been published in final form at http://onlinelibrary.wiley.com/doi/10.1002/ijc.29447/abstract?systemMessage=Wiley+Online+Library+will+be+disrupted+on+21st+March+from+10%3A30+GMT+up+to+six+hours+and+from+05%3A30+EDT+up+to+six+hours+for+essential+maintenance.++Apologies+for+the+inconvenience. 2015-03-20T08:45:04Z 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 2015-02-03T15:47:28Z 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. Description: A retrospective embargo has been placed on the pdf version of this record. The file will be added to the record on 1st December 2015. 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 Cannabinoids and reproduction Habayeb, Osama http://hdl.handle.net/2381/29866 2014-12-16T02:12:49Z 2014-12-15T10:33:57Z Title: Cannabinoids and reproduction Authors: Habayeb, Osama Abstract: The success of implantation depends on the synchronous development of the embryo and the endometrium. This process is recognised to be regulated by the endocannabinoid system, the most widely studied of which is anandamide.;The first part of this study was the development of a robust assay to measure anandamide in human plasma. The assay was then applied to measure anandamide concentrations during the menstrual cycle, pregnancy, labour and in a group of women presenting with threatened miscarriage. The mean plasma anandamide levels in the follicular phase were 1.68 nM compared to 0.87 in the luteal phase. In pregnancy, the mean levels in the first trimester were 0.89 nM and 0.44 in both the second and third trimesters. At term, the mean levels were 0.68 in the non-labour group and to 2.5 nM in labour. In the threatened miscarriage group, anandamide levels >2.0 nM were predictive of subsequent miscarriage with sensitivity of 100%, specificity of 94.4%, negative predictive value of 100% and positive predictive value of 81.8%.;Finally, to try and identify potential targets of anandamide action, the expression of the cannabinoid receptors (CB1 & CB2) and the enzyme FAAH was studied in first trimester placentas. All proteins studied were present in the tissues examined with the expression of the CB1 diminishing after 9 weeks and FAAH disappearing by 11 weeks gestation. Similarly, anandamide inhibited the growth of BeWo cells in culture. Taken together, these findings suggest that anandamide plays a role in the maintenance of early pregnancy. 2014-12-15T10:33:57Z Molecular mechanisms of epithelial to mesenchymal transition (EMT) induced by smad-interacting protein 1 (SIP1) in a squamous epidermoid carcinoma cell line Mejlvang, Jakob http://hdl.handle.net/2381/29864 2014-12-16T02:12:47Z 2014-12-15T10:33:56Z Title: Molecular mechanisms of epithelial to mesenchymal transition (EMT) induced by smad-interacting protein 1 (SIP1) in a squamous epidermoid carcinoma cell line Authors: Mejlvang, Jakob Abstract: Programmes of epithelial mesenchymal transition (EMT) are crucial for normal embryonic development and represent a potential oncogenic mechanism aberrantly exploited in oncogenesis. A hallmark of EMT is the inactivation of the E-cadherin adhesive complex, which constitutes the backbone of intercellular adhesion in epithelial tissue. Although transcriptional repressors of E-cadherin (e.g. SNAIL, SIP1, Slug, ZEB1) have been identified as potent inducers of EMT, little is still known about the EMT-programs they initiate. In this study, we show that ectopic expression of SIP1 in A431 squamous carcinoma cells induces EMT manifested by cell scattering, abrogation of E-cadherin mediated adhesion, loss of apical-basolateral bipolarity, increased invasiveness, down-regulation of epithelial (e.g. E-cadherin, Claudin-4, Keratin 13 and 15) and up-regulation of mesenchymal (e.g. Vimentin) markers. In addition to these classical features of EMT, we show that SIP1 in our model directly represses the cyclin D1 promoter activity and thereby inhibits proliferation through the pRb-pathway. Hence, a fully compromised pRb pathway is likely necessary in order for SIP1 to prosper in oncogenesis. Furthermore, using a SIP1 mutant (SIP1CIDmt) incapable of binding the co-repressor CtBP, we found that the intrinsic CtBP-interacting domain (CID) is necessary for the transcriptional repression of E-cadherin. However, expression of SIP1CIDmt successfully induces a morphological transformation in A431 cells similar to wild type SIP1 indicating that SIP1-mediated loss of epithelial phenotype is largely independent of the simultaneous down regulation of E-cadherin. 2014-12-15T10:33:56Z Pre-clinical development of anthocyanins as colorectal cancer chemopreventive agents Cooke, Darren http://hdl.handle.net/2381/29865 2014-12-16T02:12:48Z 2014-12-15T10:33:56Z Title: Pre-clinical development of anthocyanins as colorectal cancer chemopreventive agents Authors: Cooke, Darren Abstract: The hypothesis was tested that dietary Mirtoselect, a standardised mixture of 15 anthocyanins, or the isolated anthocyanin cyanidin-3-glucoside (C3G) affect adenoma development in the ApcMIN mouse, a genetic model of colorectal cancer. Anthocyanins were added to the diet at 0.03, 0.1 or 0.3% (w/w) for the animals' life time. Adenoma number and size were measured at the end of the experiment (week 16). Both interventions reduced adenoma number/burden in a dose-dependent fashion. A potential mechanism by which anthocyanins are thought to exert cancer chemoprevention is anti-oxidation. Anti-oxidant effects of the interventions were investigated in DNA from ApcMIN murine adenoma tissue by analysis of the pyrimidopurinone adduct of deoxyguanosine M1dG, a product of the reaction of DNA with malondialdehyde. At the dietary dose of 0.3% C3G and Mirtoselect significantly reduced levels of M1dG, suggesting that antioxidation may contribute to anthocyanin-mediated interference with adenoma development. C57BL mice, the ApcMIN background strain, received C3G po or I.V. at 500 or 1 mg/kg, respectively. Levels of anthocyanins in plasma and tissue were measured using a newly developed HPLC method. C3G bioavailability was 3.28%, consistent with literature values after ingestion of berries/fruits. Anthocyanin species were detected in the plasma, urine, mucosa, liver, kidneys, heart, lung, gall bladder and brain. Absorption, distribution and excretion in the urine were rapid. The mean time of peak levels for all tissues studied was ∼26 min. The work described here defines further the potential role of anthocyanins in colorectal cancer chemoprevention. It also describes pharmacokinetic and pharmacodynamic features which may help optimise future clinical chemoprevention trials. 2014-12-15T10:33:56Z Delay of breast carcinogensis by green tea catechins and black tea theaflavins in the C3(1) SV40 T/t antigen (tag) transgenic mouse model Kaur, Siminder http://hdl.handle.net/2381/29863 2014-12-16T02:12:46Z 2014-12-15T10:33:55Z Title: Delay of breast carcinogensis by green tea catechins and black tea theaflavins in the C3(1) SV40 T/t antigen (tag) transgenic mouse model Authors: Kaur, Siminder Abstract: The aim of this study was to compare the efficacies of an extract of black tea (BTT) consisting predominantly of theaflavins with that of a green tea extract (GTC), containing mainly catechins. BTT/GTC reduced proliferation and increased apoptosis and malondialdehyde DNA adduct levels (M1dG) in breast cancer cells in vitro. Pharmacological properties of BTT and GTC were explored in the C3 (1)/SV40 T/t-antigen transgenic (Tag) mouse model of mammary carcinogenesis. Female Tag mice develop mammary tumours characterised by inactivated tumour suppressor genes p53 and Rb, thus mimicking an insidious hormone-insensitive form of human breast cancer. Mice received drinking water (controls) or drinking water containing BTT or GTC (0.05%/0.01%). Intervention with either BTT or GTC (0.05%) increased Tag mouse survival accompanied with a decrease in tumour volume and tumour burden. Microscopic evaluation revealed that GTC or BTT consumption decreased the size4 of the largest lesion compared to controls.;Increased survival was accompanied by a reduction in proliferation and an increase in apoptosis, as reflected by a decrease in PCNA levels and an increase in cleaved caspase 3 levels. Both tea extracts reduced tumour M1dG adduct levels. The results intimate that both GTC and BTT delay mammary tumour development in the Tag mouse model and M1dG and apoptotic signalling molecules may perhaps constitute suitable biomarkers of efficacy. 2014-12-15T10:33:55Z Experimental models of stem cell commitment and human disease Harrison, Lea-Anne http://hdl.handle.net/2381/29862 2014-12-16T02:12:45Z 2014-12-15T10:33:54Z Title: Experimental models of stem cell commitment and human disease Authors: Harrison, Lea-Anne Abstract: The aim of this thesis was to test the hypothesis that stem cells and their progenitors are important in metaplasia in the gastrointestinal (GI) tract across species. Immunohistochemistry (IHC) was employed to identify and analyse potential stem cell markers in human tissue. Putative stem cell location was mapped using a thymidine analogue to label slow cycling cells in human tissue. Zebrafish were investigated to examine deoxycholic acid (DCA), as a model for bile acid stimulation on stem cell fate, and whole animal histology was evaluated. Finally, the liver fatty acid binding protein (L-FABP) promoter was used to direct placental cadherin (P-cadherin) expression in the GI tract in a transgenic murine model. IHC analysis showed P-cadherin was expressed in the putative stem cell compartment in human oesophageal and Barrett's mucosa. Iododeoxyuridine (IUdR) labelling identified the stem cell compartment in human tissue. Double labelling, using IUdR with Ki-67 further identified a small stem cell side population in the basal compartments. The zebrafish model showed a mucin cell phenotype in the DCA stimulated animals, which was absent from controls. In the transgenic mouse model, although neoexpression of P-cadherin was demonstrated in the small bowel by RT-PCR, there was no obvious phenotype observed. However, there was an increase in stem cell division in the small bowel. In conclusion, the data generated in this thesis support the hypothesis that stem cells play a pivotal role in human disease. It would appear that the previously undefined stem cell compartment can now be elucidated and the P-cadherin upregulation is a feature of this location. 2014-12-15T10:33:54Z Tyrosine kinase inhibitors as adjuncts to chemotherapy in bladder cancer McHugh, Lynsey A. http://hdl.handle.net/2381/29860 2014-12-16T02:12:43Z 2014-12-15T10:33:53Z Title: Tyrosine kinase inhibitors as adjuncts to chemotherapy in bladder cancer Authors: McHugh, Lynsey A. Abstract: No abstract provided. 2014-12-15T10:33:53Z Colorectal cancer - the prediction of risk Rai, Sajal http://hdl.handle.net/2381/29861 2014-12-16T02:12:43Z 2014-12-15T10:33:53Z Title: Colorectal cancer - the prediction of risk Authors: Rai, Sajal Abstract: In the year 2000, the UK government implemented the 2 Week Wait (2WW) rule and CRC guidelines to provide primary care (General Practitioners) and secondary care (hospitals) with a method to help streamline and prioritize colorectal referrals.;In spite of the best of intentions, the 2WW system has failed to perform to its expectations. The yield of CRC from the 2WW referral route has been consistently poor in most centres in the UK. The NHS guidelines even if properly implemented have been shown to be insufficiently specific to pick up most CRCs.;Selvachandran et al from Crewe, UK have developed a computer program based Patient Consultation Questionnaire (PCQ) which is easy to complete by the patient, provides a comprehensive history and a significantly improved, reliable and specific cancer risk assessment as compared to NHS guidelines. The Weighted Numerical Score (WNS) or Selva score derived from the PCQ reflects the patient's risk of having CRC. The higher the score, the better is the specificity and positive predictive value for detecting CRC. Our study has validated this questionnaire based cancer risk assessment tool in the large and ethnically diverse population of Leicester. We have shown that the PCQ based WNS improves specificity resulting in improved CRC detection rate in a significantly smaller urgently referred population. We have also assessed patient satisfaction with the use of the PCQ.;CRC presenting as an emergency continues to form a significant proportion of the total number of CRCs diagnosed in any centre. We have shown that these patients differ in their presentation and outcome from the elective patients. Lack of any specific colorectal symptoms may result in a delay in the diagnosis in some of these patients who have proximal colon cancers. Similarly, the incidence and presentation of CRC in the ethnic population differs significantly for that of the native population. 2014-12-15T10:33:53Z The value of regional intra-arterial therapy with novel sustained-release ethiodol based preparations for hepatic-neoplastic disease Dookeran, Keith A. http://hdl.handle.net/2381/29858 2014-12-16T02:12:41Z 2014-12-15T10:33:52Z Title: The value of regional intra-arterial therapy with novel sustained-release ethiodol based preparations for hepatic-neoplastic disease Authors: Dookeran, Keith A. Abstract: We continue to search for effective therapies for hepatic-metastatic disease. Loco-regional therapy is attractive because it allows for tumour directed dose intensification without associated increase in systemic toxicity, which may lead to improved efficacy. In addition, the use of sustained-release or depot preparations may obviate the need for continuous infusion via expensive implantable pumps, and may also allow treatment of patients who are unfit for surgery, or who have more advanced disease. The iodinated poppyseed oil, Ethiodol, is selectively retained in liver tumours for weeks to months after hepatic intra-arterial infusion (HAI), and demonstrates ideal properties as a vehicle for development of depot or sustained-release HAI therapies of lipophillic or oil-soluble novel anticancer agents. This project demonstrated the value of Ethiodol for this purpose. A reproducible experimental animal hepatic-metastatic model using Fisher rats and syngenic MADB106 breast adenocarcinoma cells was established, and allowed evaluation of Ethiodol based HAI preparations. Several novel anticancer agents were soluble in Ethiodol and exhibited sustained-release characteristics. These preparations included (a) the topoisomerase-1 inhibitor 9-aminocamptothecin (9AC), (b) the cytokine human recombinant interleukin-2 (Hril2), and (c) several nitric oxide (NO) donors NOR1, NOR2, NOR3, SNAP and sodium nitroprusside. These compounds were effective antitumour agents both in vitro and in vivo, and inhibited tumour engraftment after subcutaneous inoculation with admixed tumour cells. They were well tolerated and eradicated hepatic-metastatic disease when administered as loco-regional HAI therapy, with decreased toxicity compared to equivalent systemic therapy. Investigation of mechanisms of antitimour activity revealed that macrophages appeared to be important for hRIL2 activity, and that NO and cyanide ion production appeared to both contribute to the effects of sodium nitroprusside. Results suggest that clinical studies of these compounds are feasible and warrant further investigation. 2014-12-15T10:33:52Z Studies of the chemopreventive efficacy and pharmacokinetics of curcumin in a murine model of colorectal cancer Perkins, Sarah. http://hdl.handle.net/2381/29859 2014-12-16T02:12:42Z 2014-12-15T10:33:52Z Title: Studies of the chemopreventive efficacy and pharmacokinetics of curcumin in a murine model of colorectal cancer Authors: Perkins, Sarah. Abstract: Curcumin, the major yellow pigment in turmeric, prevents the development of adenomas in the intestinal tract of the C57BI/6J Min/+ mouse, a model of human colorectal cancer with an Apc gene mutation. In order to aid the rational development of curcumin as a colorectal cancer preventive agent, and the link between its chemopreventive potency in the Min/+ mouse and levels of drug and metabolites in target tissue and plasma was explored.;Curcumin was administered in the diet at a variety of dose levels and time periods to assess the effect of this phytochemical on tumour burden in Min/+ mice. The growth, survival and haematocrit of the mice were investigated. The effect of a combination of aspirin and curcumin was also explored as well as the effect of altering the dietary matrix. Long-term administration of curcumin (0.2%) was efficacious at reducing tumour multiplicity in the small intestine by 39%. This dosing regime was utilised to study the effect of curcumin on potential markers of carcinogenesis. These included cyclooxygenase-2 (COX-2) expression, malondialdehyde (MDA), and deoxyguanosine adducts, M1G and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG). The studies tested the potential application of these markers to carcinogenesis and investigated the antioxidant properties of curcumin. Potential markers of carcinogenesis and mechanisms of action of curcumin were also studied using cDNA microarray. The results highlighted significant gene changes in 420 genes, many of which are germane to chemoprevention, most notably genes involved in antioxidation. Pharmacokinetics of dietary administration of curcumin suggests that this phytochemical is poorly bioavailable with the majority of the agent excreted unchanged in the faeces.;Curcumin reduces tumour in Min/+ mouse, at least in part through reduction in oxidative DNA damage and suppression of COX-2 activity. In conclusion the results support the clinical evaluation of curcumin as a colorectal cancer chemopreventive agent. 2014-12-15T10:33:52Z The effect of carbonated soft drink components on isolated detrusor muscle contraction Dasgupta, Jaydip http://hdl.handle.net/2381/29857 2014-12-16T02:12:39Z 2014-12-15T10:33:51Z Title: The effect of carbonated soft drink components on isolated detrusor muscle contraction Authors: Dasgupta, Jaydip Abstract: The objective of our study was to investigate the hypothesis that carbonated soft drink components modulate detrusor muscle contraction. Strips of rat detrusor muscle were placed in an organ bath and stimulated with electrical field stimulation (EFS) in the absence and presence of atropine, and with alpha, beta methylene ATP, potassium, calcium and carbachol. The responses were repeated in the presence of carbonated soft drink components and the whole carbonated soft drink. The artificial sweeteners, ascorbic acid and critic acid (10-7 M to 10-2 M) enhanced the contractile response to 10 Hz EPS compared to control (p<0.01). The sweeteners (acesulfame K 10-6 M, aspartame 10-7M, sodium saccharin 10-7 M) increased the atropine resistant response to EFS marginally. Acesulfame K 10-6 M and sodium saccharin 10-7 M enhanced the maximum contractile response to alpha, beta methylene ATP, to KC1 and calcium significantly compared to control. Ascorbic acid significantly increased the atropine resistant response to EFS and inhibited contraction in response to carbachol. Both ascorbic acid and citric acid enhanced the contractile responses to alpha, beta methylene ATP, KC1 and calcium significantly compared to control. Whole carbonated soft drink (1:200) also enhanced the contractile responses to alpha,beta methylene ATP, KC1 and calcium. These results suggested that low concentrations of artificial sweeteners, ascorbic acid and critic acid, enhanced detrusor muscle contraction by augmenting Ca2+ influx by a mechanism yet to be defined. Western blot analysis suggested the presence of protein molecules in the rat bladder of similar size to the sweet taste receptor present in tongue. These data suggest that carbonated soft drink components may be acting via specific receptors to modulate the effects observed. 2014-12-15T10:33:51Z Molecular pathology as a tool to determine features of clinico-pathological progression and malignancy in melanocytic neoplasia Da Forno, Philip D. http://hdl.handle.net/2381/29856 2014-12-16T02:12:38Z 2014-12-15T10:33:50Z Title: Molecular pathology as a tool to determine features of clinico-pathological progression and malignancy in melanocytic neoplasia Authors: Da Forno, Philip D. Abstract: This thesis broadly comprises two investigations of melanocytic tumour progression.;1. The signalling ligand Wnt5a has been shown to promote motility and invasiveness in melanoma in vitro. To expand upon this data, expression of Wnt5a was examined in matched and unmatched tumour samples comprising different stages of melanoma progression. In order to place the alterations of Wnt5a in context, expression of p16INK4a and mutations of B-raf exon 15 were determined. The effect of Wnt5a expression on outcome was assessed.Wnt5a demonstrated a trend of increasing expression with progression (p = 0.013) however, expression in naevi was comparatively strong. When this was compared with p16ink4a expression and BRAF mutation, the changes supported a multi-step process of carcinogenesis. Survival analysis showed that Wnt5a is an independent marker of time to metastasis (p=0.041) and death (p=0.047).;2. B-raf and N-ras mutations are common in most melanocytic tumours, but are rare in Spitz naevi, where occasional H-ras mutations are found. The hypotheses were that Spitzoid tumours comprise a distinct subgroup of lesions that progress in the absence of B-raf and N-ras mutations, unlike most other melanocytic tumour types; but that progression from Spitz naevus to Spitzoid melanoma is unlikely because there is no subset of H-ras mutant Spitzoid melanomas. B-raf and N-ras mutations were found at lower frequency in Spitzoid melanomas (p=0.009) and in all Spitzoid tumour types (p=0.001) compared to non-Spitzoid melanomas, indicating that Spitz naevi and non-classical tumours are genetically distinct. H-ras mutations were not seen in malignant Spitzoid and non-Spitzoid melanomas indicating that H-ras mutant Spitzoid tumours do not progress to malignancy.;The findings of these investigations demonstrate the utility of molecular pathology in characterising neoplastic progression in archival melanocytic tumour tissue. 2014-12-15T10:33:50Z The effects of selective oestrogen receptor modulators in the uterus Gray, Douglas Thomas http://hdl.handle.net/2381/29855 2014-12-16T02:12:37Z 2014-12-15T10:33:50Z Title: The effects of selective oestrogen receptor modulators in the uterus Authors: Gray, Douglas Thomas Abstract: The effects of tamoxifen on uterine gene and protein expression have been examined in both women and mice treated with this drug. In the endometrium of women taking tamoxifen for up to 4 years, analysis of gene expression using cDNA arrays, indicated that tamoxifen increased expression of genes that may be involved in cell proliferation. In the endometrium of post-menopausal women, expression of the paracrine signalling protein nerve growth factor (NGF) was increased, although real time PCR showed no increase in NGF expression. Mouse models were used to compare the long-term effects of tamoxifen, oestradiol and 4-hydroxyoestradiol on uterine pathology and gene expression. In uterotrophic assays, oral dosing of oestradiol, tamoxifen or 4-hydroxyestradiol, administered to immature CD-1 mice on 3 days all led to increases in uterine weight, although not necessarily by cell proliferation. In newborn CD-1 mice the maximal uterotrophic dose of oestradiol (100 microg/kg), 4-hydroxyoestradiol (386 microg/kg) or tamoxifen (250 microg/kg) were given orally on days 1 to 5 after birth and gene and pathological changes examined in the uterus 3 months after dosing. Tamoxifen but not 4-hydroxyoestradiol or oestradiol led to adenomyosis. At this time, the expression of >250 genes were significantly altered by each treatment but changes in only nine genes were common to all three oestrogens. It is concluded that not all oestrogen agonists are the same in this model and while oestrogenicity is needed for the development of adenomyosis additional, unidentified factors, are required. 2014-12-15T10:33:50Z Effects of vitamin c on dna damage, iron homeostasis and gene expression profiling in human fibroblasts Duarte, Tiago Pereira de Lacerda Costa http://hdl.handle.net/2381/29854 2014-12-16T02:12:35Z 2014-12-15T10:33:49Z Title: Effects of vitamin c on dna damage, iron homeostasis and gene expression profiling in human fibroblasts Authors: Duarte, Tiago Pereira de Lacerda Costa Abstract: Vitamin C (ascorbic acid, AA) is an important antioxidant in human plasma. AA has, however, other important, non-antioxidant roles in cells. Of particular interest is its involvement in iron metabolism, since AA enhances dietary iron absorption, promotes Fenton reactions in vitro and was reported to have deleterious effects in individuals with iron overload. The mechanisms of AA-driven free radical generation from iron have been the subject of great discussion. However, whilst the AA-driven Fenton reaction is well established in test tube reactions, the relevance of this pro-oxidant chemistry in cells, tissues or organisms remains unclear. To understand whether AA has an overall antioxidant or pro-oxidant effect in cells, the present study investigated its ability to modulate iron homeostasis and iron-mediated oxidative injury in primary normal human diploid fibroblasts. Results show that AA increased the levels of intracellular catalytic iron and concomitantly modulated the expression of two well known iron-regulated genes, ferritin and transferrin receptor. Notably, treatment of confluent fibroblasts with physiologically relevant concentrations of AA was not harmful but sensitised cells towards iron-dependent, H2O2-induced DNA strand breakage and cell death. The possibility that AA regulates cellular function, through its effects on the intracellular redox state and/or on iron homeostasis, was also addressed by performing a genome- wide analysis of AA-induced gene expression in human fibroblasts. Results showed that AA stimulates quiescent cell populations to re-enter the cell cycle and proliferate. In summary, this work shows that AA increases iron availability and enhances ROS-mediated, iron-dependent damage in human cells. It is proposed that AA may exacerbate the deleterious effects of metals in vivo and promote normal tissue injury in situations associated with elevated ROS production. It is also suggested that AA may promote fibroblast activation during the process of wound healing, where quiescent dermal fibroblasts are required to readily undergo cell division. 2014-12-15T10:33:49Z Comparison of the mechanisms of action of three cancer chemopreventive flavonoids Al-Fayez, Mohammad A. http://hdl.handle.net/2381/29853 2014-12-16T02:12:33Z 2014-12-15T10:33:49Z Title: Comparison of the mechanisms of action of three cancer chemopreventive flavonoids Authors: Al-Fayez, Mohammad A. Abstract: Tricin (4',5,7-trihydroxy-3',5 '-dimethoxyflavone) from grass species and apigenin (4 ',5,7-trihydroxyflavone) from leafy vegetables are chemically closely related flavones. Quercetin (3,5,7,3',4 '-pentahydroxyflavone) is a flavonol contained in onions, wine, French beans and apples. These flavonoids have been reported to inhibit the growth of a variety of human-derived malignant cells in vitro at microM concentrations. Tricin, apigenin and quercetin showed differential growth-modulating abilities when they were incubated with colon-derived cells. Apigenin and more growth-inhibitory than tricin and quercetin in SW480 and HCA-7 cells. Whilst apigenin at 40 microM induced moderate apoptosis in HCA-7 cells, it arrested the cell cycle of SW480 cells without induction of apoptosis. In contrast to tricin and quercetin, which inhibited activity of COX enzymes strongly in a cell free system and decreased cellular PGE-2 levels moderately in HCA-7 and HCEC cells, apigenin lacked COX enzyme-inhibitory activity in the cell-free system. However, it reduced cellular PGE-2 moderately in HCA-7 cells and dramatically in HCEC cells, but it did not down-regulate COX-2 expression. The effect of apigenin on PGE-2 levels suggests that it may target the PGE-2 signalling pathway without effect on COX activity and expression. It is conceivable that apigenin affects cellular PGE-2 levels by induction or stimulation of the PGE-2 metabolising enzyme 15-hydroxyprostaglandin dehydrogenase. The results suggest that the pharmacological profile of apigenin renders it perhaps a more attractive chemopreventive agent than tricin or quercetin. The further exploration of the cancer chemopreventive properties of apigenin in rodent model in vivo is required. 2014-12-15T10:33:49Z Analysis of stromal changes in colorectal disease Grierson, Catherine http://hdl.handle.net/2381/29852 2014-12-16T02:12:32Z 2014-12-15T10:33:49Z Title: Analysis of stromal changes in colorectal disease Authors: Grierson, Catherine Abstract: There is accumulating evidence that the micro-environment plays a pivotal role in the progression of malignancy and that key stromal changes can be identified which influence this progress. This investigation focussed on two specific areas; fibroblast phenotype and the composition of the extracellular matrix, especially regarding the protein-tenascin-C (TN-C).;The heterogeneity within colonic fibroblast populations is becoming increasingly apparent. Using immunohistochemical techniques this study defined specific populations and identified consistent phenotypic changes in these cells in malignancy. In particular CD34 expression was lost in sub-mucosal fibroblast and alpha smooth muscle actin (alphaSMA) expression increased within tumour stroma. There was also evidence of progressive loss of high molecular weight caldesmon (hCD) expression by the pericryptal myofibroblasts with the development of malignancy. The stromal changes were specific to malignancy and were not demonstrated in pre-invasive adenomas.;TN-C is an important component of the extracellular matrix (ECM) which can occur as several different isoforms. Immunohistochemical techniques were used to demonstrate alterations in the distribution of TN-C between normal and malignant colon and the presence of isoforms containing exon 14 in both. The distribution of TN-C in pre-invasive adenomas was the same as that in normal colon but some sub-mucosal TN-C expression including exon 14 containing isoforms was observed in acute inflammation. Polymerase chain reactions (PCR) identified a total of 7 TN-C isoforms in colonic tissue with no obvious association between isoforms profile and disease.;TN-C can also be expressed by tumour cells and PCR demonstrated multiple isoforms in the colorectal tumour cell lines SW480, SW620, HCT15 and HT29. However, TN-C expression did not appear to correlate with invasive capacity as demonstrated in preliminary in-vitro invasion assays. 2014-12-15T10:33:49Z Analysis of genetic variation of the pregnane x receptor in putative cholestatic adverse drug reactions using archival liver biopsies : identification of a novel dysfuctional protein varient Carr, Daniel Frank http://hdl.handle.net/2381/29851 2014-12-16T02:12:32Z 2014-12-15T10:33:48Z Title: Analysis of genetic variation of the pregnane x receptor in putative cholestatic adverse drug reactions using archival liver biopsies : identification of a novel dysfuctional protein varient Authors: Carr, Daniel Frank Abstract: A central role in drug clearance makes PXR a strong candidate for altered functionality in adverse drug reactions (ADR), particularly drug-induced cholestasis. By screening in archive of clinical liver biopsies, 53 patients displaying evidence of cholestatic ADRs have been identified. These liver specimens were formalin-fixed, therefore nucleic acid yield and integrity is compromised. To overcome this, DNA extraction and amplification methods were optimised for sequencing of the open reading frames of the PXR gene allowing as little as lng of genomic DNA to be analysed. Simultaneous mRNA extraction and RT-PCR analysis was also developed. In the majority of cases sequence analysis revealed no significant variants. However, in two cases (3.6%) a non-synonymous T/C base substitution encoding a novel PXR protein variant, C301R, was identified as a heterozygote. In a normal control cohort of 308 no incidence of C301R was observed (p=0,023). Functional analysis by reporter gene co-transfection assay of this variant discovered an apparent attenuation of ligand (hyperforin and rifampicin)-mediated transcriptional activation, of both DR3 and ER6 CYP3A response elements compared to the wild-type human PXR. The C301R variant displayed no loss of DNA response element binding compared to wild-type PXR. The identification of this variant protein at a significantly increased frequency compared to a normal population, in a hepatic ADR cohort, would suggest that this apparently functional PXR variant may be one single predisposing factor to ADR. Similar utilisation of archival biopsies could be used to identify further candidate genes associated to ADR pre-disposition. 2014-12-15T10:33:48Z Analysis of DNA damage and cellular responses to platinum-based chemotherapeutic agents in 'in vitro' models of non-small cell lung cancer Almeida, Gabriela Martinho de http://hdl.handle.net/2381/29850 2014-12-16T02:12:31Z 2014-12-15T10:33:48Z Title: Analysis of DNA damage and cellular responses to platinum-based chemotherapeutic agents in 'in vitro' models of non-small cell lung cancer Authors: Almeida, Gabriela Martinho de Abstract: Response to cisplatin was investigated in a panel of NSCLC cell lines in terms of sensitivity/resistance to this agent, DNA crosslink formation and repair, cell cycle effects, apoptosis and gene expression changes. Measures of survival revealed a differential response between cell lines. The level of maximal DNA crosslink formation and efficiency of repair was also variable, with the most cisplatin-resistant cell lines being the ones with higher levels of crosslinks and poorer repair. Cisplatin induced a transient cell cycle arrest at S and G2/M phases in cell lines; however, only the most cisplatin-resistant ones were able to overcome this arrest. The degree of cisplatin-induced apoptosis was variable, but no direct correlation was found between cell sensitivity and apoptotic index. Cisplatin induced the expression of several apoptotic, cell cycle regulator and DNA repair genes. The fact that cisplatin-resistant cells exhibited higher crosslink formation and reduced repair was somehow unanticipated and could be attributed to increased lesion tolerance, i.e. an ability of the most resistant cells to progress through the cell cycle without completely repairing cisplatin-induced damage. Lesion tolerance may, therefore, be an important route for cisplatin resistance in the NSCLC panel. Finally, the alkaline Comet assay was used, for the first time, to detect the formation and repair of oxaliplatin-induced DNA crosslinks in cultured cells and in peripheral blood lymphocytes of patients undergoing chemotherapy. An inter-individual variability in crosslink formation and repair efficiency was observed. The Comet assay could therefore be a useful tool in future oxaliplatin-related clinical studies. 2014-12-15T10:33:48Z Sporadic breast cancer in young women : a microarray investigation Whyman, Gavin John http://hdl.handle.net/2381/29849 2014-12-16T02:12:29Z 2014-12-15T10:33:48Z Title: Sporadic breast cancer in young women : a microarray investigation Authors: Whyman, Gavin John Abstract: The aim of this thesis was to test the hypothesis that sporadic breast cancers in young women arise because of a profile of genetic alterations specific to that age group. Previous studies by our research group had identified a higher frequency of Loss of Heterozygosity (LOH) in the BRCA1, BRCA2 and p53 intervals in younger breast cancer cases (55 years); BRCA1 and P53 data had been related to protein expression. Investigations of BRCA2 protein and mRNA expression were inconclusive. A higher frequency of skewed X chromosome inactivation was identified in young women (55 years) with sporadic breast cancer in this study (P = 0.017), which might suggest the involvement of an X linked gene in the genesis of these tumours. Although it was not possible to relate cDNA expression levels to genomic copy number changes for individual tumours, the analysis of gene expression changes using Affymetrix Gene Chips identified a number of novel gene targets showing elevated expression in young breast cancers when compared to normal breast organoids. Several statistically significant candidate genes were investigated, and gene expression changes confirmed by qRT-PCR and for RBBP4 by immunohistochemistry. Of the significant gene targets that were ranked by SAM analysis DDB2 and RARRES3 are the most noteworthy. Statistically significant differences in the level of expression of these two genes might suggest that they are novel markers of young breast cancers, meriting investigation in a larger series of cases. 2014-12-15T10:33:48Z Preclinical and clinical development of biomarkers of the efficacy of curcumin in the prevention and treatment of colorectal cancer Sharma, Ricky A. http://hdl.handle.net/2381/29848 2014-12-16T02:12:28Z 2014-12-15T10:33:47Z Title: Preclinical and clinical development of biomarkers of the efficacy of curcumin in the prevention and treatment of colorectal cancer Authors: Sharma, Ricky A. Abstract: This project explored the suitability of three biological indices as potential markers of the efficacy of curcumin in preventing or treating colorectal carcinoma: glutathione S-transferase (GST) activity; a deoxyguanosine adduct (M1G) associated with oxidative DNA damage and malondialdehyde (MDA) production via lipid peroxidation (LPO); and levels of cyclooxygenase-2 (COX-2) protein and its product prostaglandin E2 (PGE2). The viability of these biomarkers was studied at three levels: malignant and non-malignant human colon cells in vitro; a rat model of LPO and the multiple intestinal neoplasia (MIN) mouse model; and a pilot study of a standardised oral preparation of Curcuma extract in patients with advanced colorectal cancer.;The results in vitro demonstrated an association between COX-2 activity and intracellular MDA levels, and a correlation in malignant cells between MDA concentration and M1G adduct levels. A diet containing 2% curcumin decreased M1G levels in rat colon mucosa and increased GST liver activity, compared to controls; the same diet completely prevented chemically induced rises in M1G levels in colon mucosa. Elevated levels of M1G were discovered in intestinal adenomas in MIN mice, relative to normal mucosa, and 0.1% dietary curcumin significantly decreased those levels. Although addition of curcumin to human blood in vitro was shown to inhibit leukocyte COX-2 activity, dose-dependent effects definitely attributable to treatment were not observed in the three biomarkers measured in the clinical trial of Curcuma extract. Compatible with the tissue levels described in the preclinical studies, curcumin's systemic bioavailability following oral dosing appears to be low in humans.;Future development of curcumin as a cancer chemopreventive agent should study its effects on M1G levels, COX-2 activity and GST isoforms in colorectal adenomas. 2014-12-15T10:33:47Z Development of novel therapies for colorectal carcinoma using dietary indoles Brookes, Alastair Forbes http://hdl.handle.net/2381/29325 2014-12-09T02:01:55Z 2014-12-08T11:42:45Z Title: Development of novel therapies for colorectal carcinoma using dietary indoles Authors: Brookes, Alastair Forbes Abstract: Colorectal carcinoma is the third commonest cancer in the UK and is the second commonest cause of cancer related death. There is a need for efficacious chemotherapeutic and chemopreventative agents against colorectal carcinogenesis. Indole-3-carbinol and its acid condensation product, 3,3 diindolylmethane (DIM), exhibit anti-tumourigenic activity in preclinical models. This study investigated their therapeutic potential in colorectal carcinoma. A panel of four colorectal carcinoma cell lines (HT29, HCT116, SW480 and SW620) was used to compare effects of DIM and I3C on cell viability, induction of apoptosis and cell cycle arrest. Treatment with DIM or I3C resulted in dose-dependent reductions in cell viability. Cell lines were sensitive to 25–75 μM DIM (GI[subscript 50] = 45 μM), but not to 200 μM I3C. DIM (40 μM) induced apoptosis in all cell lines. This was associated with downregulation of survivin and appeared to be independent of p53. Furthermore it induced G[subscript 1] arrest in HCT116, SW480 and SW620 and G[subscript 2]/M arrest in HT29. DIM induced activatory phosphorylation of Chk2 in HT29 but not in HCT116. DIM downregulated cell cycle proteins: PLK1, cdc25C, cdc2 and cyclins B1, D1 and E. Since all colon cancer cell lines expressed significant levels of Src, their response to Src inhibition was investigated. Colon cancer cells were modestly sensitive to AZD0530 (AstraZeneca), but combination with indoles reduced viability to a greater extent than single agent treatment. Combining 40 μM DIM with 1 μM AZD0530 was particularly effective, reducing viability by half after 48 hours on average. Decreases were sustained and progressive over extended timepoints. Attempts were made to establish primary cultures from resected hepatic metastases however issues were identified with cell death, fibroblast overgrowth and slow cell turnover. Potential changes to address these were identified. These data suggest chemotherapeutic potential of DIM in colon cancer, particularly in combination with Src inhibition. 2014-12-08T11:42:45Z